Increased Asymmetric Dimethylarginine In Patients With Hypertension: A Systematic Review And Meta-Analysis


 Background: Hypertension is the leading risk factor for vascular diseases, such as coronary artery disease, arterial stiffness, and stroke. Elevated asymmetric dimethylarginine (ADMA) levels have been reported to be an independent predictor of all-cause mortality. However, the role of ADMA in hypertension remains undetermined. To derive a more accurate estimate of circulating ADMA levels in hypertension patients, a meta-analysis was performed. Result: The Embase, PubMed, Cochrane Library, and Web of Science databases were searched (up to October 10, 2021) for relevant published literature. To assess study heterogeneity, Cochran’s Q and the I2 statistic were calculated. Publication bias was evaluated using a funnel plot. A total of 1951 articles were identified, of which 28 studies with 2694 subjects (1534 hypertension patients and 1160 healthy controls) were ultimately included. Plasma/serum ADMA levels were found to be higher in hypertension patients than in healthy controls (standard mean difference = 1.27, 95% confidence interval [0.99, 1.54]). Conclusion: Therefore, strategies aimed at reducing ADMA may constitute an effective therapeutic approach to prevent the development of hypertension in patients.


Introduction
Hypertension, as a chronic disease, is one of the most prevalent ve diseases and causes a large burden of disease worldwide (1). The overall prevalence of hypertension in adults is approximately 30-45%, and its prevalence increases progressively with age (2,3). In adults, hypertension is the leading risk factor for cardiovascular disease, dyslipidemia, glucose intolerance, stroke, renal events, and heart failure (3,4). The endogenous vasodilator nitric oxide (NO) has been identi ed as the endothelium-derived relaxing factor, the decrease of NO contributes to endothelial dysfunction. (5) Endothelial vasodilator dysfunction contributes to the progression of hypertension(6, 7). NO production is diminished in patients with essential hypertension(8). Therefore, preventing NO reduction in hypertensive patients may be an important way to prevent and treat hypertension.
Asymmetric dimethylarginine (ADMA) is a circulating endogenous NO synthase inhibitor (7). Increased circulating ADMA may impair the bene cial effects of NO on endothelial vasodilator function (9).
Elevation of ADMA levels has been found to be associated with carotid atherosclerosis, ischemic stroke, cardiovascular disease, and cerebrovascular disease (10,11).
Multiple studies have also shown that hypertension patients have elevated ADMA levels compared to controls (12)(13)(14)(15). However, some studies have reported differing results(16, 17). Therefore, a metaanalysis was performed to resolve inconsistencies in the literature and overcome the limitations of small sample sizes of individual studies. We systematically reviewed the available data to form a more accurate representation of the differences in ADMA levels between individuals with hypertension and those with normotension.

Search strategy
We performed a systematic search strategy to identify all relevant publications without any language restrictions. Two reviewers (X.-H.C. and H.W.) independently searched the Embase, PubMed, Cochrane Library, and Web of Science databases from inception to October 10, 2021. The reference lists of the retrieved articles were also screened for related research. The search strategies for the key databases are presented in Appendix 1.

Study selection
We selected studies that investigated the relationship between ADMA levels and hypertension. The title and abstract of each article were examined independently by the two reviewers (X.-H.C. and T.X.). After eliminating duplicates, the titles and abstracts of the identi ed articles were screened to exclude irrelevant studies. The full-text article was obtained if determined eligible by both reviewers. Any disagreements were discussed and resolved by consensus.
Studies were required to meet the following criteria to be included in the meta-analysis: 1) included patients were ≥ 18 years old; 2) a cross-sectional, cohort, or case-control study design was applied, with data on both patients diagnosed as having hypertension and normotension controls; and 3) detailed data regarding ADMA levels were provided for both groups. If any study was reported in more than one publication, the most comprehensive study employing the largest sample size was included.

Data extraction
A data extraction form was designed by the two reviewers (X.-H.C. and L.X.). The reviewers extracted the relevant data from all eligible studies, and any disagreements were resolved by consensus. The following data was extracted: the name of the rst author, year of publication, type of study, mean or range age of participants, proportion of male participants, de nition of hypertension, sample size, ADMA measure, and the obtained ADMA values.

Quality assessment
The Newcastle-Ottawa Scale was used to assess the quality of the included studies(18). Studies with a score of ≥ 5 out of 9 were considered high-quality, while those with a score < 5 were deemed low-quality.
Any disagreements among the reviewers were discussed and resolved by consensus.

Statistical analysis
All statistical analyses were conducted via Revman software (version 5.3.0, Copenhagen, Denmark). Across several studies (13,17,(19)(20)(21)(22)(23)(24), the data were presented as mean ± standard error of the mean (SEM). We used the following formula to transfer the SEM to standard deviation (SD) (µmol/L): SD = SEMx √ N. In addition, for some studies(21, 22, 24), we used a calculator to transfer median and range into mean ± SD (25). The units for ADMA levels differed between studies, including nmol/L(16), ng/L(26), and ng/ml (27). The molecular formula of ADMA is C 8 H 18 N 4 O 2 , and the molar mass (M) of ADMA is 202.2565 g/mol; therefore, we employed the following formula to transfer the original units of After adjusting the ADMA levels into the same units (µmol/L), standard mean difference (SMD) and 95% con dence interval (CI) were calculated as summary statistics. The I 2 and Cochran's Q tests were performed to assess the heterogeneity between the studies, with I 2 > 50% or p < 0.05, respectively, indicating a statistically signi cant heterogeneity(28). If signi cant heterogeneity was found (p < 0.05) across the studies, the random-effect model would be performed. Otherwise, a xed-effect model would be employed. Finally, a funnel plot was generated to detect possible publication bias (29). 3 Results

Study selection
A total of 1951 studies were identi ed using manual and electronic search methods. After reviewing the abstract and/or the full text, 1918 articles were excluded. Thus, 28 studies met the inclusion criteria for the current meta-analysis ( Figure 1).

Study characteristics
Characteristics of the included trials are summarized in Table 1. The reviewed trials were published from 1999 to 2021, with sample sizes ranging from 19 to 400 subjects and a total of 2694 subjects (1534 hypertension patients and 1160 normotension controls) included in the meta-analysis. Age ranged from 18 to 87 years. Sixteen trials excluded hypertensive patients who had used anti-hypertensive drugs, 11 trials included hypertensive patients who were receiving anti-hypertensive therapy, and one trial did not report the use of anti-hypertensive drugs. All the included studies had a case-control study design. Concerning the applied quality assessment, 3 trials received 6 points on the applied Newcastle-Ottawa Scale, 17 trials received 7 points, whereas 8 trials received ≥ 8 points on this scale. In 16 (57.1%) trials, ADMA measurements were taken from plasma, 11 (39.3%) were taken from serum, and one trial did not report the origin of their ADMA measurement. In the hypertension group, the median of the recorded mean ADMA levels was 0.705 µmol/L. However, in the normotension group, the median of the recorded mean ADMA levels was 0.476 µmol/L.

Main ndings
The forest plot displaying the mean ADMA concentrations in hypertension patients and controls across the included trials is shown in Figure 2. Due to the marked heterogeneity between studies (I 2 = 88%, p < 0.001), random-effects models were used to perform the statistical analysis. The pooled results showed that ADMA concentrations were signi cantly higher in patients with hypertension than in normotension controls (SMD = 1.27 µmol/L, 95% CI [0.99, 1.54 µmol/L]; p < 0.001).

Publication bias
There was no evidence of publication bias following inspection of the funnel plot ( Figure 3).

Discussion
This is the rst meta-analysis to evaluate the potential association between ADMA levels and hypertension. Our meta-analysis found that circulating levels of the endogenous NO synthase inhibitor ADMA were positively correlated with hypertension, indicating that: (1) circulating ADMA levels are a novel marker of subclinical hypertension and may be a predictor for the development of hypertensive disease; (2) elevated ADMA levels and/or mechanisms underlying increases in ADMA may play a pivotal role in the development of hypertension.
Hypertension can originate from early life, known as the developmental origins of health and disease (30). Blood pressure (BP) is strongly and directly related to vascular mortality, increases in BP lead to increased mortality rate (31). Large artery stiffening is the most important pathophysiological determinant of hypertension and age-dependent increases in pulse pressure (3). Anti-hypertensive treatment can normalize BP in a few weeks, but does not improve atherosclerotic lesions or arterial stiffness (32).
Endothelial dysfunction is associated with increased arterial stiffness and the development of hypertension(2), and is a predictor of subsequent adverse cardiovascular events (33). NO is a powerful regulator of endothelial function, and abnormalities in the production or actions of NO can lead to endothelial dysfunction and abnormal vascular remodeling(6).
The synthesis of NO requires NO synthase (NOS) (34). There are two types of NOS in blood vessels, endothelial NOS (eNOS) and inducible NOS (iNOS). ADMA is an endogenous NOS inhibitor that inhibits the activities of both eNOS and iNOS (35). Therefore, elevated ADMA levels reduce NO production. As NO plays a crucial role in maintaining endothelial function, theoretically, elevated circulating ADMA levels would cause endothelial dysfunction, aggravate arterial stiffness, and promote the development of hypertension and cardio-cerebrovascular diseases by reducing the production of NO.
ADMA has been associated with several vascular diseases, such as coronary artery disease(36), preeclampsia (37), chronic kidney diseases, and carotid intima-media thickness(38), and is considered a prognostic marker of all-cause mortality(39) and a predictor of early-onset atherosclerosis (40).
Correspondingly, in the present meta-analysis, the circulating levels of ADMA were strongly elevated in patients with hypertension compared to normotension controls.
However, this meta-analysis presents several limitations. First, heterogeneity of the included studies might reduce the reliability of the pooled estimate. Second, several studies included patients with hypertension who had already received anti-hypertensive drugs before the trial, yet they failed to report the effects of anti-hypertensive treatment on the ADMA outcome. Third, only case-control studies examining the relationship between ADMA and hypertension were included in this meta-analysis, demonstrating the need for additional prospective studies in future research.

Conclusions
In summary, the results of this meta-analysis display a signi cant positive association between elevated circulating ADMA concentrations and hypertension. Therefore, strategies aimed at reducing ADMA may constitute an effective therapeutic approach to prevent the development of hypertension in patients.

Availability of data and materials
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Competing interests
The authors declare that they have no competing interests.

Author contributions
HW: conception and design of study. XHC and TX: independently reviewed the title, abstract, or full text of all the identi ed articles. XHC and LX: acquisition of data and assess eligibility. HW and LX: analysis and/or interpretation of data. XHC and TX: drafting and revision of manuscript. Any disagreements regarding the eligibility of a study were resolved by mutual discussion (HW) or consultation with TX. All authors contributed to the article and approved the submitted version.

Funding
This work was not supported by funding agencies.

Acknowledgements
Not applicable.
8 References Figure 1 Flow chart of study selection for inclusion in the systematic review andmeta-analysis. Meta-analysis of 28 studies reporting on ADMA in hypertension compared with healthy controls. The random-effect model showed that hypertension patients have signi cantly elevated plasma/serum ADMA level than healthy controls. SMD, standard mean difference. Figure 3