Cardiovascular (CVD) and metabolic diseases (such as obesity, insulin resistance, and diabetes) are major health problems worldwide. Such conditions are physiologically related through mechanisms that involve endocrine, nervous, and immune system cross-talk [1
]. Several studies demonstrated that diabetic patients have two- to fourfold propensity to develop coronary artery disease (CAD), myocardial infarction, and other heart diseases [2
]. Importantly, CVD and diabetes impose large economic burdens on the individual patient and on national healthcare systems. For this reason, the search for complementary and alternative therapies is of major interest.
Among non-pharmacological therapies, physical exercise and nutrition were extensively studied due to their potential benefits [3
]. Particularly from the nutritional standpoint, supplementation of people with CVD and metabolic complications with amino acids [7
], vitamins [8
], fatty acids [9
], proteins, and others [3
] were used as a tool to improve immune, neural, cardiovascular, metabolic, and endocrine function. Interestingly, one of the major connections between these diseases is the availability of nitric oxide (NO), a nitrogen free radical that is continuously produced from the semi-essential amino acid, l
]. This molecule participates in several regulatory processes such as relaxation and proliferation of vascular smooth muscle cells, angiogenesis, immune response, insulin secretion and signaling, and cell communication [1
Decreased production of NO∙ may result in cellular dysfunction, decreased blood flow, glucose transport, insulin resistance, insulin secretion, hypertension, and diabetes. In fact, several studies reported that NO∙ production is blunted (at the plasma or cellular environment levels) in cardiovascular and metabolic diseases, causing important physiological disturbances [11
]. Among the mechanisms that underlie the lower NO∙ production/availability in metabolic and cardiovascular diseases, we can include the decreased blood levels of insulin, the increased production of angiotensin II (AngioII), hyperhomocysteinemia, increased asymmetric dimethylarginine (ADMA) synthesis, and the low plasma concentration of l
] (Please see Figure 1
A for details).
-arginine supplementation is considered a potential therapy for the treatment of cardiovascular and metabolic diseases, targeting to normalize the NO∙ levels and other metabolites produced from l
-arginine, such as polyamines (Please see Figure 1
B for details). Different studies tested l
-arginine for improving metabolic and cardiovascular function [12
]. However, methodological variations in study populations and interventions limit the interpretability on the efficiency of l
-arginine supplementation on different biomarkers of cardiovascular health or metabolic diseases.
Therefore, the aim of this systematic review and meta-analysis was to summarize randomized controlled trials (RCTs) that assessed the effects of l
-arginine supplementation compared to placebo in people presenting CVD, obesity, and/or diabetes. The primary outcome was flow-mediated dilation, chosen by its properties to indicate subclinical atherosclerotic disease (in non-CVD patients), as well as vascular function when disease is already manifested. Secondary outcomes were nitric oxide metabolite formation (nitrites and nitrates, named tNOx) and ADMA. Figure 1
A,B illustrate how our research question was constructed, connecting cellular mediators (NOx and ADMA) of l
-arginine synthesis to a clinical outcome (blood flow), and the rationale for using l
This review was conducted following the Cochrane Handbook for Systematic Reviews of Interventions (Collaboration 2011) and reporting adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [20
] This study was registered at PROSPERO international prospective register of systematic reviews (CRD42017077289).
2.1. Search Strategy and Study Selection
Eligible studies were identified through a systematic search without language restrictions of the following electronic databases, from the earliest to 21 July 2017: Medical Literature Analysis and Retrieval System Online MEDLINE (accessed by Pubmed), Excerpta Medica dataBASE (Embase), Cochrane Wiley (Central Register of Controlled Trials), ClinicalTrials.gov, Physiotherapy Evidence Database (PEDRO), and “Literatura Latino-Americana e do Caribe em Ciências da Saúde” (LILACS). We additionally carried out manual searches in reference lists of selected published studies.
A combination of terms was used to identify relevant publications: (1) cardiovascular disease, arginine, and vascular or insulin sensitivity outcomes, and (2) obesity, arginine, and vascular or insulin sensitivity outcomes. The exploded versions of the Medical Subject Headings (MeSH) were (1) (Heart Diseases OR Coronary Disease Coronary OR Heart Disease OR Coronary Artery Disease OR Myocardial Infarction OR Myocardial Revascularization), and (2) (Obesity OR Overweight OR Abdominal Obesity OR Visceral Obesity OR Central Obesity OR Obese OR excess body weight) AND (Arginine OR l-Arginine OR Arginine*) AND (Endothelium OR endothelial OR vascular OR “intima-media” OR “brachial artery” OR “flow mediated dilation” OR “flow mediated dilatation” OR “fmd” OR “hyperemia” OR vasomotor OR vasodilation OR vasodilatory) OR (Insulin Resistance OR Insulin sensitivity OR HOMA OR Clamp OR Glucose tolerance OR Glucose challenge). The search strategy was peer-formulated by an information specialist (D.U.).
2.2. Eligibility Criteria
We included RCTs that compared arginine supplementation with a placebo control group in subjects with any type of cardiovascular disease (coronary artery disease, peripheral artery disease, chronic heart failure, myocardium infarction, angina, etc.), obesity (mean body mass index (BMI) >30 kg/m2, waist circumference >88 cm), and/or type 2 diabetes mellitus (T2DM; glycated hemoglobin (HbA1c) ≥6.5%; fasting glycaemia ≥126 mg/dL; glycemia 2 h glucose tolerance test (GTT) ≥200 mg/dL).
The interventions included oral supplementation of l-arginine (capsule, biscuit, barre, shake, syrup, etc.) with a minimum duration of three days, but no limit set for maximum duration or dose of l-arginine supplementation. The comparisons included a control group with the same health condition, receiving an oral supplementation of a placebo substance for the same duration as the arginine supplementation.
Exclusion criteria included healthy patients, intravenous arginine administration, combined interventions (l-arginine plus other active substances, or diet/exercise associated), placebo groups with an active component, a duration of the intervention shorter than three days, non-randomized controlled trials, and studies without outcomes of interest. To better standardize the units of measurement for later data extraction and unit transformation, studies including outcomes of interest, but with different measurement techniques or body regions of assessment (i.e., plethysmography) were excluded.
The main clinical outcome of this review was blood flow after post-ischemic hyperemia, assessed by flow-mediated dilation (FMD), using the ultrasound technique. Secondary biochemical outcomes were nitrite/nitrate (NOx) rate and asymmetric dimethylarginine (ADMA), assessed by known enzymatic colorimetric assays. Data were extracted as means or differences between means and dispersion values at the onset (baseline) and at the end of interventions. Articles in English, Portuguese, or Spanish were considered for review.
2.3. Data Extraction
Two independent investigators (J.R-K. and A.P.T.F.) evaluated titles and abstracts of retrieved articles. Abstracts that did not provide enough information about eligible reasons for exclusion were left for the full-evaluation stage. The same reviewers independently assessed full-text articles, defining eligible studies, and continued the flow work for data extraction. Disagreements were solved by consensus or by a third reviewer (I.M.G.R). Specific characteristics of intervention (duration of the intervention, dose of l-arginine, type of oral administration, etc.), age and gender of participants, clinical condition, specific outcomes and methods of assessments, adherence, and/or dropout rates were also extracted.
2.4. Risk of Bias Assessment
Risk of bias of individual studies was evaluated according to the Cochrane Collaboration tool for assessing risk of bias in randomized trials [21
], a seven-item instrument, as follows: (1) adequate sequence generation; (2) allocation concealment; (3) blinding of participants and personnel; (4) blinding of outcomes assessors; (5) incomplete outcome data; (6) selective reporting; and (7) other bias. Risk of bias of each of the seven domains was expressed in the format “low”, “high”, or “unclear” risk. The standards of assessment for risk of bias (by consensus of the authors) were as follows: low risk of bias detected for seven items = “low” risk of bias, unclear risk for one to two items = “some” risk of bias, unclear risk for more than three items = “high” risk of bias.
2.5. Data Analysis
Estimates of pooled effects were obtained by comparing absolute post-intervention means for each group for NOx and ADMA outcomes (μmol/L), and standardized mean differences (SMD) for blood flow outcome. Results were expressed as means ± standard deviation (SD) between the groups arginine vs. placebo. Unit transformation for post-intervention mean values and respective dispersion values were performed when appropriate, in accordance with Cochrane Collaboration recommendations (Collaboration 2011). Calculations were performed using a random-effect model. An α value = 0.05 was considered statistically significant.
The inconsistency index (I2) was used to quantify statistical heterogeneity in meta-analyses, and values greater than 50% were considered indicative of high heterogeneity [21
]. Heterogeneity was explored as follows: (i) by carrying out additional meta-analysis removing each study at a time to check if a particular study was solely affecting heterogeneity; and (ii) conducting a sensitivity analysis based on a review to evaluate previous relevant clinical information. All analyses were conducted using Reviewer Manager Software version 5.3.
This systematic review with meta-analysis aimed to verify the effects of l
-arginine supplementation compared to placebo on blood flow, NOx, and ADMA responses in people with CVD, obesity, and/or diabetes using RCTs. We firstly demonstrated that there was no difference in blood flow responses comparing l
-arginine with placebo supplementation in patients with cardiovascular and/or metabolic disorders. However, under sensitivity analysis, the exclusion of studies with extreme responses favoring arginine [12
] or placebo [22
] suggested superiority of arginine supplementation regarding blood flow improvements. Secondly, there was no difference in NOx and ADMA responses comparing arginine with placebo supplementation in these patients, although speculation on subgroup responses indicates that obese/T2DM patients could improve NOx response and further endothelial function as a result of arginine supplementation. Although we pre-specified subgroup analyses, we underscored that (i) NOx and ADMA subgroup meta-analyses were based on few studies, and (ii) distinct sample sets of individuals precluded us from exploring subgroup estimates of blood flow.
Recently, a few meta-analyses were published on oral l
-arginine supplementations in patients with cardiovascular disease. In a meta-analysis of randomized controlled trials assessing the effect of l
-arginine supplementation on clinical outcomes (all-cause mortality, myocardial reinfarction, successful resuscitation, shock/pulmonary edema, recurrent myocardial ischemia, and hospitalization for heart failure) in patients with acute myocardial infarction, interventions (both providing 9 g of l
-arginine per day orally) were not associated with significant change in the risk of total events [29
]. Neither of the two studies included in this previous review were included in our study due to the absence of outcomes of interest. Another meta-analysis aimed to examine the effect of oral l
-arginine supplementation on blood pressure. Dong et al. summarized 11 randomized controlled trials with oral l
-arginine interventions with dosages from 4 to 24 g/day [30
]. Compared with placebo, l
-arginine supplementation significantly lowered systolic blood pressure by 5.39 mm Hg (95% CIs = −8.54 to −2.25) and diastolic blood pressure by 2.66 mm Hg (95% CIs = −3.77 to −1.54). Sensitivity analyses for studies with a minimum duration of four weeks and studies in which participants did not use antihypertensive medications yielded similar results. These discrepant results and different populations demonstrate that the effects of l
-arginine supplementation are not yet fully understood in patients with cardiovascular disease.
However, even though oral l
-arginine supplementation seems to be a plausible strategy for improving endothelial dysfunction in patients with coronary risk, primary studies showed mixed results [12
]. Such high heterogeneity is a common characteristic of meta-analyses that involve chronic interventions, such as diet or supplementation, and may be influenced by a number of factors such as characteristics of patients, dosage of substances, types of medical treatment, and sample size [21
]. In the present study, we could only observe positive effects of arginine supplementation on blood flow responses when removing the more heterogeneous studies of the meta-analysis.
In this regard, Adams et al. [12
] evaluated a small number of individuals (n
= 10); thus, the statistical power (not reported) may have been low to provide estimate differences. Also, Wilson et al. [22
] did not show positive effects of l
-arginine supplementation (3 g/day) during six months on blood flow responses, possibly because larger doses of 5–15 g/day would be required to improve endothelial function in humans [31
]. In fact, the average daily consumption of l
-arginine in the American diet is 5.4 g, although human studies increased the oral intake of l
-arginine supplementation from two to five times. In addition, the study by Wilson included patients with several cardiovascular risk factors, such as diabetes and hypertension, which may cause both intra- and inter-study heterogeneity.
In addition to sample size and the arginine dose, it should be considered that vasodilation is mediated by several mechanisms (independent of l
-arginine availability) and molecules (potassium, oxygen, purines, and prostaglandins), and not only by NO [32
]. This may also be considered when interpreting a non-significant meta-analytical estimate of the effects of l
-arginine supplementation on blood flow. In this regard, we point out that trials herein summarized often evaluated physiological or clinical outcomes without assessing whether the expected biochemical effect was triggered (increase in bioactive NO). Therefore, there is uncertainty if l
-arginine supplementation yields any influence on blood flow or endothelial function even when increases in bioactive NO are achieved.
Regarding NOx and ADMA responses, there were no changes overall in comparing l
-arginine with placebo supplementation in the selected group of patients taken together. Large confidence intervals indicate high heterogeneity in the individual studies alone and among studies, which may influence polled results. In addition, biochemical markers have great variability depending on the measurement technique, time of the day, and type of patients, etc. [33
]. For example, a recent meta-analysis showed that ELISA measurements overestimated plasma levels of ADMA compared to high-performance liquid chromatography (HPLC) [34
On the other hand, in performing a subgroup analysis for type of patients, we found that, by only considering obese/T2DM patients or cardiovascular disease patients in separate subgroups, there was a positive polled effect of arginine supplementation on NOx response. For example, Alizadeh et al. [23
] and Bogdanski et al. [28
] found that l
-arginine supplementation increased NOx levels in obese people, and Martina [25
] found the same result in T2DM patients. The same occurred in only analyzing the patients with cardiovascular diseases [13
]. This particular positive response may be associated with the fact that l
-arginine, along with other amino acids (l
-arginine precursors), such as l
-glutamine, are chronically decreased in the plasma of insulin-resistant/obese people [36
]. Thus, correction of the l
-arginine availability may restore NOx (NO production), but supplementation above the physiological levels may not induce any further NO increase.
Even though the analysis of very few studies may limit conclusions, it supports a physiological speculation on possible mechanisms that may be particularly involved in the effects of arginine supplementation on endothelial function of patients with metabolic or cardiovascular disorders. In comparing ADMA and NO metabolism (Figure 1
A), NO production can be affected by several signaling molecules, including ADMA, insulin sensitivity, level of inflammation, and l
-arginine availability, while ADMA levels are mainly controlled by dimethylarginine dimethylhydrolase (DDAH) activity and protein hydrolysis [1
]. Thus, restoration of l
-arginine availability through supplementation may directly induce NO production, which may explain why NOx response to l
-arginine supplementation and ADMA did not change in the included studies. Changes in ADMA levels may require longer periods of the disease to take place (thus, longer exposure to oxidative stress damage), and to interfere in NO metabolism and vasodilation. Thus, it is unlikely to reduce with short periods of l
Hypercholesterolemia is also known to increase ADMA, but endothelial dysfunction in the setting of hypertension or diabetes is not accompanied by increased ADMA. Thus, the clinical and mechanistic data suggest that subjects with hypercholesterolemia may be more likely to benefit from l
-arginine than normal subjects or those with other forms of vascular disease [38
]. In the present meta-analysis, the disease does not appear to influence the ADMA response to l
-arginine supplementation. Although ADMA appears to be a potential mediator of oxidative stress, the association between higher levels of ADMA and increased cardiovascular risk is still unclear.
The results of this meta-analysis should be interpreted with caution due to some limitations. Firstly, we included studies with individuals with different cardiovascular risk factors, resulting in a heterogeneous sample. Secondly, the sample sizes of individual trials were small, which might more easily suffer from sample imbalances and an influence of baseline confounding factors. Thirdly, the validity of the present meta-analysis depended upon the quality of the individual studies. It should also be considered that risk of bias in individual studies was detected. Although all studies were randomized and placebo-controlled trials, allocation concealment, quality of randomization, and details of blinding were not always reported (See Table 2
). Moreover, a large variation in the supplementation duration was observed among the studies (three days to 18 months), which may impact adverse effects and safety (reported in very few studies) of l
-arginine supplementation. Fourthly, the small number of limited studies limits us in the analysis of publication bias, as we planned and stated in this synthesis registration. Fifthly, varied sample groups could include patients who were likely taking medications that would impact the primary and secondary outcomes; however, we were unable to assess whether this may have happened. Lastly, the studies usually assess a clinical outcome without checking if the expected biochemical effect actually happened, and limitations can exist in interpreting if l
-arginine is able to change the outcomes even when bioactive NO is increased.
Finally, although the inclusion of studies with individuals presenting different cardiovascular risk factors and undergoing different supplementation strategies may result in high heterogeneity, to the best of our knowledge, this is the first review that systematically looked at the overview of arginine supplementation on clinical (blood flow) and biochemical markers (NOx and ADMA) of endothelial dysfunction in people experiencing cardiovascular risk. We might also speculate that the benefits of the intervention may outweigh the risks when subgroups of patients are analyzed, at least regarding the mechanisms involving NOx responses related to improvements in blood flow and endothelium function. Future research should focus on the development of higher-quality RCTs in specific subgroups of patients, and comparison with other types of intervention and supplementation duration. Also, analyzing a number of other biomarkers would clarify how l-arginine may induce or not induce positive effects on endothelial function and associated cardiovascular risk.