Objective: The aim of this study was to investigate the effect of oral supplementation with L-arginine on serum biochemical profile, blood pressure, microcirculation, and vasoreactivity/endothelial function in young controls, and elderly women with and without type 2 diabetes mellitus (T2DM). Methods: Healthy young (n = 25), healthy elderly (n = 25), and elderly women with type 2 diabetes mellitus (T2DME, n = 23, glycated Hb ≥6.4% and mean of 7.7 years for duration of the disease), aged 18–30 and older than 65 years, respectively, were included in the study. All patients underwent biochemical analysis (fasting glycemia and lipidogram), arterial blood pressure, nailfold videocapillaroscopy (capillary diameters, functional capillary density [FCD], peak red blood cell velocity [RBCVmax] after 1 min ischemia, time to reach peak RBCV [TRBCVmax]), and venous occlusion plethysmography (vasoreactivity), before and after 14 days of oral supplementation with L-arginine (5 g/day). Results: L-Arginine did not change fasting glycemia and lipidogram, but it decreased systolic, diastolic, and mean arterial pressure in elderly women, increased RBCVmax in all groups, and did not decrease TRBCVmax in T2DME. Capillary diameters and FCD remained unchanged in all groups. L-Arginine improved vasoreactivity during reactive hyperemia and after sublingual nitroglycerin (0.4 mg) in all groups. Conclusion: L-Arginine supplementation (5g/day during 14 days) was able to improve vascular/microvascular health in the elderly women with or without T2DM.
The World Health Organization in a global analysis estimated that the economic burden of diabetes mellitus in adults aged 20–79 years will increase to USD 2.2 trillion by 2030. Data suggest that the number of adults with diabetes aged up to 79 years will increase to 642 million in 2040, which means 1 in every 10 subjects, even if age-specific prevalence remains constant . Type 2 diabetes mellitus (T2DM) progresses silently, and 90% of these patients have dyslipidemia, obesity, and arterial hypertension, important risk factors for coronary heart disease . Macro- and microangiopathy of T2DM are associated with development of serious clinical complications including retinopathy, nephropathy, neuropathy, and increased risk of stroke and myocardial infarction . Endothelial dysfunction is a hallmark of chronic hyperglycemia, and it has been attributed to a loss of nitric oxide (NO) synthesis and bioavailability with increased generation of reactive oxygen species. The endothelium plays a pivotal role in the maintenance of vascular tone and structural integrity. Moreover, in diabetic patients, capillary pressure is higher than that in normal individuals . Furthermore, it is well known that patients with diabetes mellitus have a lower percentual of maximum increment of red blood cell velocity (RBCV) than controls and longer time to get it after post-ischemia reperfusion .
Aging also impairs NO bioavailability, frequently associated with L-arginine deficiency. L-Arginine is a semi-essential amino acid derived from endogenous production or dietary intake. It is metabolized by NO synthase (NOS) into L-citrulline and NO that has age-related changes . Oral L-arginine supplementation has been shown to improve flow-mediated dilatation of the brachial artery in older healthy seniors . In another study, long-term L-arginine treatment improved endothelial function and had additive effect on glucose metabolism and sensitivity in middle-aged obese type 2 diabetic patients . L-Arginine supplementation (6.4 g orally/day) during 18 months in middle-aged subjects could delay the development of T2DM for a longer period, probably by reduction of oxidative stress . L-Arginine has positive effects over some diseases such as hypertension, diabetes mellitus, heart disease, and peripheral artery disease . Based on these data, we hypothesized that the use of L-arginine during a short period of time perhaps could bring improvements to the microcirculation.
Microcirculation and endothelial function can be assessed by nailfold videocapillaroscopy (NVC) and venous occlusion plethysmography (VOP) . NVC shows the capillary structure through capillary diameters and microcirculatory function through RBCV and functional capillary density (FCD), while with VOP, it is possible to investigate vasoreactivity. After 5 min of ischemia and its release, reactive hyperemia occurs where myogenic, neural, and local factors such as adenosine, prostaglandins, and NO take part in the process . From the participation of NO in reactive hyperemia, we can infer about the participation of the endothelium in carrying out vasodilation. Sublingual nitroglycerin infusion allows the assessment of endothelium-independent vasodilation by direct NO donation to vascular smooth muscle. The vasodilatation magnitude reflects microvascular function, and it is an important marker of overall vascular health and cardiovascular disease risk [11, 13].
As T2DM may cause an important impairment of the cardiovascular system, the aim of this study was to evaluate in vivo if short-term use of oral L-arginine supplementation (14 days) would improve the microcirculation and endothelial function/vasoreactivity observed through NVC and VOP, blood pressure, and glucose/lipid biochemical profile in elderly persons with T2DM, and to compare them with healthy young and elderly women.
Material and Methods
Study Design, Approval, and Ethics
Seventy-five women were allocated into 3 groups: healthy young subjects (HY, 20–30 years old), healthy elderly (HE) control, and elderly women with T2DM (T2DME with fasting glycemia > 99 mg/dL and glycated Hb ≥ 6.4%, over 65 years old). HE was defined as the absence of chronic cardiometabolic diseases except mild hypertension treated with 1 antihypertensive drug. Subjects were recruited from ambulatories of geriatric and diabetology clinics at the State University of Rio de Janeiro (UERJ) and a private supplementary health program. HY was composed mainly of students from the UERJ between April 2016 and April 2019. All subjects signed a written informed consent form enclosed in the protocol approved by the Ethics Committee from the UERJ according to the Helsinki Declaration revised in 2008 and registered at Clinical.Trials.gov (NCT04112875).
Patients with type 1 diabetes mellitus, T2DM using insulin, cognitive impairment, frailty, cardiometabolic disorders, liver and/or kidney dysfunction, autoimmune diseases, smoking less than a year detected through anamnesis and physical exam (signs of nicotine in skin and beard), and uncontrolled hypertension with pressure levels >140/90 mm Hg were excluded.
There were 3 medical appointments: first: written informed consent signature, anamnesis, and physical exam that included blood pressure at rest, weight and height, and body mass index (weight in kilograms/square of height in meters); second: venous blood sample collection after 12 h fasting, measuring glucose, total cholesterol, triglyceride, high-density lipoprotein (HDL-cholesterol), low-density lipoprotein, followed by NVC and VOP. Afterward, each subject received a box with 14 sachets containing 5 g L-arginine supplementation, and it was recommended to ingest it with water, 30 min before breakfast for 14 days; third: blood sample, NVC, VOP, and empty box returned after 14 days of oral supplementation.
Venous blood samples were collected in tubes containing EDTA-sodium (1 mg/mL) and in polystyrene tubes without an anticoagulant. EDTA-containing tubes were promptly chilled. Plasma was immediately separated by centrifugation at 3,000 rpm and 4°C for 10 min, and serum was isolated by centrifugation at 1,000 rpm at room temperature for 10 min. Samples were stored at −80°C until they were assayed. Fasting plasma glucose was determined by the oxidase method and total cholesterol, triglycerides, and HDL-cholesterol by the oxidase/peroxidase method. Low-density lipoprotein was calculated using Friedewald’s equation.
Upon arrival at the laboratory, the subject waited in an acclimatized room with controlled temperature of 24 ± 1°C for 20 min, and then, she was transferred to another acclimatized room (same temperature), where she sat comfortably in a high chair, and the fourth left finger was placed, at the heart level, on an acrylic platform of a Leica MZFLIII stereoscopic microscope (Wetzlar, Germany) equipped with an epi-illumination system (100 W Xenon lamp). Coupled to this system, there was a video camera Leica DFC365 FX and an HP Z420 workstation (Intel Xeon CPU E5-1620 3.60 GHz, 8.0 GB RAM, 500 GB hard drive) with a 30’’ NEC monitor. The finger skin temperature was kept at 25 ± 2°C and monitored throughout the exam with an YSI Precision 4000 A digital thermometer (Dayton, OH, USA) with the thermistor probe taped within 1 cm proximal to the nailfold. A drop of mineral oil was placed on the nailfold bed to improve image quality. A pressure cuff connected to a mercury manometer was placed on the evaluated finger for functional testing of the microcirculation (reactive hyperemia response). FCD (number of capillaries in the microscopic field with flowing red blood cells per unit tissue area) was evaluated using 250× magnification. At a magnification of 680×, we obtained capillary diameters (AFD – afferent, APD – apical, and EFD – efferent; in micrometers), basal RBCV (in mm/s), maximum RBCV after 1 min of arterial occlusion release in the evaluated finger (RBCVmax; in mm/s) during the reactive hyperemia response, time to reach RBCVmax in seconds (TRBCVmax), and RBCVmax/RBCV. Analyses of 3 fields of the same periungueal bed were performed: medial, central, and lateral. The average of these measurements was determined to increase exam reliability by reducing measurement variability.
Venous Occlusion Plethysmography
A Hokanson ® AI6 plethysmograph (AD Instruments, Castle Hill, NSW, Australia) was used coupled to a software for processing and analyzing forearm volume curves and calculating forearm blood flow (FBF). Lead II ECG was used to measure the heart rate continuously. A mercury-filled Silastic strain-gauge was placed on the upper third of the left forearm at maximal circumference.
For evaluation of VOP, patients were accommodated in a bed with the left forearm to the heart level, in an acclimatized room with temperature 21 ± 1°C for 20 min before procedures. The arm collection cuff pressure was 40 mm Hg and wrist cuff occlusion pressure 200 mm Hg. The wrist cuff was inflated immediately before each blood flow measurement. By blocking venous efflux with an upper arm cuff, the slope of change in forearm volume expresses FBF. FBF is calculated by software from forearm volume curves with values in mL min-1 100 mL tissue. Arterial blood pressure was obtained before each FBF measurement at the right arm.
VOP examination was performed in 4 stages, in sequence: baseline 1, reactive hyperemia response (partial endothelium-dependent vasodilatation and FBF after release of 5 min of brachial artery ischemia), baseline 2, and after 5 min sublingual spray of nitroglycerin, 0.4 mg (Nitrolingual BurnsAdler Pharmaceuticals Inc., Charlotte, NC, USA) used to assess endothelium-independent vasodilatation. FBF was measured for 2 min in each stage and each forearm volume curve recorded for 18 s. An interval of 15 min was allowed between reactive hyperemia response and baseline 2.
Statistical power was calculated using G*Power 184.108.40.206 (Kiel, Universität Kiel, Germany), considering a sample power of 0.95, probabilistic error of 5%, and effect size of 0.6, for microcirculatory-dependent variables of the study, resulting in a total sample size of 21 subjects per group. STATISTICA® 7.0 (StarSoft Inc., Dayton, OK, USA) was used for statistical analysis. The Shapiro-Wilks test was used for normality evaluation. Kruskal-Wallis and Dunn’s tests for multiple comparison test were used to compare data of groups HY, HE, and T2DME baseline and compare data posttreatment. The effect of L-arginine supplementation in each group was analyzed using Wilcoxon matched pair tests. Results are given as mean ± SD or median (1st–3rd quartiles). Statistical significance with p < 0.05 was adopted.
Of 120 interviewed elderly women, 48 were included in the study (HE = 25 and T2DME = 23), and 25 HY were recruited without dropouts.
Patients with T2DM had 7.7 years of disease on average, presented HbA1c of 7.2 ± 0.8% (55 ± 3 mmol/L), and mainly used metformin (87%) as treatment (Table 1). No patients with T2DM have used statins or other drugs during the study except oral hypoglycemic ones.
Table 1 shows anthropometric data from HY, HE, and T2DME groups. Elderly subjects were 72.8 ± 7.3 (HE) and 72.6 ± 6.0 (T2DME) years old, while HY were 25.9 ± 3.2 years old. HE and T2DME groups were overweight and obese patients with BMI 28.1 ± 3.65 and 31.2 ± 5.39 kg/m2, respectively.
Levels of fasting glucose and lipid profile did not change significantly after L-arginine supplementation in all studied groups. Fasting glucose in the T2DME group before and after L-arginine supplementation presented higher levels than that in HY and HE groups as expected. On the other hand, HDL-cholesterol had lower levels in the T2DME than the HY and HE groups (T2DME: before 53.4 ± 14.2, after 50.8 ± 15.5; HY: before 61.5 ± 12.9, after 63.0 ± 13.7; HE: before 62.3 ± 17.5, after 60.8 ± 18.9, mg/dL) (Table 2). L-Arginine did not increase HDL-cholesterol in any group.
L-Arginine did not modify capillary diameters (apical, afferent, and efferent) and FCD in any group. However, L-arginine supplementation increased RBCVmax in all groups (HY: before 0.72 [0.69–0.77], after 0.77 [0.70–0.81]; HE: before 0.69 [0.65–0.71], after 0.74 [0.71–0.80]; T2DME: before 0.69 [0.67–0.71], after 0.75 [0.74–0.77]). TRBCVmax presented higher values in the T2DME (before 18 [12–20], after 15.50 [14.50–20.50]) than the HY and HE groups which often occurs in this disease. One can note that L-arginine did not decrease TRBCVmax in T2DME.
Capillary diameters, FCD, RBCV, and RBCVmax presented no intergroup differences before or after treatment (Table 3). In summary, L-arginine improved the microcirculatory function profile through increase of RBCVmax in all groups but not decreased TRBCVmax in T2DME.
VOP and Blood Pressure
In the HE and T2DME groups, systolic blood pressure (HE: before 129 [119–145], after 123 [114–134]; T2DME: before 132 [120–145], after 126 [112–132], mm Hg), diastolic blood pressure (HE: before 70 [64–76], after 67 [63–73]; T2DME: before 69 [66–75], after 66 [60–74], mm Hg), and mean arterial pressure (HE: before 91 [83–98], after 87 [82–92]; T2DME: before 89 [84–99], after 87 [77–93], mm Hg) decreased after L-arginine intervention, while in HY, there was no blood pressure change. FBF baseline 2 increased in T2DME by L-arginine (before 1.63 [0.98–2.22], after 2.08 [1.50–2.62], mL/min/100 mL tissue). Maximum FBF during reactive hyperemia reflecting 30% endothelial-dependent vasodilation increased in all groups after L-arginine use (HY: before 10.25 [8.72–11.72], after 13.37 [10.84–15.91]; HE: before 9.70 [7.75–14.48], after 13.18 [10.05–15.81]; T2DME: before 7.77 [6.64–10.29], after 9.43 [8.29–11.11], mL/min/100 mL tissue). Such increases correspond to 30%, 36%, and 21% enhancement of FBF, respectively (Fig. 1; Table 4). Prior to treatment with L-arginine, T2DME had lower maximum FBF during reactive hyperemia than HY, which did not occur between HE and HY. Concerning endothelial-independent vasodilatation with nitroglycerin, L-arginine was able to improve vasoreactivity in all groups as well (HY: before 1.71 [1.32–2.64], after 2.55 [2.03–2.86]; HE: before 1.59 [1.32–2.23], after 2.25 [1.67–3.45]; T2DME: before 2.19 [1.50–3.10], after 2.78 [2.13–3.52], mL/min/100 mL tissue). Post-nitroglycerin flow % increase was 49%, 41%, and 27% in all studied groups, respectively. There were no inter-group differences before and after treatment concerning endothelial-independent vasodilatation (Fig. 2; Table 4).
In this study, the effects of 5 g L-arginine supplementation daily during 14 days were observed on microcirculation using NVC and vasoreactivity using VOP. Also, glucose and lipid profile and blood pressure have been analyzed in young healthy controls, healthy women, and elderly women with T2DM. The main findings were unchanged glucose and lipid profile, blood pressure decrease in HE and T2DME groups, preservation of microcirculatory structure (capillary diameters) and perfusion (FCD), increase of maximum RBCV in capillaries, unchanged TRBCVmax in T2DME, increase in FBF baseline 2 in the T2DME group, and increased vasoreactivity during reactive hyperemia (endothelium, neural, and myogenic factor participation) and after sublingual infusion of nitroglycerin (endothelial-independent vasodilatation) observed in the forearm in all groups.
Studies with healthy men showed improvement of glucose metabolism up to 7 days of L-arginine supplementation with doses ranging from 8.0 to 10.0 g/day [14-16]. Perhaps, duration of treatment and dose of arginine may be correlated with its beneficial effects on glucose metabolism. Further research will be needed to discover this fact. Also, L-arginine action may be different in healthy people and those with the disease. Lucotti et al.  have shown that oral L-arginine reduces plasma glucose in middle-aged type 2 diabetic patients associated with hypocaloric diet and exercise training. We could not demonstrate the same effects in our patients. One explanation could be that hypocaloric diet and physical training alone can lower fasting glucose levels. Probably, diet and physical exercise have potentiated the effect of L-arginine in Lucotti et al.’s  work. In our study, patients with T2DM were sedentary and did not have any special diet and were treated mainly with metformin. Another difference between our study and the one by Lucotti et al.  is the long-term oral L-arginine treatment (21 days) associated with a higher load of L-arginine (8.3 g/day). We used 5.0 g/day for 14 days.
L-Arginine was not able to improve the lipid profile in any group. Georgescu et al.  in 2000 showed that production of advanced glycation end products was associated with increased plasma glucose and cholesterol concentrations. Sepandi et al.  in 2019 reviewed several studies on the effects of oral L-arginine supplementation on lipid profile and inflammatory markers showing that findings are inconsistent on this aspect. In many studies, L-arginine supplementation significantly reduced triglyceride levels, but in others lipids and inflammatory indices were not changed .
Concerning blood pressure, Kelly et al.  described its improvement in renal transplant and hemodialysis patients with L-arginine use. Also, Dong et al.  conducted a meta-analysis of 11 randomized, double-blinded, placebo-controlled trials, with 387 patients, showing that it reduced blood pressure with a median daily dose of 9 g of L-arginine. They found that systolic blood pressure lowered by 5.39 mm Hg (95% CI: 2.25–8.54, p = 0.001) and diastolic blood pressure by 2.66 mm Hg (95% CI: 1.54–3.77, p < 0.001) compared to the placebo. In our study, systolic, diastolic, and mean arterial pressure showed improvement in HE and T2DME with normal median arterial pressure and lower dose of L-arginine (Table 4).
James et al.  found higher capillary pressure in elderly women than young controls with normal arterial blood pressure values. However, capillary density did not differ between groups. Our study showed similar results concerning FCD and diameters using NVC.
RBCV response to ischemia is well studied among patients with diabetes through NVC as well. Pazos-Moura et al.  described lower RBCVmax post-occlusion, and the mean time to reach it was significantly prolonged with enlarged capillaries in T2DM (mean age 58.4 ± 8.2 years) than in controls (37.4 ± 15.0 years). Halfoun et al.  showed prolonged time to reach peak capillary blood flow during reperfusion in diabetes as well, but the projected area of transverse segment of hand nailfold capillary loops, using computerized videophotometry, remained unaltered. Some theories were raised about the behavior of microcirculation in diabetes. Tooke et al.  in 1980 considered a hemodynamic hypothesis to justify flow disturbances in diabetic microcirculation. T2DM impairs endothelial-dependent vasodilatation which may contribute to high prevalence of vascular diseases in such patients , and insulin resistance plays a key role in the development of the disease. Insulin resistance and endothelial dysfunction share many signaling pathways, including hyperinsulinemia, glucotoxicity, lipotoxicity, and inflammation. These mechanisms impair PI3K-dependent insulin signaling pathways in the vascular endothelium, which leads to endothelial imbalance, leading to a close association between metabolic impairment and cardiovascular disease . Thus, improvement of RBCVmax is desirable in patients with T2DM as their microcirculation fails to respond adequately to ischemic stress.
Aging and insulin resistance are associated with impaired microvascular responses to insulin in skeletal muscles , and L-arginine has a direct endothelial vasodilator effect on the insulin pathway as well. Arginine can induce insulin secretion from pancreatic beta cells, and insulin receptors are stimulated, generating an insulin-dependent vasodilatation . Insulin has a protective effect over arginine mobility compromised in diabetes . For example, in diabetic rats, oral administration of arginine reversed endothelial dysfunction . Another clinical trial demonstrated that the use of L-arginine 2 g/day during 3 months decreased lipid peroxidation which could reduce microangiopathic long-term complications in diabetes mellitus . Therefore, patients with T2DM would benefit from the use of L-arginine. Indeed, in our study, resting RBCV was similar among young, healthy, and T2DM elderly women and was not influenced by L-arginine. However, maximum RBCV during the reactive hyperemia response increased in all studied groups, but the time to reach RBCVmax did not decrease with the use of L-arginine in the T2DME group. Although Pazos-Moura et al.  found enlarged capillaries in diabetes, our study did not show any changes in capillary diameters with or without L-arginine, and FCD was not different in the T2DME compared to HY and HE groups. On the other hand, Groen et al.  found reduced skeletal muscle capillary density with aging and T2DM, accompanied by impairments in endothelial glycocalyx function. Perhaps the short duration of the disease, mean 7.7 years, may not be enough to show structural microcirculatory changes or premature perfusion problems.
L-Arginine plays a critical role in vascular function. Low plasma concentration of L-arginine results in impaired L-arginine-NO signaling and may promote endothelial dysfunction . Also, L-arginine has been effective in decreasing oxidative metabolism in water polo players . Another important carbohydrate-like substance, vitamin C, has an antioxidant effect and protects vascular endothelial function as well. D’Uscio et al.  showed increases in tetrahydrobiopterin and eNOS activity in the aortas of apoE-deficient and control mice after vitamin C use.
Both vitamin C and L-arginine are known to improve endothelial function and reduce vascular permeability during infectious disorders. We can speculate their association could be synergistic in tackling infectious diseases. For example, COVID-19 presents endotheliopathy. Oral L-arginine with liposomal vitamin C might be helpful to treat COVID-19 and other infectious diseases .
In our study, L-arginine was able to increase FBF baseline 2 but not baseline 1 evaluated with VOP in the T2DME group. As there are natural fluctuations in basal blood flow over time, it may be a point effect of L-arginine. NO is responsible for 30–50% of vasodilating effect during the hyperemia response with noninvasive POV. Adenosine, prostaglandins, endothelium-derived hyperpolarizing factor, and vascular musculature are also involved [12, 36]. Considering that factors other than NO may change significantly among studied groups, we can only estimate endothelial function with peak FBF during hyperemia. The exact percentage of the influence of the endothelium on vasoreactivity has not been measured. Many reports evaluated endothelial dysfunction with aging and diabetes/hyperglycemia [12, 36]. Age-related endothelial dysfunction is reported in humans and animals to explain alterations in vasodilatory function of endothelial cells in many situations [7, 37]. The hyperglycemia is directly related to vascular modifications that persist for years in prediabetic and type 2 diabetic patients with uncontrolled blood glucose. Among several pathological factors induced by hyperglycemia which affect the vasculature, the presence of adiposity associated with insulin resistance plays a key role in the decline of the vasodilator capacity . Reports have shown that overweight and obesity modify microvascular function, and it is often associated with aging and T2DM [8, 39]. In this study, the T2DME group had overweight and obese patients with compromised endothelial-dependent vasodilatation compared with the HE and HY groups. Bode-Böger et al.  demonstrated the benefits of using L-arginine to improve endothelial function in healthy elderly people. When we used L-arginine in elderly women with T2DM and overweight/obesity, we have shown improvement in endothelial-dependent vasodilatation with similar results. In aging people with hyperglycemia, endothelial function is impaired by decrease in NO bioavailability due to decrease in circulating L-arginine . L-Arginine supplementation retours plasma arginine and, in our study, improved vascular function in young, healthy elderly, and type 2 diabetic subjects similarly. Lucotti et al.  in 2006 also demonstrated that long-term oral L-arginine supplementation improved insulin sensitivity, endothelial function, and oxidative stress in middle-aged obese type 2 diabetic people associated with exercise and hypocaloric diet.
Although endothelial-independent vasodilatation, after sublingual nitroglycerin, was not different between groups, all of them have increased FBF after L-arginine use. This result was unexpected as L-arginine is a precursor of NO synthesis, improving endothelial-dependent vasodilatation. L-Arginine has been described to decrease oxidative and inflammatory stress during physical exercise in aging rats . Perhaps, L-arginine decreasing oxidative stress may have some influence on independent endothelial vasodilatation, but it still remains to be elucidated. To our knowledge, it is the first time that an improvement of both endothelial-dependent and independent vasodilatation is shown at the same time in young controls, and elderly with and without T2DM after short-term L-arginine oral supplementation. For example, Kimura et al.  found impaired L-arginine/NO pathways and the possibility of capillary rarefaction without sublingual nitroglycerin-mediated dilatation in elderly with T2DM. Trott et al.  showed reversal of age-related decrements on endothelium-dependent dilatation in skeletal muscle feeding arteries with exercise by increasing NO bioavailability. It was possible to demonstrate that short-term L-arginine oral supplementation can be very effective in improving the microcirculatory profile in elderly people with T2DM as in healthy elderly and young controls.
Study Limitation and Strengths
This study was carried out only with elderly women. As 80% of the patients in our clinic have been female, to avoid possible differences between the sexes, we chose to do the study only with women. Another limitation is the study of endothelial function specifically. We have studied reactive hyperemia after the release of 5 min of brachial artery ischemia. This process depends on many factors such as myogenic, neural, and local ones such as NO released by endothelium. As we have not measured NO, eNOS, or ET-1 in blood, we cannot determine the exclusivity of the participation of the endothelium. Indeed, Tagawa et al.  in 1994 used NO-monomethyl-L-arginine, a blocker of NO synthesis, that significantly decreased total reactive hyperemic flow (flow debt repayment) by 20%–30% after 3 and 10 min of arterial occlusion. Their results suggested that NO played a minimal role in peak reactive hyperemia and a modest significant role in maintaining vasodilation after peak vasodilation. The strength of this work is the use of low-dose arginine (5 g/day) in a relatively short period of time, 2 weeks, in elderly women with diabetes mellitus with good effects over microcirculation. A cohort study showed higher risk of T2DM with higher dietary L-arginine levels (4.05 + 1.50 g/day) after 5.8 years of follow-up . Thus, long-term treatment with L-arginine is not advisable. L-Arginine has been also effective in improving microcirculation in healthy young and elderly women. Maybe, the use of L-arginine for short periods of time in healthy elderly women can protect their microcirculation in order to attenuate the harmful effects of DM if it occurs, but this remains to be evaluated.
Some potential endothelial function biomarkers may be studied to evaluate cardiovascular health. For example, L-arginine is a common substrate for both NOS and arginase. This last compound produces urea and L-ornithine, and when upregulated, it decreases NO synthesis. Higher levels of arginase have been associated with diabetic retinopathy . Another biomarker could be asymmetric dimethylarginine (ADMA). ADMA acts as a competitive inhibitor of the NOS. According to Keller et al. , arginine and arginine/ADMA ratio were independently associated with 90-day mortality and other adverse outcomes in patients after out-of-hospital cardiac arrest. Finally, serine/threonine kinase is important for eNOS activation in response to many signal transduction pathways and may be associated with L-arginine oral supplementation. Threonine kinase alone restored endothelial vasorelaxations in spontaneously hypertensive rats .
In conclusion, short-term oral L-arginine supplementation (5 g/day for 14 days) improved both vasoreactivity during reactive hyperemia (partially endothelial-dependent) and independent vasodilatation through sublingual nitroglycerin, maximum RBCV in healthy young, and healthy and type 2 diabetic elderly women. Moreover, it decreased blood pressure in the elderly, but it did not modify the glucose/lipid profile or microcirculatory structure/perfusion in any group. L-Arginine may be used in elderly women with T2DM for a short time to improve vascular function and decrease cardiovascular risk.
Statement of Ethics
All studies with human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration.
Conflict of Interest Statement
The authors declare no conflict of interest.
This study received funding from the Carlos Chagas Filho Foundation for Research Support in the State of Rio de Janeiro (FAPERJ).
Data collection was performed by Costa G. and Shushanof M. Data analysis was performed by Costa G., Bouskela E., and Bottino, D. Manuscript writing was performed by Costa G., Bouskela E., and Bottino, D. Manuscript review was performed by Costa G., Shushanof M., Bouskela E., and Bottino D.
Data Availability Statement
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.