L-arginine as dietary supplement for improving microvascular function

Abstract

BACKGROUND: Reduced availability of nitric oxide leads to dysfunction of endothelium which plays an important role in the development of cardiovascular diseases.

OBJECTIVE: The aim of the present study was to determine whether the dietary supplement L-arginine improves the endothelial function of microvessels by increasing nitric oxide production.

METHODS: We undertook experiments on 51 healthy male volunteers, divided into 4 groups based on their age and physical activity since regular physical activity itself increases endothelium-dependent vasodilation. The skin laser Doppler flux was measured in the microvessels before and after the ingestion of L-arginine (0.9 g). The endothelium-dependent vasodilation was assessed by acetylcholine iontophoresis and the endothelium-independent vasodilation by sodium nitroprusside iontophoresis. In addition, we measured endothelium-dependent and endothelium-independent vasodilation in 81 healthy subjects divided into four age groups.

RESULTS: After the ingestion of L-arginine, the endothelium-dependent vasodilation in the young trained subjects increased (paired t-test, p < 0.05), while in the other groups it remained the same. There were no differences in the endothelium-independent vasodilation after ingestion of L-arginine. With aging endothelium-independent vasodilation decreased while endothelium-dependent vasodilation remained mainly unchanged.

CONCLUSION: Obtained results demonstrated that a single dose of L-arginine influences endothelium-dependent vasodilation predominantly in young, trained individuals.

Keywords

L-arginine nitric oxide skin microcirculation laser Doppler flux endothelium-dependent vasodilation endothelium-independent vasodilation physical activity aging

1 Introduction

The innermost layer of the vascular wall – endothelium plays an important role in the development of cardiovascular and rheumatologic diseases [7 , 44]. Under mechanical or chemical stimuli it releases vasoconstrictors and vasodilators. Vasodilation produced by vasodilators from the endothelium is termed endothelium-dependent vasodilation. If activated, sympathetic vasodilator nerves release acetylcholine (ACh) and an unknown cotransmitter. It is believed that ACh stimulates the release of different substances from endothelial cells: endothelium-derived hyperpolarizing factor (EDHF), NO and prostacyclin [30, 48]. EDHF is a major vasodilator of the small resistance arteries and thus an important modulator of blood pressure and local blood flow, but its chemical structure is still unknown. NO, synthetised from L-arginine by endothelial NO synthase (eNOS), is also involved in control of vasodilation. Dysfunctional NO signalling in resistance arteries and the myoendothelial junction contributes to disease manifestation such as hypertension and peripheral artery disease [36]. Since L-arginine is the only substrate for NO synthesis, it is essential for at least part of the endothelium-dependent vasodilation. In young, healthy adults L-arginine is produced endogenously in sufficient quantities, but factors such as ageing and various pathological conditions (inflammation, injury, starvation, stress) can lead to its deficiency [35, 41].

Aged humans exhibit attenuated cutaneous vasodilatory responses to heating, resulting from a diminished release of neurogenic cotransmitter, associated with decreased NO bioavailability [25 , 28]. For this reason it is hypothesized that dietary supplementation of L-arginine should have a beneficial effect on vascular health especially in older age.

Many researchers have demonstrated improvement in the endothelium-dependent vasodilation of subjects who were regularly physically active [13 , 51]. Increased shear force due to chronically increased blood flow in the active tissues is the main stimulus for the synthesis and secretion of NO from endothelial cells. It was found that regular physical activity leads to a significantly increased expression of the enzyme eNOS and consequently to a greater availability of NO [19].

The objective of the present study was to determine whether the effect of a single dose of the dietary supplement L-arginine can improve endothelial function by increasing NO availability in small resistance vessels. Since regular physical activity independently increases endothelium-dependent vasodilation, we have tested the hypothesis that the effect of L-arginine depends on individual age and on their regular physical activity.

2 Materials and methods

2.1 Subjects

We enrolled 51 healthy male volunteers divided into four groups based on their age and physical activity: young sedentary (YS), young trained (YT), older sedentary (OS) and older trained (OT). The trained subjects participated in aerobic sports (long distance running, cycling, or triathlon) at least 5 hours per week. In each subject the maximal oxygen capacity (VO₂max) was determined by a treadmill test. All the selected subjects were males in order to avoid the effect of sex hormone fluctuations on cardiovascular functioning, which is typical for women during their reproductive period of life [8]. All subjects avoided smoking and drinking alcohol, coffee and drinks containing caffeine or theine for at least eight hours prior to the measurements being performed. They also avoided dietary supplements for at least 10 days before the measurements. None of the subjects were suffering from any acute illness or was being treated with antihypertensive therapy. The measurements were carried out after a 15-min acclimatisation in the supine position in a quiet and comfortable area at room temperature kept between 23 and 25°C. The subjects were instructed to move as little as possible during the measurements to avoid movement artefacts. Measurements were performed in a supine position. The participant characteristics are presented in Table 1.

In order to clarify the differences between young and older subjects we measured endothelium-dependent and endothelium-independent vasodilation in 81 subjects, divided into four age groups. In the first group there were 32 subjects aged 20–28 years, in the second 19 subjects aged 29–39 years, in the third 19 subjects aged 40–49 years and in the fourth 17 subjects 50–74 years old.

The study was approved by the Republic of Slovenia National Medical Ethics Committee. A Statement of Informed Consent to participate in the study was signed by each subject.

2.2 Task force monitor

Using the automated biomedical device Task Force Monitor (CNSystems Medizintechnik, Austria), which is discussed in more detail elsewhere [39], electrocardiograms, continuous (beat-to-beat) systolic (SAP), diastolic (DAP) and mean arterial pressure, stroke volume (SV) and cardiac output (CO) at rest were recorded. Impedance cardiography was used for the assessment of cardiac function by means of an alternating electric current that passes from the electrodes through the chest. From changes in resistance during the cardiac cycle, which occurs due to changes in the blood volume of the heart, the device calculates the stroke volume with the help of a specific algorithm [16]. A small cuff for the continuous measurement of arterial pressure was placed on the index and middle fingers of the subject’s right hand. Placed on the right arm was a larger cuff for brachial artery measurement to which the values of arterial pressure from the smaller cuff were calibrated. This method helps avoid incorrect measured values of arterial pressure as a result of measurements in digital arteries.

2.3 LD fluxmetry and iontophoresis

Cutaneous microvascular flow was measured on the volar side of the forearm with LD fluxmetry, which is discussed in more detail elsewhere [34, 52]. In the present study, a two channel LD fluxmeter (Periflux 4001 Master/4002 Satellite; Perimed, Järfälla, Sweden) was used. LD probes (PF481) with a patch and a pad for iontophoretical substance delivery were attached to the ventral side of the left forearm. The LD probe on the left forearm was moved to a different place for each measurement, being attached to parts of the forearm with no visible surface veins.

Iontophoresis is a non-invasive method that enables the local introduction of charged molecules through the skin using a weak electrical current [34, 38]. Endothelium-dependent vasodilation was assessed using ACh, while endothelium-independent vasodilation was determined by use of sodium nitroprusside (SNP). Following ACh and SNP iontophoresis, vasodilation typically occurs and the effect of these vasodilators lasts for several minutes, which can be monitored by measuring LD flux. In the present study, the device Perimed’s PeriIont PF 382 (Perimed, Stockholm, Sweden) was used. A 1% ACh solution in deionized water (Merck, Germany) and a 1% SNP solution in deionized water (Merck, Germany) was applied. The ACh and SNP iontophoresis protocols as described by Morris and Shore [34] were followed. ACh was introduced with a positive DC electric current of 0.1 μA for 30 s, repeated every 60 s, until a plateau was reached, when the LD flux was no longer increasing. When introducing SNP, a negative DC current of 0.1 μA for 30 s was used. This was repeated every 120 s until a plateau was reached.

2.4 Protocol

Before the ingestion of L-arginine, measurements of cardiovascular parameters by a Task Force Monitor and cutaneous LD flux were recorded at rest for 6 minutes. This was followed by a measurement of the LD flux response to the iontophoresis of ACh (150 μL, 1% solution) and SNP (150 μL, 1% solution). The subject then ingested a solution of 0.9 g arginine hydrochloride, dissolved in 200 mL of water. This was followed by a 30-minute break, during which the level of C-reactive protein (CRP) in each subject was measured by taking a drop of blood from the tip of the finger - this in order to exclude infection. After 30 minutes all measurements were repeated.

2.5 Data evaluation and statistical analysis

Power and sample size program was used to determine sample size of subject groups enrolled in present study [14]. A study was planned with 10 subjects in each test group. Prior data indicate that the difference in the LD flux response before and after ingestion of L-arginine is normally distributed with standard deviation 30. If the true difference in the mean LD flux response is 30, we would be able to reject the null hypothesis that this response difference is zero with probability (power) 0.8. The error probability associated with this test of the null hypothesis is 0.05.

All analogue signals were converted to a digital form using an analog-to-digital converter and stored on a personal computer for further analysis. All obtained signals were analysed via the computer package Nevrokard (Kiauta Slovenia). The cardiovascular parameters were compared among groups by analysis of the variance (ANOVA, Dunnett’s test). The LD flux at rest was expressed as the average value of the 6-minute recording. The LD flux values during iontophoresis were expressed as a percentage of the basal LD flux for each subject. At first, we tested the influence of ageing on microvascular response to L-arginine. Then we tried to determine whether the effect of L-arginine depends on regular physical activity within the same age group.

The LD flux responses to the provocation tests were compared to baseline values by analysis of variance for repeated measurements (RM-ANOVA, Dunnett’s test). The values obtained before and after the administration of L-arginine in each group were compared by way of a paired t-test or Wilcox Signed Rank test. All results are given as mean values and standard errors (SE) of mean values. A Pearson correlation coefficient test was performed to evaluate the correlation between the changes of LD flux response to ACh and SNP after ingestion of L-arginine and the VO₂ max in each group of subjects.

3 Results

3.1 Resting values before ingestion of L-arginine

The level of CRP in all participants was below 8 mg/L so major inflammatory processes in the vessel walls of participants were excluded.

The YT subjects had statistically significantly lower DAP compared to the YS (Dunett’s test, p = 0.009) and the OS subjects (p = 0.034). The OT subjects had statistically significantly lower heart rate (HR) compared to the OS subjects (p = 0.011). The significantly higher LD flux was noticed in the YT compared to the YS subjects (p = 0.046). There were no significant differences among groups in SAP, SV and CO.

3.2 Resting values after ingestion of L-arginine

After the ingestion of L-arginine there was a statistically significant decrease in HR of all four groups of subjects (paired t-test, p < 0.05). The YT subjects had a statistically significantly lower HR compared to YS (Dunnett’s test, p < 0.05); the OT compared to the OS subjects also (p < 0.05). A statistically significant decrease of SAP and DAP was observed only in the YS group (paired t-test, p < 0.05) (Fig. 1).

CO significantly decreased in the YT, OS and OT. SV decreased significantly in the YT (paired t-test, p < 0.05). A LD flux increase was observed only in the group of YS subjects (paired t-test, p < 0.05) (Fig. 2).

3.3 Microvascular response to iontophoresis

A statistically significant increase in the LD flux response to ACh iontophoresis after the ingestion of L-arginine was observed only in the group of YT subjects (paired t-test, p < 0.05) (Fig. 3).

There were no statistically significant differences in the LD flux response to SNP iontophoresis after ingestion of L-arginine (Fig. 4).

In the group of YT subjects a correlation (R = –0.509; p = 0.0369) was observed between VO₂ max and a change in the LD flux response to the application of ACh after the ingestion of L-arginine.

3.4 Microvascular response to ACh and SNP iontophoresis in different age groups

The endothelium-dependent vasodilation in three out of four age groups of subjects did not change. In the group of subjects, aged 40–49 years, it was statistically significantly attenuated. The endothelium-independent vasodilation was statistically significantly attenuated in the oldest group compared to the younger groups. There was also a significant difference between the old and the medium aged group (29–39 years and 40–49 years) and between the medium aged and the young. The vasodilating response to ACh and SNP as a function of aging is presented in Fig. 5.

4 Discussion

The key findings of the present study are that after the ingestion of L-arginine: 1) the heart rate and the cardiac output decreased while blood pressure and resting LD flux mainly did not change; 2) the endothelium-dependent vasodilation increased only in the group of young trained men and 3) the endothelium-independent vasodilation did not change in any of the groups. 4) The endothelium-dependent vasodilation was diminished only in the age group of 40 to 49 years, while the endothelium-independent vasodilation was reduced with ascending age.

4.1 Systemic effect of L-arginine

Following administration of L-arginine HR decreased. This and the decrease of CO could be the result of the increase of participant calmness during the course of the experiment. Some other researchers did not find any change in HR following the ingestion of L-arginine in healthy subjects aged 18 to 37 years [1] nor after an intra-arterial infusion of L-arginine [43].

In our study, blood pressure and resting LD flux after ingestion of L-arginine mainly did not change. Many researchers have studied the effect of L-arginine on arterial pressure in healthy normotensive volunteers but their results are contradictory. Nakaki et al. [37], Bode-Boger et al. [2], and Giugliano et al. described the reduction of arterial pressure in normotensive subjects following an intravenous infusion of various concentrations of L-arginine. On the contrary, Chin-Dusting et al. [10] failed to show L-arginine-induced changes in arterial pressure. The differences in the observed results could be due to the different doses of L-arginine used.

4.2 Microvascular response to ACh iontophoresis

In the YT subjects we observed a significant increase in the endothelium-dependent vasodilation of cutaneous microvessels after the administration of L-arginine. As demonstrated by various studies [13, 33], in subjects who have a sufficient amount of available NO, regular physical activity adds to an increase in endothelium-dependent vasodilation. During exercise shear stress activates eNOS. eNOS-derived NO formed from L-arginine diffuses from endothelial cells into underlying smooth muscle cells and induces vasodilation by stimulating NO-sensitive guanylyl cyclase [32]. Training also results in decreased sensitivity to the vasoconstrictor effects of norepinephrine [12] and reduced oxidative stress, thus improving endothelial function [29, 45]. Furthermore, regular physical activity improves endothelium-dependent vasodilation of the brachial artery not only in healthy and young individuals but also in older subjects [45] and in people with cardiovascular diseases such as hypertension [22], heart failure [21], coronary artery disease [23] and acute myocardial infarction [50]. However, in our study L-arginine did not improve endothelium-dependent microvascular vasodilation in YS, OS or OT subjects. Similar results were found on the conductive arteries. The application of higher daily doses of L-arginine in healthy individuals (aged less than 37 years) did not have any effect on endothelium-dependent vasodilation of the brachial artery [1 , 46].

In healthy young individuals L-arginine should be endogenously synthesized in sufficient quantities, and the availability of NO should be large enough to maintain good dilation capability of their vasculature [35, 41]. However, in sedentary subjects it is possible that there is not enough eNOS available to bind the additional amount of substrate, that vessel wall is not normally responsive to released NO or that there is greater sensitivity to the vasoconstrictor effects of norepinephrine [12] or greater effect of oxidative stress.

In older subjects there are several additional possible explanations for the obtained results. Endothelial dysfunction is a primary phenotypic change in the process of ageing [15 , 47] and can be reversed with the addition of L-arginine. With advanced age, functional changes progress to structural changes, and irreversible defects of vasculature occur, for instance, vascular smooth muscle hypertrophy, reduction in capillary density, thickening of the microvascular basement membrane [15, 24]. In such cases, application of L-arginine would not have a measurable effect on blood pressure or skin microvascular blood flow. Numerous studies have shown that aging is associated with a decrease in endothelium-dependent vasodilation of conduit arteries [5 , 47]. The deficiency of NO and consequent endothelium dysfunction may be the result of reduced expression, impaired activity of the enzyme eNOS [4, 49], deficiency or reduced cellular uptake of L-arginine, elevated arginase activity, increased degradation of NO by endogenous inhibitors or by reactive oxygen species. One of the processes that may be associated with ageing is the occurrence of minimal chronic inflammation, which leads to the development of cardiovascular diseases [15] and degradation of L-arginine into ornithine and urea by the enzyme arginase [41].

There are many different tests used to study endothelial function, among others thermal hyperemia, postocclusive reactive hyperemia, pressure-induced vasodilation, flow-mediated arterial dilation and iontophoresis of the vasoactive drug. Those tests differ in the mechanisms, underlying vasodilation. Many studies concerning the effect of L-arginine and aging on endothelial function differs either in provocation used for the assessment of the vasodilation or in the type of vessels investigated. Bode-Boger et al. [3] showed that a daily 2-week-long consumption of L-arginine improved flow-mediated arterial dilation (FMD) in elderly healthy subjects. In contrast, Gates et al. [18] noted age-associated decline in FMD despite of increasing L-arginine concentration and similar concentration of asymmetric dimethylarginine (ADMA, competitive inhibitor of eNOS) and dimethylarginine dimethylaminohydrolase II, the enzyme isoform that controls ADMA degradation.

Holowatz and coworkers [26] measured effects of L-arginine (applied by microdialysis) on the reflex cutaneous vasodilation during whole-body heating, but the mechanism of this phenomenon is only partially dependent on ACh action [9, 31]. Their results showed greater vasodilation in young compared to older subjects. Vasodilation in older subjects improved to the level of young ones afterL-arginine addition [26]. Similarly inhibition of arginase improved vasodilation capability during heating in older subjects [26].

Our finding in the present study that endothelium-dependent vasodilation of cutaneous microvessels did not improve after single ingestion of L-arginine in the medium aged subjects could be explained by the assumption that vasculature of a medium aged subjects was already less responsive to NO due to functional changes which might be reversed with prolonged use of L-arginine. It is possible that the dose of L-arginine ingested in our experimental setting was not high enough.

4.3 Microvascular response to SNP iontophoresis

After the ingestion of L-arginine LD flux in response to SNP iontophoresis did not change in any of the tested group. Similar results were described by many researchers who studied the effect of L-arginine on the endothelium-independent vasodilation of arteries of subjects with a family history of hypertension [43], as well as those who had hypertension [43], hypercholesterolaemia [11] or chronic heart failure irrespective of physical activity [21].

4.4 Microvascular response to endothelium-dependent and endothelium-independent iontophoresis in different age groups

To obtain a clearer view of differences between young and older subjects we measured endothelium-dependent and endothelium-independent vasodilation in subjects, divided into four age groups. In three out of four groups the endothelium-dependent vasodilation did not differ. In contrast, in the group of subjects, aged 40–49 years, the endothelium-dependent vasodilation was significantly smaller than in younger or in older individuals. The reasons for this irregularity in present results are not known, perhaps they are linked to relatively sudden changes in hormonal status of the subjects in this age group [9] and for that reason they are transitory. On the contrary, the endothelium-independent vasodilation was diminished with advancing age. These results are in line with some results, already reported [6 , 48]. Possible explanation is that NOS and cyclooxygenase pathways are capable of enzymatic cross-talk. The inhibition of one vasodilator pathway may lead to upregulation of another [6, 48]. Since endothelium-independent vasodilation is not dependent on L-arginine concentration and endothelium-dependent vasodilation mainly does not decrease with ageing, we might conclude that the attenuated response to SNP in aged individuals is not due to diminished functioning of eNOS, but rather due to attenuated response of smooth muscles to messenger NO. Another possibility is alteration in the release of EDHF, which is particularly important in small resistance arteries involved in flow distribution and blood pressure modulation [17].

There are some limitations of our study. Our study was restricted to a single relatively low dose of L-arginine ingestion and only acute response to it. Using the impedance cardiography the CO and UV values can be determined only indirectly and therefore the results are only approximate. Errors due to this limitation were diminished by comparison of the values obtained in the same subject before and after ingestion of L-arginine. In addition, the result of iontophoretic application of ACh and SNP is also influenced by skin resistance, which differs among subjects. Therefore, the ACh or SNP vasodilatory effect before and after ingestion of L-arginine was compared in the same participant.

5 Conclusions

To our knowledge this is the first study to examine the effect of dietary supplement L-arginine on endothelial-dependent and endothelial-independent vasodilation at the level of microcirculation in young and medium aged healthy adults. It has been demonstrated that the dietary supplement L-arginine has a positive effect on the endothelial function of microvessels of young, physically active individuals. In medium aged subjects L-arginine supplementation did not improve endothelial function. From the observed results it could be concluded that as a dietary supplement L-arginine has the greatest acute impact on the young trained individuals probably through greater expression of eNOS and/or better responsiveness of their vascular smooth muscles to NO. To confirm beneficial effect of long term L-arginine supplementation in elderly subjects additional studies at the level of microcirculation are needed.

Conflict of interest

The authors confirm that they have no conflict of interest.

Footnotes

Acknowledgments

Present work was supported by the Grant No.: P3-0019, Ministry of Higher Education, Science and Technology, Slovenia.

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