Introduction

Primary aldosteronism (PA) is the most common cause of secondary hypertension. Previous studies have shown that the rate of cardiovascular events is higher in patients with PA than in patients with essential hypertension (EHT) [1, 2]. Endothelial dysfunction is the initial step in the development of atherosclerosis [3]. Furthermore, it has been established that endothelial function is an independent predictor of cardiovascular events [4]. It is well known that hypertension causes endothelial dysfunction [5,6,7,8]. Aldosterone would play important roles in the pathogenesis, development, and maintenance of hypertension [9,10,11,12,13,14].

Several studies have shown that aldosterone impairs endothelial function [15,16,17]. In a previous study, we demonstrated that endothelial function assessed by flow-mediated vasodilation (FMD) was impaired in patients with aldosterone-producing adenoma (APA) compared with that in patients with idiopathic hyperaldosteronism (IHA) or patients with EHT [17]. However, there is no information on the relationship between IHA and grade of vascular function in the macrovasculature and microvasculature.

It is well established that brachial FMD and log-transformed reactive hyperemia index (log RHI) measured by RH-peripheral arterial tonometry (RH-PAT) are useful for assessment of endothelial function. Both FMD and log RHI are associated with cardiovascular risk factors and cardiovascular events. However, there is no significant correlation between FMD and log RHI [18]. It is thought that FMD reflects macrovascular endothelial function and that log RHI reflects microvascular endothelial function. Thus, FMD and log RHI may indicate endothelial function in different vascular beds.

The purpose of this study was to evaluate the grade of vascular function, endothelium-dependent vasodilation assessed by FMD and log RHI, and endothelium-independent vasodilation assessed by nitroglycerine-induced vasodilation (NID) in patients with APA and patients with IHA compared with those in patients with EHT.

Methods

Subjects

We studied 53 patients with APA (35 men and 18 women; mean age: 49 ± 13 years), 52 patients with IHA (30 men and 22 women; 53 ± 12 years), and 52 age-, sex-, and blood pressure-matched patients with EHT (30 men and 22 women; 53 ± 10 years). Subjects were enrolled from the Hiroshima University Hypertension Database. This was a single center study. The ethical committees of our institutions approved the study protocol. Witten informed consent for participation in the study was obtained from all of the subjects.

Patients with EHT: Hypertension was defined as systolic blood pressure of more than 140 mm Hg or diastolic blood pressure of more than 90 mm Hg, in a sitting position, on at least three different occasions in the outpatient clinic of Hiroshima University School of Medicine. Secondary hypertensive patients were excluded on the basis of a complete history and physical examination, ramm hdiologic and ultrasound examinations, and urinalysis. Plasma renin activity (PRA), plasma aldosterone concentration (PAC), and serum creatinine, potassium, calcium, and free thyroxine concentrations were determined, and 24 h urinary excretion of catecholamines, 17-hydroxycorticosteroids, 17-ketogenic steroids, and vanillylmandelic acid was also measured. Patients with a history of coronary heart disease, cerebrovascular disease, or peripheral arterial disease were excluded from this study.

Patients with PA: PA, including the classification of PA, was defined according to the report of the guidelines for diagnosis and treatment of primary aldosteronism: the Japan Endocrine Society 2009 [19]. Briefly, a diagnosis of PA was confirmed by the captopril-challenge test, upright furosemide-loading test, and saline-loading test after screening for an aldosterone-to-renin ratio [ARR; PAC (pg/mL)/PRA (ng/mL/hr)] of more than 200. Then, to identify the lateralization of aldosterone secretion, PAC and plasma cortisol concentration were measured in adrenal venous blood using an adrenal vein sampling technique under adrenocorticotropic hormone stimulation in all patients. No patient had multiple endocrine neoplasias. Patients with familial hyperaldosteronism type 1 or 2 and patients with aldosterone-producing carcinoma were also not included in this study. No antihypertensive agents were taken by the patients for >2 weeks before the study.

Study protocol

Subjects fasted the previous night for at least 12 h. The study began at 08:30 am. The subjects were kept in the supine position in a quiet, dark, air-conditioned room (constant temperature of 22 –25 °C) throughout the study. A 23-gauge polyethylene catheter was inserted into the left deep antecubital vein to obtain blood samples. Thirty minutes after maintaining the supine position, log RHI, FMD, and NID were measured. The observers were blind to the form of examination.

Subjects fasted the previous night for at least 12 h. Thirty minutes after remaining in the supine position and fasting serum concentrations of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, blood urea nitrogen, creatinine, and glucose were measured.

Measurement of RHI

Peripheral arterial pulse amplitude was measured using a PAT device that was placed on each first finger (Endo-PAT2000, Itamar Medical, Caesarea, Israel). The inflation pressure of the PAT device was set to 10 mm Hg below diastolic blood pressure or at least 70 mm Hg. After baseline pulse amplitude had been recorded from each finger for 5 min, the blood pressure cuff was inflated in the test arm for 5 min to whichever inflated pressure would be higher: 200 mm Hg or 60 mm Hg plus systolic blood pressure. Pulse amplitude was recorded simultaneously from both fingers. After the cuff had been deflated, pulse amplitude was recorded for up to 5 min. The ratio of PAT was calculated automatically through a computer algorithm (Itamar Medical). Log RHI was calculated for subsequent analysis. Since the raw values of RHI had a heteroscedastic error structure, we used a natural logarithmic transformation. Inter- and intra-coefficients of variation for the pulse wave amplitude were 5.5% and 6.0%, respectively, in our laboratory.

Measurements of FMD and NID

Vascular response to reactive hyperemia in the brachial artery was used for assessment of endothelium-dependent FMD. A high-resolution linear artery transducer was coupled to computer-assisted analysis software (UNEXEF18G, UNEX Co, Nagoya, Japan) that used an automated edge detection system for measurement of brachial artery diameter [20].

A blood pressure cuff was placed around the forearm. The brachial artery was scanned longitudinally 5–10 cm above the elbow. When the clearest B-mode image of the anterior and posterior intimal interfaces between the lumen and vessel wall was obtained, the transducer was held at the same point throughout the scan by a special probe holder (UNEX Co) to ensure consistency of the image. Depth and gain setting were set to optimize the images of the arterial lumen wall interface. When the tracking gate was placed on the intima, the artery diameter was automatically tracked, and the waveform of diameter changes over the cardiac cycle was displayed in real time using the FMD mode of the tracking system. This allowed the ultrasound images to be optimized at the start of the scan and the transducer position to be adjusted immediately for optimal tracking performance throughout the scan. Pulsed Doppler flow was assessed at baseline and during peak hyperemic flow, which was confirmed to occur within 15 s after cuff deflation. Blood flow velocity was calculated from the color Doppler data and was displayed as a waveform in real time. The baseline longitudinal image of the artery was acquired for 30 s, and then the blood pressure cuff was inflated to 50 mm Hg above systolic pressure for 5 min. The longitudinal image of the artery was recorded continuously until 5 min after cuff deflation. Pulsed Doppler velocity signals were obtained for 20 s at baseline and for 10 s immediately after cuff deflation. Changes in brachial artery diameter were immediately expressed as percentage change relative to the vessel diameter before cuff inflation. FMD was automatically calculated as the percentage change in peak vessel diameter from the baseline value. Percentage of FMD [(Peak diameter-Baseline diameter)/Baseline diameter] was used for analysis. Blood flow volume was calculated by multiplying the Doppler flow velocity (corrected for the angle) by heart rate and vessel cross-sectional area (-r2). Reactive hyperemia was calculated as the maximum percentage increase in flow after cuff deflation compared with baseline flow.

The response to nitroglycerine was used for assessment of endothelium-independent vasodilation. NID was measured as described previously [20]. Briefly, after acquiring baseline rest images for 30 s, a sublingual tablet (75 μg nitroglycerine) was given, and images of the artery was recorded continuously until the dilation reached a plateau after administration of nitroglycerine. Subjects who had received nitrate treatment and subjects in whom the sublingually administered nitroglycerine tablet was not dissolved during the measurement were excluded from this study. NID was automatically calculated as a percent change in peak vessel diameter from the baseline value. Percentage of NID [(peak diameter−baseline diameter)/baseline diameter] was used for analysis. Inter- and intra-coefficients of variation for the brachial artery diameter were 1.6% and 1.4%, respectively, in our laboratory.

Statistical analysis

Results are presented as means ± SD for continuous variables and as percentages for categorical variables. Statistical significance was set at a level of P < 0.05. Continuous variables were compared by using ANOVA with the Tukey-HSD post hoc test for multiple groups. Categorical variables were compared by means of the χ2 test. Relationships between variables were determined by Spearman correlation coefficients analysis. Multivariable regression analysis was performed to identify factors associated with log RHI and FMD in risk factors and laboratory data. The data were processed using the software package Stata, version 9 (Stata Co, College Station, TX).

Results

Baseline clinical characteristics

The baseline clinical characteristics of the 52 patients with EHT, 52 patients with IHA, and 53 patients with APA are summarized in Table 1. Diastolic blood pressure was significantly higher in patients with APA than in patients with IHA. There was no significant difference in diastolic blood pressure between patients with IHA or APA and patients with EHT. Serum potassium concentration was significantly lower in patients with IHA or APA than in patients with EHT and was lower in patients with APA than in patients with IHA. PAC was significantly higher in patients with IHA or APA than in patients with EHT and was higher in patients with APA than in patients with IHA. PRA was significantly lower in patients with IHA or APA than in patients with EHT. There was no significant difference in PRA between patients with IHA and patients with APA. ARR was significantly higher in patients with IHA or APA than in patients with EHT and was higher in patients with APA than in patients with IHA. The percentage of patients with IHA or APA who used calcium channel blockers and alpha blockers was significantly higher than the percentage of patients with EHT who used calcium channel blockers and alpha blockers. The other parameters were similar in the groups.

Table 1 Clinical characteristics of the subjects
Full size table

Vascular function

Log RHI was significantly lower in the IHA and APA groups than in the EHT group (0.54 ± 0.25 and 0.55 ± 0.23 versus 0.79 ± 0.28; P < 0.01, respectively), whereas there were no significant differences in log RHI between the IHA and APA groups (Fig. 1a). FMD was significantly lower in the APA group than in the EHT groups (3.4 ± 2.1% versus 4.8 ± 2.8%; P = 0.02), whereas there was no significant difference in FMD between the IHA and the APA and EHT groups (Fig. 1b). NID was significantly lower in the APA group than in the EHT groups (10.0 ± 4.5% versus 12.5 ± 5.7%; P = 0.03), whereas there was no significant difference in NID between the IHA, APA, and EHT groups (Fig. 1c).

Fig. 1
figure 1

Bar graphs show Log RHI (a), flow-mediated vasodilation (b), nitroglycerine-induced vasodilation (c), in patients with essential hypertension (EHT), patients with idiopathic hyperaldosteronism (IHA) and patients with aldosterone-producing adenoma (APA)

Full size image

Log RHI correlated with systolic blood pressure (r = 0.26; P < 0.01), diastolic blood pressure (r = 0.19; P = 0.02), PAC (r = −0.19; P = 0.03), and PRA (r = 0.23; P < 0.01). FMD correlated with age (r = −0.24; P < 0.01), HbA1c (r = −0.22; P = 0.02), and PAC (r = −0.21; P = 0.01). NID correlated with age (r = −0.27; P < 0.01) and serum potassium (r = 0.20; P = 0.02) (Table 2).

Table 2 Univariate analysis of relationships among Log RHI, FMD, NID, and variables
Full size table

After adjustment for age and sex, the association of log RHI with PAC (t = −2.24; P = 0.03) and the associations of FMD with PAC (t = −3.07; P < 0.01) remained significant (Table 3).

Table 3 Multivariate analysis of FMD and RHI with clinical variables
Full size table

Discussion

In this study, we demonstrated for the first time that endothelial function in a microvasculature, but not that in a macrovasculature, was impaired in patients with IHA compared with that in patients with EHT. Endothelial function was impaired in both the macrovasculature and microvasculature in patients with APA compared with that in patients with EHT. Microvascular endothelial function assessed by log RHI was similarly impaired in patients with APA and patients with IHA, while macrovascular endothelial function assessed by FMD was impaired in patients with APA but not in patients with IHA. NID was impaired in patients with APA compared with that in patients with IHA. Endothelial function was impaired in the digital microcirculation but not in the brachial macrocirculation in patients with IHA compared with that in patients with EHT.

Microvascular endothelial function was impaired in the digital artery in patients with IHA compared with that in patients with EHT, whereas macrovascular endothelial function in the brachial artery was similar in patients with IHA and patients with EHT. In a previous study, we showed that FMD is significantly lower in patients with APA than in patients with IHA and patients with EHT, whereas there is no significant difference in FMD between patients with IHA and patients with EHT [17]. We and several investigators showed that there was a significant relationship between PAC and FMD in all patients, and surgical resection of APA improved FMD in patients with APA [15, 17]. However, there is no information on the relationship between IHA and microvascular vascular function. Measurement of vascular function by RH-PAT is a useful method for assessing microvascular vascular function. Recently, several studies have shown that log RHI is related to multiple factors of atherosclerosis [18, 21,22,23]. In the present study, we found that there was a significant relationship between PAC and log RHI. After adjustment for age and sex, the association of log RHI with PAC remained significant.

In the present study, FMD was similar in patients with IHA and patients with EHT. Both FMD and log RHI are used to assess endothelial function. Several investigators have shown that FMD and log RHI are associated with cardiovascular risk factors and cardiovascular events [22,23,24,25,26,27]. However, it has been shown that there is no significant relationship between FMD and log RHI [18]. There is a possibility that different endothelial functions are assessed by FMD and log RHI [18, 28]. Microvascular endothelial function in patients with IHA was impaired compared with that in patients with EHT, whereas macrovascular endothelial function was similar in patients with IHA and patients with EHT. These findings suggest that moderately elevated PAC may impair microvascular endothelial function and that greatly elevated PAC may impair endothelial function in both macrovasculature and microvasculature. It is likely that endothelial function is different depending on the vascular size and vascular bed, especially under the condition of elevation of circulating aldosterone levels. It remains unclear whether the prevalence of cardiovascular events is higher in patients with IHA than in patients with EHT. Form the aspect of microvascular endothelial function, patients with IAH may have more progressive atherosclerosis and a higher prevalence of cardiovascular events compared with those in patients with EHT.

Recently, some possible mechanisms by which aldosterone impairs endothelial function have been postulated. Aldosterone increases reactive oxygen species through activation of NADPH oxidase in mice and rats in a mineralocorticoid receptor-dependent manner [29, 30]. Griol-Charhbili et al. [31]. reported that aldosterone-induced endothelial dysfunction was due to a decrease in epidermal growth factor receptor (EGFR) activity. EGFR activity is required for the cross-talk between aldosterone and angiotensin II. EGFR-related vascular dysfunction may be involved in vasoconstriction. Aldosterone directly inhibits endothelial NO synthase (eNOS) phosphorylation, leading to a decrease in eNOS activity in human umbilical vein endothelial cells [32, 33]. In addition, aldosterone causes vascular structural changes through increases in inflammation and fibrosis in the vasculature, resulting in endothelial dysfunction [34, 35]. These findings suggest that aldosterone per se impairs endothelial function through various mechanisms, including the eNOS/NO pathway and proinflammatory pathway.

In the present study, the percentages of patients with IHA and APA taking calcium channel blockers and alpha blockers were significantly higher than the percentage of patients with EHT taking calcium channel blockers and alpha blockers. Some studies have shown that calcium channel blockers reduce oxidative stress, increase NO release and improve endothelial dysfunction [36]. In addition, beneficial effects of alpha-blockers on endothelial function has been shown [37]. Therefore, it is considered that endothelial function in patients with IHA and APA has already been improved compared with that in patients with EHT. Nevertheless, log RHI was significantly lower in the IHA and APA groups than in the EHT group and FMD was significantly lower in the APA group than in the EHT group.

In this study, the number of patients with PA was relatively small. However, we observed that microvascular endothelial function was impaired in patients with IHA compared with that in patients with EHT. Future studies are needed to confirm the role of aldosterone in microvascular endothelial function in patients with IHA in a large population.

In conclusion, microvascular endothelial function was impaired in patients with IHA compared with that in patients with EHT. Patients with IHA may have more cardiovascular events in the future than those in patients with EHT. Therefore, we should pay attention when caring for patients with IHA.