Abstract

Background:

A deficiency of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide (NO) synthase, decreases NO synthesis and increases superoxide production. Supplementation of BH4 has been postulated to improve endothelial function in atherosclerotic patients. The purpose of this study was to determine whether BH4 restores endothelium-dependent vasodilation in patients with essential hypertension.

Methods:

We evaluated the effects of BH4 on forearm vascular responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and isosorbide dinitrate (ISDN), an endothelium-independent vasodilator, both in patients with essential hypertension and in age- and sex-matched normal control subjects. Forearm blood flow (FBF) was measured using strain gauge plethysmography.

Results:

The response of FBF to ACh was less in hypertensive patients (n = 8) than in normal control subjects (n = 8). There was no significant difference in FBF response to ISDN in the two groups. During coinfusion of BH4 (500 mg/min), the FBF response to ACh in hypertensive patients increased significantly (14.8 ± 4.6 to 25.6 ± 7.3 mL/min/100 mL tissue, P < .05) to the level of normal control subjects. In the control subjects, also, BH4 augmented the FBF response to ACh (27.8 ± 8.7 to 36.1 ± 9.6 mL/min/100 mL tissue, P < .05). The increase in FBF after ISDN was not altered by BH4 in either group (each group, n = 6).

Conclusion:

Supplementation of BH4 augments endothelium-dependent vasodilation in both normotensive and hypertensive individuals.

Am J Hypertens 2002;15: 326–332 © 2002 American Journal of Hypertension, Ltd.

Nitric oxide (NO) plays an important role in the regulation of vascular tone, inhibits the aggregation and adhesion of platelets, and participates in the suppression of smooth muscle cell proliferation.1–3 Altered NO release from endothelium has been demonstrated in patients with vascular disease, including hypertension.4–6 Several lines of evidence have shown that endothelium-dependent vasodilation evoked by the stimulation of NO release in brachial,7–9 coronary,10 renal,11–13 femoral,14 and small arteries15 is impaired in patients with essential hypertension.

Endothelial NO synthase (eNOS) requires several cofactors such as heme, flavin adenine dinucleotide, and flavin mononucleotide as well as tetrahydrobiopterin (BH4), for full enzymatic activity.16,17 The substance BH4 is an allosteric effector in the coupling of the oxidase and reductase domains of eNOS.16 Recently it has been reported that a deficiency of BH4 decreases NO synthesis and increases superoxide production.18 In prehypertensive spontaneous hypertensive rats, dysfunctional eNOS with insufficient BH4 produces superoxide generation, leading to a decrease in NO activity compared with that in normotensive Wistar-Kyoto rats.19 Reduced availability of BH4 may contribute to the maintenance and development of hypertension. In addition, it has been demonstrated, in vivo and in vitro, that supplementation of BH4 improves endothelial function.20,21 However, there is no information regarding the role of BH4 in the moderation of endothelial function in humans with hypertension.

We evaluated whether BH4 restores endothelium-dependent vasodilation in patients with essential hypertension. For this purpose, we studied the effect of BH4 on forearm vascular responses to vasoactive agents such as acetylcholine (ACh), an endothelium-dependent vasodilator, and isosorbide dinitrate (ISDN), an endothelium-independent vasodilator.

Methods

Subjects

We studied 14 Japanese patients with essential hypertension (11 men and three women; mean age 47 ± 10 years) and 14 normotensive subjects (10 men and four women; mean age 44 ± 7 years). Hypertension was defined as a systolic blood pressure (BP) of >140 mm Hg or a diastolic BP of >90 mm Hg, while seated, on at least three different occasions. Measurements were obtained in the outpatient clinic of Hiroshima University Faculty of Medicine. Patients with secondary forms of hypertension were excluded. No patient had a history of antihypertensive treatment before the study. Normotension was defined as a systolic BP of <130 mm Hg and a diastolic BP of <80 mm Hg. Subjects with a history of cardiovascular or cerebrovascular disease, hypercholesterolemia, diabetes mellitus, liver disease, renal disease, or smoking were excluded. The study protocol was approved by the ethical committee of the Hiroshima University Faculty of Medicine. Informed consent for participation was obtained from all subjects.

Measurement of forearm blood flow

Forearm blood flow (FBF) was measured using a mercury-filled silicone elastomer (Silastic) strain-gauge plethysmograph (EC-5R, D.E. Hokanson, Bellevue, WA) as previously described.7 The FBF was expressed as milliliters per minute per 100 mL of forearm tissue volume. Four plethysmographic measurements were averaged for analysis of FBF at baseline and during administration of drugs. Forearm vascular resistance (FVR) was calculated as the mean arterial pressure divided by FBF.

In the preliminary study, we evaluated the effect of the intra-arterial infusion of BH4 (0.1, 0.5, 1.0, 5.0, and 25 mg/min for 5 min, respectively) on forearm hemodynamics (n = 4). None of the tested dosages of BH4 altered FBF, arterial BP, or heart rate. Plasma biopterin concentrations measured after infusion of graded dose BH4 (0.1, 0.5, 1.0, 5.0, and 25 mg/min) increased from 2.1 ± 0.6 to 18 ± 4.5, 108 ± 47, 1120 ± 596, and 5219 ± 1768 ng/mL, respectively. In the present study we used 500 μg/min of BH4, as in previous studies.20

Study protocol 1: effect of BH4 on endothelium-dependent vasodilation

Forearm vascular responses to ACh alone and in combination with BH4 were evaluated in eight patients with essential hypertension (six men and two women; mean age 48 ± 11 years) and eight age- and sex-matched normal control subjects (six men and two women; mean age 44 ± 9 years). The study began at 8:30 AM. Subjects fasted the previous night for at least 12 h. They were kept in the supine position in a quiet, dark, air-conditioned room (constant temperature 22° to 25°C) throughout the study. A 23-gauge polyethylene catheter (Hakkow, Okayama, Japan) was inserted into the left brachial artery for the infusion of ACh and BH4 and for the recording of arterial pressure with an AP-641G pressure transducer (Nihon Kohden, Tokyo, Japan) under local anesthesia (1% lidocaine). Another catheter was inserted into the left deep antecubital vein to obtain blood samples.

After the patients were placed for 30 min in the supine position, FBF and arterial BP were measured. The effect of the endothelium-dependent vasodilator ACh on forearm hemodynamics was then measured. The ACh (3.75, 7.5, and 15 μg/min) was infused intra-arterially for 5 min at each dose using a constant rate infusion pump (Terfusion STG-523; Terumo, Tokyo, Japan). The FBF was measured during the last 2 min of the infusion. After a 30-min rest period, ACh (3.75, 7.5, and 15 μg/min) was infused for 5 min at each dose in combination with BH4 (500 μg/min), and the FBF was measured.

Study protocol 2: effect of BH4 on endothelium-independent vasodilation

The forearm vascular responses to ISDN alone and in combination with BH4 were evaluated in a protocol identical to study protocol 1 in six patients with essential hypertension (five men and one woman; mean age 46 ± 8 years) and six age- and sex-matched normal control subjects (four men and two women; mean age 44 ± 6 years). The effects of the endothelium-independent vasodilator ISDN on forearm hemodynamics were measured. We infused ISDN (0.75, 1.5, and 3.0 μg/min) intra-arterially for 5 min at each dose, and FBF was measured during the last 2 min of the infusion. After a 30-min rest period, ISDN (0.75, 1.5, and 3.0 μg/min) was infused for 5 min at each dose in combination with BH4 (500 μg/min), and the FBF was measured.

Study drugs

In this study we used ACh chloride (Daiichi Pharmaceutical, Tokyo, Japan), ISDN (Eisai Pharmaceutical, Tokyo, Japan), and (6R)-5,6,7,8-tetrahydrobiopterin (BH4; Sigma Chemical Co., St. Louis, MO). All drugs were obtained commercially and were dissolved in oxygen-free saline immediately before use.

Analytical methods

Routine chemical methods were used to determine serum concentrations of total cholesterol, HDL cholesterol, triglycerides, creatinine, glucose, insulin, and electrolytes. Serum concentration of low density lipoprotein (LDL) was estimated using Friedewald's method.22 The plasma concentration of biopterin was measured by high performance liquid chromatography (BML Co., Tokyo, Japan).

Statistical analysis

Results are presented as mean ± SD. P values < .05 were considered statistically significant. The Mann-Whitney U test was used to evaluate differences between the hypertensive and normal control subjects for baseline parameters. Comparisons of dose-response curves of parameters during drug infusion were analyzed by analysis of variance (ANOVA) for repeated measures. The data were processed using either the software package StatView IV (SAS Institute Inc., Cary, NC) or Super ANOVA (Abacus Concepts, Berkeley, CA).

Results

Clinical characteristics

The baseline clinical characteristics of the normal control subjects and the patients with essential hypertension are shown in Tables 1 and 2. Systolic and diastolic BPs as well as FVR were significantly higher in the hypertensive patients than in the normal control subjects. Other parameters were similar in the two groups.

Table 1

Baseline clinical characteristics in the normal control subjects and hypertensive patients in acetylcholine study

Variable Normal Control Subjects (n = 8) Hypertensive Patients (n = 8) 
Body mass index (kg/m224.4 ± 1.8 24.3 ± 1.9 
Systolic blood pressure (mm Hg) 115.2 ± 8.9 155.3 ± 9.7* 
Diastolic blood pressure (mm Hg) 68.2 ± 6.5 97.5 ± 4.9* 
Heart rate (bpm) 69.0 ± 6.4 71.1 ± 7.7 
Total cholesterol (mmol/L) 4.99 ± 0.71 5.02 ± 0.74 
Triglycerides (mmol/L) 0.96 ± 0.51 1.02 ± 0.57 
HDL cholesterol (mmol/L) 1.42 ± 0.28 1.34 ± 0.25 
LDL cholesterol (mmol/L) 3.32 ± 0.48 3.46 ± 0.65 
Serum glucose (mmol/L) 4.8 ± 0.5 4.8 ± 0.9 
Serum insulin (pmol/L) 53.2 ± 13.1 60.1 ± 17.2 
FBF (mL/min/100 mL tissue) 4.6 ± 1.3 4.5 ± 1.3 
FVR (mm Hg/mL/min/100 mL tissue) 18.2 ± 4.1 25.6 ± 3.7* 
Variable Normal Control Subjects (n = 8) Hypertensive Patients (n = 8) 
Body mass index (kg/m224.4 ± 1.8 24.3 ± 1.9 
Systolic blood pressure (mm Hg) 115.2 ± 8.9 155.3 ± 9.7* 
Diastolic blood pressure (mm Hg) 68.2 ± 6.5 97.5 ± 4.9* 
Heart rate (bpm) 69.0 ± 6.4 71.1 ± 7.7 
Total cholesterol (mmol/L) 4.99 ± 0.71 5.02 ± 0.74 
Triglycerides (mmol/L) 0.96 ± 0.51 1.02 ± 0.57 
HDL cholesterol (mmol/L) 1.42 ± 0.28 1.34 ± 0.25 
LDL cholesterol (mmol/L) 3.32 ± 0.48 3.46 ± 0.65 
Serum glucose (mmol/L) 4.8 ± 0.5 4.8 ± 0.9 
Serum insulin (pmol/L) 53.2 ± 13.1 60.1 ± 17.2 
FBF (mL/min/100 mL tissue) 4.6 ± 1.3 4.5 ± 1.3 
FVR (mm Hg/mL/min/100 mL tissue) 18.2 ± 4.1 25.6 ± 3.7* 

HDL = high-density lipoprotein; LDL = low-density lipoprotein; FBF = forearm blood flow; FVR = forearm vascular resistance.

All results are presented as the mean ± SD.

*

P < .05 v normal control subjects.

Table 2

Baseline clinical characteristics in the normal control subjects and hypertensive patients in isosorbide dinitrate study

Variable Normal Control Subjects (n = 6) Hypertensive Patients (n = 6) 
Body mass index (kg/m224.7 ± 1.9 24.2 ± 2.0 
Systolic blood pressure (mm Hg) 116.8 ± 9.8 155.8 ± 10.6* 
Diastolic blood pressure (mm Hg) 69.0 ± 6.9 95.1 ± 6.3* 
Heart rate (bpm) 67.3 ± 7.1 69.2 ± 8.1 
Total cholesterol (mmol/L) 5.03 ± 0.82 5.05 ± 0.88 
Triglycerides (mmol/L) 0.98 ± 0.57 1.05 ± 0.64 
HDL cholesterol (mmol/L) 1.47 ± 0.35 1.37 ± 0.46 
LDL cholesterol (mmol/L) 3.12 ± 0.67 3.53 ± 0.72 
Serum glucose (mmol/L) 4.9 ± 0.7 4.5 ± 0.8 
Serum insulin (pmol/L) 51.1 ± 15.6 54.7 ± 16.9 
FBF (mL/min/100 mL tissue) 4.7 ± 1.4 4.6 ± 1.3 
FVR (mm Hg/mL/min/100 mL tissue) 18.1 ± 4.3 25.3 ± 4.9* 
Variable Normal Control Subjects (n = 6) Hypertensive Patients (n = 6) 
Body mass index (kg/m224.7 ± 1.9 24.2 ± 2.0 
Systolic blood pressure (mm Hg) 116.8 ± 9.8 155.8 ± 10.6* 
Diastolic blood pressure (mm Hg) 69.0 ± 6.9 95.1 ± 6.3* 
Heart rate (bpm) 67.3 ± 7.1 69.2 ± 8.1 
Total cholesterol (mmol/L) 5.03 ± 0.82 5.05 ± 0.88 
Triglycerides (mmol/L) 0.98 ± 0.57 1.05 ± 0.64 
HDL cholesterol (mmol/L) 1.47 ± 0.35 1.37 ± 0.46 
LDL cholesterol (mmol/L) 3.12 ± 0.67 3.53 ± 0.72 
Serum glucose (mmol/L) 4.9 ± 0.7 4.5 ± 0.8 
Serum insulin (pmol/L) 51.1 ± 15.6 54.7 ± 16.9 
FBF (mL/min/100 mL tissue) 4.7 ± 1.4 4.6 ± 1.3 
FVR (mm Hg/mL/min/100 mL tissue) 18.1 ± 4.3 25.3 ± 4.9* 

Abbreviations as in Table 1.

All results are presented as the mean ± SD.

*

P < .05 v normal control subjects.

The response of FBF to the infusion of the endothelium-dependent vasodilator ACh was smaller in the hypertensive patients than in the normal control subjects (Fig. 1). The vasodilating effect of the endothelium-independent vasodilator ISDN was similar in the two groups (Fig. 1). No significant change was observed in arterial BP or heart rate with intra-arterial infusion of either ACh or ISDN in either group.

Effects of acetylcholine and isosorbide dinitrate on forearm blood flow in the hypertensive patients and normal control subjects. The response of the forearm vasculature to acetylcholine was less in hypertensive patients (n = 8) than in normal control subjects (n = 8). There were no significant differences in the vascular responses to isosorbide dinitrate between the two groups (n = 6, respectively).

Effects of the coinfusion of BH4, ACh, and ISDN on forearm hemodynamics

Infusion of BH4 improved impaired endothelium-dependent vasodilation in hypertensive patients to the level of normal control subjects (Fig. 2). The FBF response to the infusion of the endothelium-dependent vasodilator ACh increased significantly with coinfusion of BH4 in the control as well as the hypertensive group (Fig. 2). The increase in FBF during infusion of the endothelium-independent vasodilator ISDN was not altered by coinfusion of BH4 in normal or hypertensive individuals (Fig. 2).

Effects of acetylcholine and isosorbide dinitrate on forearm blood flow before and after coinfusion of BH4 in hypertensive patients and normal control subjects. Coinfusion of BH4 augmented endothelium-dependent vasorelaxation in both groups (n = 8, respectively). However, BH4 did not alter endothelium-independent vasorelaxation in either group (each group, n = 6). BH4 = tetrahydrobiopterin; NS = not significant.

No significant change was observed in arterial BP or heart rate after intra-arterial infusion of either ACh or ISDN in combination with BH4 in either group.

Discussion

The present findings demonstrate that BH4, an essential cofactor for eNOS, enhances ACh-induced vasodilation in normotensive as well as essential hypertensive subjects, and that the augmentation of endothelium-dependent vasodilation evoked by BH4 may be due to an increase in NO availability. In fact, BH4 did not significantly alter endothelium-independent vasodilation in either normotensive or hypertensive subjects.

In the present study, endothelium-dependent vasodilation was reduced in essential hypertensive patients compared with normotensive control subjects, whereas endothelium-independent vasodilation was similar in both groups. These findings are consistent with previous studies showing that endothelial function in brachial,7–9 coronary,10 renal,11–13 femoral,14 and small artery15 circulation was impaired in hypertensive patients. Recently it was reported that the endothelial dysfunction seen in hypertensive patients is reversed by treatment with antihypertensive agents13,15,23 and with lifestyle modification.24 Endothelial dysfunction is an early feature of atherosclerosis and vascular complications in patients with essential hypertension. Thus, it is clinically important to restore endothelial dysfunction in conditions where there is reduced NO availability.

Several investigators have reported that when the levels of BH4 are suboptimal, endothelium-dependent vasodilation is impaired in coronary arteries of dogs, and that addition of BH4 can restore this vasodilation.21 Also, BH4 supplementation improves impaired endothelium-dependent vasodilation in brachial arteries of patients with hyperecholesterolemia20 and in saphenous vein rings from patients who smoke.25 These findings suggest that BH4 deficiency may contribute to endothelial dysfunction in these patients. However, in the present study, supplementation of BH4 augmented ACh-induced vasodilation in normotensive as well as hypertensive individuals. It is unlikely that the deficiency of BH4 is linked to impaired endothelium-dependent vasodilation in patients with essential hypertension.

Several reasons may explain why BH4 augments endothelium-dependent vasodilation. The biologic effects of NO are regulated not only by the amounts of production but also by the degree of superoxide inactivation.

First, BH4 when added may directly stimulate eNOS activity, leading to an increase in NO production. In fact, BH4 is an important cofactor for activity of all NOS isoforms, including eNOS.16,17 It is known that cytokine-induced NO production requires an increase in intracellular BH4 levels, and that exogenous BH4 supplementation enhances NO production.26 These findings suggest that the cofactor BH4 alone can regulate NOS activity. In rat aortic rings, administration of exogenous BH4 causes endothelium-dependent vasorelaxation.27 This vasorelaxation was abolished by Nω-monomethyl-L-arginine, suggesting that BH4-evoked, endothelium-dependent vasodilation may be due to stimulated eNOS activity and an increase in NO production.

Second, there is also a possibility the BH4 does not play an important role in the regulation of NOS activity, but contributes to the generation of reactive oxygen species, especially superoxide in vascular endothelium.16,17 It has recently been reported that the degradation of BH4 by reactive oxygen species, including peroxynitrite, superoxide, and hydrogen peroxide, is associated with the downregulation of eNOS.28 Therefore, BH4 supplementation may increase the intracellular content of BH4 and augment ACh-induced vasodilation through the inhibition of NO inactivation. However, in the present study, the precise mechanism by which supplementation of BH4 increases NO availability was not elucidated.

Finally, it is possible that the increase in BH4 causes vasodilation directly. However, in the preliminary study, we performed intra-arterial administration of BH4 in a dose-dependent manner to evaluate the direct effect of BH4 on forearm blood flow. Although the maximum dose of BH4 (25 mg/min) caused a 2500-fold increase in the biopterin concentration, BH4 did not significantly alter forearm hemodynamics including FBF, arterial BP, and heart rate. This finding is consistent with previous reports that BH4 does not alter basal FBF in either hypercholesterolemic patients or normal individuals,20 suggesting that the infusion of exogenous BH4 may not cause vasodilation directly in the brachial artery.

In our study, BH4 significantly augmented ACh-induced vasodilation in normal control subjects. In contrast, Stores et al20 reported that BH4 administered at the same dose used in the present study, had no effect on endothelium-dependent vasodilation in healthy control subjects. In the present study, our control group did not have risk factors for endothelial dysfunction such as high BP, abnormal lipid and glucose metabolism, postmenopause, and smoking. Although the reason for this discrepancy is unknown, it may be due to age differences (our control group: 44 ± 9 [SD] years v the control group of Stores et al: 28 ± 2 [SE] years), because aging can impair endothelium-dependent vasodilation.12,29

The number of subjects included is relatively small. Therefore, we cannot exclude the possibility that there is selection bias in the results.

In conclusion, BH4 augmented the endothelium-dependent vasodilation in the forearm in normotensive as well as hypertensive individuals through an increase in NO generated by eNOS. Our results also suggest that BH4 supplementation may represent a new therapeutic intervention for endothelial dysfunction in patients with essential hypertension. In future studies, it will be important to determine whether BH4 restores impaired endothelial function in the settings of atherosclerosis, chronic heart failure, diabetes mellitus, and estrogen withdrawal.

Acknowledgments

The authors thank Drs. Shigeaki Arai and Masahiko Sakai for preparation of the BH4 and oxygen-free saline, and Yuko Omura for secretarial assistance.

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Author notes

*
This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (T. Oshima) and Japan Heart Foundation Grant for Research on Hypertension and Metabolism (Y. Higashi) and a Grant for Research Foundation for Community Medicine (Y. Higashi).