Tetrahydrobiopterin Enhances Forearm Vascular Response to Acetylcholine in Both Normotensive and Hypertensive Individuals

AJH 2002; 15:326–332

Yukihito Higashi, Shota Sasaki, Keigo Nakagawa, Yukihiro Fukuda, Hideo Matsuura, Tetsuya Oshima, and Kazuaki Chayama Downloaded from https://academic.oup.com/ajh/article/15/4/326/217588 by guest on 29 September 2021

Background: A deﬁciency of tetrahydrobiopterin subjects (n ϭ 8). There was no signiﬁcant difference in

(BH4), an essential cofactor for nitric oxide (NO) syn- FBF response to ISDN in the two groups. During coinfu- thase, decreases NO synthesis and increases superoxide sion of BH4 (500 mg/min), the FBF response to ACh in Ϯ production. Supplementation of BH4 has been postulated hypertensive patients increased signiﬁcantly (14.8 4.6 to improve endothelial function in atherosclerotic patients. to 25.6 Ϯ 7.3 mL/min/100 mL tissue, P Ͻ .05) to the level The purpose of this study was to determine whether BH4 of normal control subjects. In the control subjects, also, Ϯ restores endothelium-dependent vasodilation in patients BH4 augmented the FBF response to ACh (27.8 8.7 to with essential hypertension. 36.1 Ϯ 9.6 mL/min/100 mL tissue, P Ͻ .05). The increase Methods: We evaluated the effects of BH on forearm in FBF after ISDN was not altered by BH4 in either group 4 ϭ vascular responses to acetylcholine (ACh), an endotheli- (each group, n 6). um-dependent vasodilator, and isosorbide dinitrate Conclusion: Supplementation of BH4 augments endo- (ISDN), an endothelium-independent vasodilator, both in thelium-dependent vasodilation in both normotensive and patients with essential hypertension and in age- and sex- hypertensive individuals. Am J Hypertens 2002;15: matched normal control subjects. Forearm blood ﬂow 326Ð332 © 2002 American Journal of Hypertension, Ltd. (FBF) was measured using strain gauge plethysmography. Results: The response of FBF to ACh was less in Key Words: Tetrahydrobiopterin, nitric oxide, acetyl- hypertensive patients (n ϭ 8) than in normal control choline, endothelial function, essential hypertension.

itric oxide (NO) plays an important role in the is an allosteric effector in the coupling of the oxidase and regulation of vascular tone, inhibits the aggrega- reductase domains of eNOS.16 Recently it has been re-

tion and adhesion of platelets, and participates in ported that a deficiency of BH4 decreases NO synthesis N 1Ð3 18 the suppression of smooth muscle cell proliferation. and increases superoxide production. In prehypertensive Altered NO release from endothelium has been demon- spontaneous hypertensive rats, dysfunctional eNOS with strated in patients with vascular disease, including hyper- insufficient BH4 produces superoxide generation, leading tension.4Ð6 Several lines of evidence have shown that to a decrease in NO activity compared with that in nor- endothelium-dependent vasodilation evoked by the stim- motensive Wistar-Kyoto rats.19 Reduced availability of 7Ð9 10 ulation of NO release in brachial, coronary, re- BH4 may contribute to the maintenance and development nal,11Ð13 femoral,14 and small arteries15 is impaired in of hypertension. In addition, it has been demonstrated, in patients with essential hypertension. vivo and in vitro, that supplementation of BH4 improves Endothelial NO synthase (eNOS) requires several co- endothelial function.20,21 However, there is no information factors such as heme, flavin adenine dinucleotide, and regarding the role of BH4 in the moderation of endothelial flavin mononucleotide as well as tetrahydrobiopterin function in humans with hypertension. 16,17 (BH4), for full enzymatic activity. The substance BH4 We evaluated whether BH4 restores endothelium-de-

Received May 30, 2001. First Decision August 23, 2001. (T. Oshima) and Japan Heart Foundation Grant for Research on Hyper- Accepted November 6, 2001. tension and Metabolism (Y. Higashi) and a Grant for Research Founda- From the First Department of Internal Medicine (YH, SS, KN, YF, tion for Community Medicine (Y. Higashi). HM, KC) and the Department of Clinical Laboratory Medicine (TO), Address correspondence and reprint requests to Dr. Yukihito Higashi, Hiroshima University Faculty of Medicine, Hiroshima, Japan. First Department of Internal Medicine, Hiroshima University Faculty of This study was supported in part by a Grant-in-Aid for Scientiﬁc Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan; e- Research from the Ministry of Education, Science and Culture of Japan mail: [email protected]

0895-7061/02/$22.00 © 2002 by the American Journal of Hypertension, Ltd. PII S0895-7061(01)02317-2 Published by Elsevier Science Inc. AJH–April 2002–VOL. 15, NO. 4, PART 1 TETRAHYDROBIOPTERIN AND ENDOTHELIAL FUNCTION 327

pendent vasodilation in patients with essential hyperten- 48 Ϯ 11 years) and eight age- and sex-matched normal Ϯ sion. For this purpose, we studied the effect of BH4 on control subjects (six men and two women; mean age 44 forearm vascular responses to vasoactive agents such as 9 years). The study began at 8:30 AM. Subjects fasted the acetylcholine (ACh), an endothelium-dependent vasodila- previous night for at least 12 h. They were kept in the tor, and isosorbide dinitrate (ISDN), an endothelium-inde- supine position in a quiet, dark, air-conditioned room pendent vasodilator. (constant temperature 22¡ to 25¡C) throughout the study. A 23-gauge polyethylene catheter (Hakkow, Okayama, Methods Japan) was inserted into the left brachial artery for the infusion of ACh and BH4 and for the recording of arterial Subjects pressure with an AP-641G pressure transducer (Nihon We studied 14 Japanese patients with essential hyperten- Kohden, Tokyo, Japan) under local anesthesia (1% lido- Ϯ caine). Another catheter was inserted into the left deep sion (11 men and three women; mean age 47 10 years) Downloaded from https://academic.oup.com/ajh/article/15/4/326/217588 by guest on 29 September 2021 and 14 normotensive subjects (10 men and four women; antecubital vein to obtain blood samples. mean age 44 Ϯ 7 years). Hypertension was deﬁned as a After the patients were placed for 30 min in the supine systolic blood pressure (BP) of Ͼ140 mm Hg or a diastolic position, FBF and arterial BP were measured. The effect of BP of Ͼ90 mm Hg, while seated, on at least three different the endothelium-dependent vasodilator ACh on forearm occasions. Measurements were obtained in the outpatient hemodynamics was then measured. The ACh (3.75, 7.5, clinic of Hiroshima University Faculty of Medicine. Pa- and 15 ␮g/min) was infused intra-arterially for 5 min at tients with secondary forms of hypertension were ex- each dose using a constant rate infusion pump (Terfusion cluded. No patient had a history of antihypertensive treat- STG-523; Terumo, Tokyo, Japan). The FBF was measured ment before the study. Normotension was deﬁned as a during the last 2 min of the infusion. After a 30-min rest systolic BP of Ͻ130 mm Hg and a diastolic BP of Ͻ80 period, ACh (3.75, 7.5, and 15 ␮g/min) was infused for 5 ␮ mm Hg. Subjects with a history of cardiovascular or min at each dose in combination with BH4 (500 g/min), cerebrovascular disease, hypercholesterolemia, diabetes and the FBF was measured. mellitus, liver disease, renal disease, or smoking were

excluded. The study protocol was approved by the ethical Study Protocol 2: Effect of BH4 on committee of the Hiroshima University Faculty of Medi- Endothelium-Independent Vasodilation cine. Informed consent for participation was obtained from The forearm vascular responses to ISDN alone and in all subjects. combination with BH4 were evaluated in a protocol iden- tical to study protocol 1 in six patients with essential Measurement of Forearm Blood Flow hypertension (five men and one woman; mean age 46 Ϯ 8 Forearm blood flow (FBF) was measured using a mercury- years) and six age- and sex-matched normal control sub- filled silicone elastomer (Silastic) strain-gauge plethysmo- jects (four men and two women; mean age 44 Ϯ 6 years). graph (EC-5R, D.E. Hokanson, Bellevue, WA) as previ- The effects of the endothelium-independent vasodilator ously described.7 The FBF was expressed as milliliters per ISDN on forearm hemodynamics were measured. We in- minute per 100 mL of forearm tissue volume. Four ple- fused ISDN (0.75, 1.5, and 3.0 ␮g/min) intra-arterially for thysmographic measurements were averaged for analysis 5 min at each dose, and FBF was measured during the last of FBF at baseline and during administration of drugs. 2 min of the infusion. After a 30-min rest period, ISDN Forearm vascular resistance (FVR) was calculated as the (0.75, 1.5, and 3.0 ␮g/min) was infused for 5 min at each ␮ mean arterial pressure divided by FBF. dose in combination with BH4 (500 g/min), and the FBF In the preliminary study, we evaluated the effect of the was measured. intra-arterial infusion of BH4 (0.1, 0.5, 1.0, 5.0, and 25 mg/min for 5 min, respectively) on forearm hemodynam- Study Drugs ϭ ics (n 4). None of the tested dosages of BH4 altered FBF, arterial BP, or heart rate. Plasma biopterin concen- In this study we used ACh chloride (Daiichi Pharmaceu- tical, Tokyo, Japan), ISDN (Eisai Pharmaceutical, Tokyo, trations measured after infusion of graded dose BH4 (0.1, 0.5, 1.0, 5.0, and 25 mg/min) increased from 2.1 Ϯ 0.6 to Japan), and (6R)-5,6,7,8-tetrahydrobiopterin (BH4; Sigma 18 Ϯ 4.5, 108 Ϯ 47, 1120 Ϯ 596, and 5219 Ϯ 1768 Chemical Co., St. Louis, MO). All drugs were obtained ng/mL, respectively. In the present study we used 500 commercially and were dissolved in oxygen-free saline ␮ 20 immediately before use. g/min of BH4, as in previous studies.

Study Protocol 1: Effect of BH4 on Analytical Methods Endothelium-Dependent Vasodilation Routine chemical methods were used to determine serum Forearm vascular responses to ACh alone and in combi- concentrations of total cholesterol, HDL cholesterol, trig-

nation with BH4 were evaluated in eight patients with lycerides, creatinine, glucose, insulin, and electrolytes. essential hypertension (six men and two women; mean age Serum concentration of low density lipoprotein (LDL) was 328 TETRAHYDROBIOPTERIN AND ENDOTHELIAL FUNCTION AJH–April 2002–VOL. 15, NO. 4, PART 1

Table 1. Baseline clinical characteristics in the normal control subjects and hypertensive patients in acetylcholine study

Normal Control Hypertensive Subjects Patients (8 ؍ n) (8 ؍ Variable (n Body mass index (kg/m2) 24.4 Ϯ 1.8 24.3 Ϯ 1.9 Systolic blood pressure (mm Hg) 115.2 Ϯ 8.9 155.3 Ϯ 9.7* Diastolic blood pressure (mm Hg) 68.2 Ϯ 6.5 97.5 Ϯ 4.9* Heart rate (bpm) 69.0 Ϯ 6.4 71.1 Ϯ 7.7 Total cholesterol (mmol/L) 4.99 Ϯ 0.71 5.02 Ϯ 0.74 Triglycerides (mmol/L) 0.96 Ϯ 0.51 1.02 Ϯ 0.57 HDL cholesterol (mmol/L) 1.42 Ϯ 0.28 1.34 Ϯ 0.25 LDL cholesterol (mmol/L) 3.32 Ϯ 0.48 3.46 Ϯ 0.65 Downloaded from https://academic.oup.com/ajh/article/15/4/326/217588 by guest on 29 September 2021 Serum glucose (mmol/L) 4.8 Ϯ 0.5 4.8 Ϯ 0.9 Serum insulin (pmol/L) 53.2 Ϯ 13.1 60.1 Ϯ 17.2 FBF (mL/min/100 mL tissue) 4.6 Ϯ 1.3 4.5 Ϯ 1.3 FVR (mm Hg/mL/min/100 mL tissue) 18.2 Ϯ 4.1 25.6 Ϯ 3.7*

HDL ϭ high-density lipoprotein; LDL ϭ low-density lipoprotein; FBF ϭ forearm blood flow; FVR ϭ forearm vascular resistance. All results are presented as the mean Ϯ SD. * P Ͻ .05 v normal control subjects. estimated using Friedewald’s method.22 The plasma con- Results centration of biopterin was measured by high performance Clinical Characteristics liquid chromatography (BML Co., Tokyo, Japan). The baseline clinical characteristics of the normal control Statistical Analysis subjects and the patients with essential hypertension are Results are presented as mean Ϯ SD. P values Ͻ .05 were shown in Tables 1 and 2. Systolic and diastolic BPs as well considered statistically significant. The Mann-Whitney U as FVR were significantly higher in the hypertensive pa- test was used to evaluate differences between the hyper- tients than in the normal control subjects. Other parame- tensive and normal control subjects for baseline parameters were similar in the two groups. ters. Comparisons of dose-response curves of parameters The response of FBF to the infusion of the endotheli- during drug infusion were analyzed by analysis of vari- um-dependent vasodilator ACh was smaller in the hyper- ance (ANOVA) for repeated measures. The data were tensive patients than in the normal control subjects (Fig. processed using either the software package StatView IV 1). The vasodilating effect of the endothelium-independent (SAS Institute Inc., Cary, NC) or Super ANOVA (Abacus vasodilator ISDN was similar in the two groups (Fig. 1). Concepts, Berkeley, CA). No significant change was observed in arterial BP or heart

Table 2. Baseline clinical characteristics in the normal control subjects and hypertensive patients in isosorbide dinitrate study

Normal Control Hypertensive Subjects Patients (6 ؍ n) (6 ؍ Variable (n Body mass index (kg/m2) 24.7 Ϯ 1.9 24.2 Ϯ 2.0 Systolic blood pressure (mm Hg) 116.8 Ϯ 9.8 155.8 Ϯ 10.6* Diastolic blood pressure (mm Hg) 69.0 Ϯ 6.9 95.1 Ϯ 6.3* Heart rate (bpm) 67.3 Ϯ 7.1 69.2 Ϯ 8.1 Total cholesterol (mmol/L) 5.03 Ϯ 0.82 5.05 Ϯ 0.88 Triglycerides (mmol/L) 0.98 Ϯ 0.57 1.05 Ϯ 0.64 HDL cholesterol (mmol/L) 1.47 Ϯ 0.35 1.37 Ϯ 0.46 LDL cholesterol (mmol/L) 3.12 Ϯ 0.67 3.53 Ϯ 0.72 Serum glucose (mmol/L) 4.9 Ϯ 0.7 4.5 Ϯ 0.8 Serum insulin (pmol/L) 51.1 Ϯ 15.6 54.7 Ϯ 16.9 FBF (mL/min/100 mL tissue) 4.7 Ϯ 1.4 4.6 Ϯ 1.3 FVR (mm Hg/mL/min/100 mL tissue) 18.1 Ϯ 4.3 25.3 Ϯ 4.9*

Abbreviations as in Table 1. All results are presented as the mean Ϯ SD. * P Ͻ .05 v normal control subjects. AJH–April 2002–VOL. 15, NO. 4, PART 1 TETRAHYDROBIOPTERIN AND ENDOTHELIAL FUNCTION 329 Downloaded from https://academic.oup.com/ajh/article/15/4/326/217588 by guest on 29 September 2021

FIG. 1. 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 ϭ 8) than in normal control subjects (n ϭ 8). There were no significant differences in the vascular responses to isosorbide dinitrate between the two groups (n ϭ 6, respectively). rate with intra-arterial infusion of either ACh or ISDN in pendent vasodilator ISDN was not altered by coinfusion of either group. BH4 in normal or hypertensive individuals (Fig. 2). No signiﬁcant change was observed in arterial BP or

Effects of the Coinfusion of BH4, ACh, and heart rate after intra-arterial infusion of either ACh or ISDN on Forearm Hemodynamics ISDN in combination with BH4 in either group.

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 Discussion infusion of the endothelium-dependent vasodilator ACh The present ﬁndings demonstrate that BH4, an essential increased signiﬁcantly with coinfusion of BH4 in the con- cofactor for eNOS, enhances ACh-induced vasodilation in trol as well as the hypertensive group (Fig. 2). The in- normotensive as well as essential hypertensive subjects, crease in FBF during infusion of the endothelium-inde- and that the augmentation of endothelium-dependent va- 330 TETRAHYDROBIOPTERIN AND ENDOTHELIAL FUNCTION AJH–April 2002–VOL. 15, NO. 4, PART 1 Downloaded from https://academic.oup.com/ajh/article/15/4/326/217588 by guest on 29 September 2021

FIG. 2. Effects of acetylcholine and isosorbide dinitrate on forearm blood flow before and after coinfusion of BH4 in hypertensive patients and ϭ normal control subjects. Coinfusion of BH4 augmented endothelium-dependent vasorelaxation in both groups (n 8, respectively). However, ϭ ϭ ϭ BH4 did not alter endothelium-independent vasorelaxation in either group (each group, n 6). BH4 tetrahydrobiopterin; NS not significant.

sodilation evoked by BH4 may be due to an increase in NO of BH4 are suboptimal, endothelium-dependent vasodila- availability. In fact, BH4 did not significantly alter endo- tion is impaired in coronary arteries of dogs, and that 21 thelium-independent vasodilation in either normotensive addition of BH4 can restore this vasodilation. Also, BH4 or hypertensive subjects. supplementation improves impaired endothelium-depen- In the present study, endothelium-dependent vasodila- dent vasodilation in brachial arteries of patients with hy- tion was reduced in essential hypertensive patients com- perecholesterolemia20 and in saphenous vein rings from 25 pared with normotensive control subjects, whereas endo- patients who smoke. These findings suggest that BH4 thelium-independent vasodilation was similar in both deficiency may contribute to endothelial dysfunction in groups. These findings are consistent with previous studies these patients. However, in the present study, supplemen- 7Ð9 showing that endothelial function in brachial, coro- tation of BH4 augmented ACh-induced vasodilation in nary,10 renal,11Ð13 femoral,14 and small artery15 circulation normotensive as well as hypertensive individuals. It is was impaired in hypertensive patients. Recently it was unlikely that the deficiency of BH4 is linked to impaired reported that the endothelial dysfunction seen in hyperten- endothelium-dependent vasodilation in patients with es- sive patients is reversed by treatment with antihyperten- sential hypertension. 13,15,23 24 sive agents and with lifestyle modification. Endo- Several reasons may explain why BH4 augments endothelial dysfunction is an early feature of atherosclerosis thelium-dependent vasodilation. The biologic effects of and vascular complications in patients with essential hy- NO are regulated not only by the amounts of production pertension. Thus, it is clinically important to restore en- but also by the degree of superoxide inactivation. dothelial dysfunction in conditions where there is reduced First, BH4 when added may directly stimulate eNOS NO availability. activity, leading to an increase in NO production. In fact,

Several investigators have reported that when the levels BH4 is an important cofactor for activity of all NOS AJH–April 2002–VOL. 15, NO. 4, PART 1 TETRAHYDROBIOPTERIN AND ENDOTHELIAL FUNCTION 331

isoforms, including eNOS.16,17 It is known that cytokine- plementation may represent a new therapeutic intervention induced NO production requires an increase in intracellu- for endothelial dysfunction in patients with essential hy-

lar BH4 levels, and that exogenous BH4 supplementation pertension. In future studies, it will be important to deter- 26 enhances NO production. These ﬁndings suggest that the mine whether BH4 restores impaired endothelial function cofactor BH4 alone can regulate NOS activity. In rat aortic in the settings of atherosclerosis, chronic heart failure, rings, administration of exogenous BH4 causes endotheli- diabetes mellitus, and estrogen withdrawal. um-dependent vasorelaxation.27 This vasorelaxation was ␻ abolished by N -monomethyl-L-arginine, suggesting that Acknowledgments BH4-evoked, endothelium-dependent vasodilation may be due to stimulated eNOS activity and an increase in NO The authors thank Drs. Shigeaki Arai and Masahiko Sakai production. for preparation of the BH4 and oxygen-free saline, and Second, there is also a possibility the BH does not play Yuko Omura for secretarial assistance. 4 Downloaded from https://academic.oup.com/ajh/article/15/4/326/217588 by guest on 29 September 2021 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 References

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