AJH 2002; 15:326–332 Tetrahydrobiopterin Enhances Forearm Vascular Response to Acetylcholine in Both Normotensive and Hypertensive Individuals 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 326–332 © 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 1–3 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.4–6 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 7–9 10 ulation of NO release in brachial, coronary, re- BH4 may contribute to the maintenance and development nal,11–13 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.