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Home , Nitroglycerin, Tetrahydrobiopterin

Journal of Human (2008) 22, 401–407 & 2008 Nature Publishing Group All rights reserved 0950-9240/08 $30.00 www.nature.com/jhh ORIGINAL ARTICLE : a novel antihypertensive therapy

M Porkert1, S Sher1, U Reddy1, F Cheema1, C Niessner1, P Kolm2, DP Jones3, C Hooper4, WR Taylor1, D Harrison1 and AA Quyyumi1 1Division of Cardiology, Emory University, Atlanta, GA, USA; 2Christiana Care Center for Outcomes Research, Newark, DE, USA; 3General Clinical Research Center, Emory University, Atlanta, GA, USA and 4Centers for Disease Control, Atlanta, GA, USA

Tetrahydrobiopterin (BH4) is a for the nitric after 5 weeks and persisted for the 8-week study period. oxide (NO) synthase , such that its insuffi- Study 2: subjects given 400 mg BH4 had decreased ciency results in uncoupling of the , leading to systolic (P ¼ 0.03) and mean BP (P ¼ 0.04), with a peak release of rather than NO in disease decline of 16±19 mm Hg (P ¼ 0.04) at 3 weeks. BP states, including hypertension. We hypothesized that returned to baseline 4 weeks after discontinuation. oral BH4 will reduce arterial (BP) and Significant improvement in endothelial function was improve endothelial function in hypertensive subjects. observed in Study 1 subjects and those receiving

Oral BH4 was given to subjects with poorly controlled 400 mg BH4. There was no significant change in subjects hypertension (BP 4135/85 mm Hg) and weekly measure- given the 200 mg dose. This pilot investigation indicates ments of BP and endothelial function made. In Study 1, 5 that oral BH4 at a daily dose of 400 mg or higher has À1 À1 or 10 mg kg day of BH4 (n ¼ 8) was administered a significant and sustained antihypertensive effect orally for 8 weeks, and in Study 2, 200 and 400 mg of in subjects with poorly controlled hypertension, an

BH4 (n ¼ 16) was given in divided doses for 4 weeks. effect that is associated with improved endothelial NO Study 1: significant reductions in systolic (P ¼ 0.005) . and mean BP (P ¼ 0.01) were observed with both doses Journal of Human Hypertension (2008) 22, 401–407; of BH4. Systolic BP was 15±15 mm Hg (P ¼ 0.04) lower doi:10.1038/sj.jhh.1002329; published online 6 March 2008

Keywords: tetrahydrobiopterin; endothelium;

Introduction is synthesized in the vascular endothelium by endothelial NO synthase (eNOS) Nitric oxide (NO) is a potent endothelium-derived and tetrahydrobiopterin (BH ) is an essential cofac- vasodilator that regulates blood pressure (BP) and 4 1–6 tor for eNOS. During NO catalysis, is bound regional blood flow. Decreases in NO production by the ferrous in the domain of or bioavailability impair endothelium-dependent eNOS. BH4 plays a crucial role in donating an dilation that can be both a consequence as well as electron that to scission of the O–O bond, a cause of hypertension. Among other protective leading to formation of an –oxy complex that roles of the endothelium, mediated in part by NO, participates in the of L-, are the inhibition of vascular smooth muscle cell leading to formation of NO. In the absence of BH4, proliferation and growth, and inhibition of pro- the Fe2 þ O–O complex dissociates to release inflammatory cytokine expression, platelet aggrega- superoxide (OKÀ). This phenomenon is referred to tion and oxidative modification of low-density 2 7 as eNOS uncoupling. Accumulating evidence sug- lipoprotein. Importantly, the presence of endothe- gests that hypertension increases vascular produc- lial dysfunction heralds increased risk of future tion of reactive oxygen species, which can serve to adverse cardiovascular events in patients including oxidize BH and to eNOS uncoupling.9,10 Our those with hypertension.8 4 previous experimental studies have shown that BH4 is oxidized to in the vasculature of the deoxycorticosterone acetate salt hypertensive mouse, and supplementation with oral BH4 can Correspondence: Dr AA Quyyumi, Division of Cardiology, Emory reduce BP and improve oxidative stress in this University, 1364 Clifton Road, Ste D403C, Atlanta, GA 30322, model.11 USA. Several studies have assessed the effects of BH on E-mail: [email protected] 4 Received 25 July 2007; revised 21 November 2007; accepted 1 endothelial function. , a substrate for December 2007; published online 6 March 2008 BH4 synthesis, improved endothelium-dependent Tetrahydrobiopterin for hypertension M Porkert et al 402 in coronary arterioles obtained from uncontrolled hypertension (BP 4180 mm Hg systo- patients undergoing coronary artery bypass graft lic and/or 110 mm Hg diastolic), renal or hepatic 12 surgery (CABG). Acute infusion of BH4 improved dysfunction, and bleeding disorders. All subjects endothelial function in angiographically normal- gave informed consent and the study was approved appearing segments of coronary arteries in subjects by the Emory University Investigational Review with or hypercholesterolae- Board. mia.13,14 A similar improvement was observed in the forearm circulation of diabetics and smokers.15–17 These studies have used either parenteral infusion Study design of BH4 or a single oral dose, and it has not been Both studies were designed as pilot investigations to shown that chronic oral administration of BH4 could (a) evaluate the effects of BH4 on BP and vascular cause a sustained improvement in endothelial function, (b) the dose–response of this effect. For function. Moreover, no prior study has examined this reason, they were not placebo controlled, but the effect of BH4 in subjects with hypertension. We included a withdrawal phase. After screening, hypothesized that chronic oral therapy with BH4 in suitable subjects were admitted to the Emory hypertensive patients will result in reduction of University General Clinical Research Center. All arterial BP as a result of improvement in endothelial BP and rate measurements were made after dysfunction. For this purpose, we conducted two having the patient rest in a seated position for at pilot studies where (a) the duration of action of oral least 10 min, by coordinators unaware of study BH4 and (b) its dose–response were studied in stage. Mean BP was calculated as: (systolic hypertensive subjects. BP þ 2 Â diastolic BP)/3. Tetrahydrobiopterin (Schircks Laboratories, Jona, Switzerland) was compounded with an equivalent Methods amount of C to ensure stability of the drug. Patient selection Subjects were advised to keep the pills in a Subjects between the ages of 18 and 75 years were refrigerator. recruited if they had uncontrolled hypertension on traditional stable antihypertensive therapy (BP X135/85) or newly diagnosed hypertension (BP Study 1: investigation of the time of onset and duration

X140/90). Patients were continued on their current of action of oral BH4 antihypertensive therapy throughout the study. Eight hypertensive subjects (Table 1) were assigned Exclusion criteria included female subjects with to either 5 mg kgÀ1 dayÀ1 (n ¼ 4) or 10 mg kgÀ1 dayÀ1

childbearing potential, history of recently sympto- (n ¼ 4) of BH4, given in two divided doses orally for matic coronary or peripheral vascular disease, 8 weeks. This regimen was based on BH4 dosing in known secondary causes for hypertension, severe . Weekly BP measurements were

Table 1 Baseline characteristics

Study 1 (n ¼ 8) Study 2 (n ¼ 16)

400 mg (n ¼ 8) 200 mg (n ¼ 8)

Age (years) 60.5±11 (48–74) 57±9 (43–72) 62±8 (45–69) Males 3 4 2 Mean number of antihypertensive 1.4±0.9 (0–3) 2.0±1.9 (0–6) 1.9±1.0 (1–3) Treated with ACE-I or ARB 4 4 7 Treated with diuretics 3 6 2 Treated with calcium antagonists 2 3 3 Treated with beta blockade 2 2 3 Treated with 2 3 3 Systolic blood pressure (mm Hg) 144±6.6 (138–154) 149±10.3 (140–171) 154±12.3 (137–178) Diastolic blood pressure (mm Hg) 81±14.4 (54–100) 88±11.1 (73–108) 79±13.0 (51–92) Mean arterial pressure (mm Hg) 102±9.7 (82–115) 109±10.0 (96–129) 104±9.6 (88–118)

Past medical history Coronary artery disease 0 0 2 High cholesterol (4240 mg per 100 ml) 2 5 4 0 2 1 Current tobacco 0 1 1 Former tobacco 2 3 3

Abbreviations: ACE-I, angiotensin converting ; ARB, angiotensin receptor blocker. Continuous values are represented as mean±s.d. with range in parentheses.

Journal of Human Hypertension Tetrahydrobiopterin for hypertension M Porkert et al 403 Study 1 after discontinuation of therapy. In Study 2, it was measured at the end of the 2-week run-in BH4 period, after 4 weeks of BH4 therapy and 4 weeks after discontinuation of BH4. The technique has been described in detail previously.18 Briefly, stu- 0 1 2 3 4 5 6 7 8 9 14 Weeks dies were performed in a temperature-controlled BP room, and brachial diameter was measured above the antecubital fossa in the non-dominant arm using FMD an 11 MHz high resolution ultrasound transducer (Acuson Inc., Malvern, PA, USA). Flow-mediated vasodilation (FMD) was determined by inflating a BP cuff on the forearm to 4200 mm Hg for 5 min, Study 2 deflating rapidly, and recording brachial diameter at 1 min after onset of hyperaemia. Three diameter measurements were averaged on end-diastolic Vitamin C BH4 frames each time. FMD was calculated as: (average diameter with hyperaemiaÀaverage baseline dia- meter)/average baseline diameter  100. Endothe- -1 -2 0 1 2 3 4 5 8 Weeks lium-independent vasodilator response was measured as brachial artery diameter change 5 min BP after 0.4 mg sublingual using the formula: (average diameter post nitroglycerinÀ FMD average baseline diameter)/average baseline Figure 1 Protocol for Studies 1 and 2. BP, blood pressure, FMD, diameter  100. flow-mediated vasodilation.

Statistical analysis made throughout the treatment period and at 1 and 6 The primary end point was the change in systolic, weeks after discontinuation (Figure 1). mean and diastolic BP during BH4 therapy. Second- ary end points included change in FMD with BH4. Linear mixed effects models for repeated measures data were used to analyse the change in BP from Study 2: investigation of dose–response of oral BH4 Of the 16 subjects, 6 were male and mean age was baseline to the end of the treatment period and 60±8 years (Table 1). Eight subjects received 100 mg differences in change between 100 and 200 mg dose groups. A quadratic term for week was included in of oral BH4 twice a day and 8 received 200 mg twice daily. There were no significant differences in the model to account for nonlinear trends over the baseline characteristics between the two groups treatment period. Dose, week and week squared were the main effect terms in the model; dose by (Table 1). Because BH4 is compounded in vitamin C in a 1:1 ratio, all subjects received 100 or 200 mg of week and dose by week squared were the interaction vitamin C twice a day during the first 2-week run-in terms in the model. Significant interaction terms period to control for possible changes in BP with indicate a difference in the pattern of change vitamin C therapy. After this 2-week run-in period, between the two dose groups. FMD data were analysed using the two-tailed Student’s t-test. All subjects received BH4 at their assigned dosage for the next 4 weeks. This study was shorter because the results are expressed as mean±s.d., and P-values peak effect was noted by the third week in the first o0.05 are considered statistically significant. study. Weekly heart rate and BP measurements were The authors had full access to the data and take responsibility for its integrity. All authors have read made during the run-in and treatment periods. BH4 was then stopped, and follow-up was performed at 1 and agree to the paper as written. and 4 weeks after discontinuation where BP and heart rate measurements were made (Figure 1). A complete metabolic panel and profile was Results performed prior to initiation of , at the Baseline patient characteristics are included in end of BH4 therapy, and 1 month after discontinua- Table 1. tion.

Effect of BH4 on BP Measurement of endothelial function Study 1. In the combined group, there was a Brachial artery endothelium-dependent and -inde- significant reduction in systolic (P ¼ 0.005 quadratic pendent function was measured using ultrasound. trend) and mean arterial BP (P ¼ 0.01) with BH4 In Study 1, brachial reactivity was measured at treatment (Figure 2). Systolic BP was lowered by a baseline, after 8 weeks of BH4 treatment and 1 week mean of 13±9 mm Hg after 3 weeks (P ¼ 0.004) and

Journal of Human Hypertension Tetrahydrobiopterin for hypertension M Porkert et al 404 160 BH4 160 BH4 150 140 140 p=0.005

130 SBP 120 SBP DBP 120 MBP DBP 110 MBP 100 p=0.01 100

Blood Pressure mmHg 80 90 Blood Pressure mmHg 80 60 70 02451 38 Weeks 60 0 12345678914 Figure 4 BP response with 200 mg BH4 in Study 2. Values are ± Weeks mean s.e.m. P-value by ANOVA not significant. BP, blood pressure; DBP, diastolic blood pressure; MBP, mean blood Figure 2 BP response in Study 1. Values are mean±s.e.m. pressure; SBP, systolic blood pressure. P-value by ANOVA. ANOVA, analysis of variance; BP, blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure; SBP, systolic blood pressure. 86±11, and 106±12 mm Hg, respectively, after 2 weeks of vitamin C (all P ¼ NS). Subjects given 400 mg of oral BH4 had a signifi- cant decrease in systolic (P ¼ 0.03) and mean BP BH4 160 (P ¼ 0.04 by linear trend analysis) (Figure 3). The p=0.03 decrease in diastolic BP did not reach statistical 140 significance (P ¼ 0.08). Mean BP was significantly SBP lower after 1 week (P ¼ 0.02). There was a further DBP 120 p=0.04 MBP significant reduction in BP during the subsequent weeks reaching a nadir after 3 weeks when systolic 100 BP was a mean 16 mm Hg lower; P ¼ 0.04 (Figure 3).

Blood Pressure mmHg A week after termination of therapy, BP remained 80 lower, but 4 weeks after discontinuation of BH4,BP 60 rose back up to baseline levels. No significant 01 23 4 58 change in BP was observed in subjects given Weeks 200 mg of BH4 (Figure 4). There was no significant Figure 3 BP response with 400 mg BH4 in Study 2. Values are alteration in heart rate with BH4 in either group. mean±s.e.m. P-value by ANOVA. BP, blood pressure; DBP, diastolic blood pressure; SBP, systolic blood pressure, MBP, mean blood pressure. Effect of BH4 on endothelial function Study 1. Flow-mediated vasodilation of the bra- chial artery increased from a mean of 3.4±1to 15±15 mm Hg (P ¼ 0.04) after 5 weeks. This reduc- 8.2±3.4% after 8 weeks of BH4 (P ¼ 0.05, n ¼ 6) and tion persisted for the 8 weeks of treatment. Mean BP returned to baseline levels 6 weeks after termination ± was also significantly reduced by BH4, but the of therapy (3.7 1.3%, P ¼ NS compared to base- change in diastolic BP did not reach statistical line). Nitroglycerin-mediated vasodilation remained significance (Figure 2). BP returned towards pre- unchanged (11.9±3% before and 16.1±5% after treatment levels 6 weeks after discontinuation of BH4, P ¼ 0.1). therapy (P ¼ NS, compared to baseline). There was no significant change in heart rate with BH4. There Study 2. There was a significant improvement in were also no statistically significant differences FMD with 400 mg of BH4 (n ¼ 7). FMD improved À1 ± ± between the 5 and 10 mg kg doses of BH4. from 3.7 3% before to 7.1 4.9% after 4 weeks of BH4 therapy (P ¼ 0.016). One month after disconti- Study 2. In this study, we aimed to determine nuation of BH4 therapy, FMD returned to baseline ± whether lower doses of BH4 will have a similar levels (3.2 1.1%, P ¼ 0.6 compared to baseline). effect on BP. There was no significant change in There was no significant change in FMD in subjects heart rate or BP during the run-in phase with given 200 mg of BH4 (5.3±2.5 vs 6.2±3.5%; vitamin C in either group; systolic, diastolic and P ¼ 0.55). There was also no significant change in mean arterial BP in the 16 subjects were 152±11, nitroglycerin-mediated, endothelium-independent 84±13, 106±10 mm Hg before, and 148±19, vasodilation during the study in either group. There

Journal of Human Hypertension Tetrahydrobiopterin for hypertension M Porkert et al 405 Table 2 Effects of BH4 on lipid and metabolic markers BP. Nevertheless, we cannot exclude the possibility that the reduction in BP per se contributed to Baseline 4 weeks P-value improvement of vascular endothelial function.

Sodium (mequiv lÀ1) 139.8±2.3 137.5±3.1 0.002 Potassium (mequiv lÀ1) 3.8±0.5 4.1±0.3 0.06 BUN (mg per 100 ml) 15.1±5.6 17.6±5.2 0.12 Potential mechanisms Creatinine (mg per 100 ml) 1.06±0.28 1.07±0.30 0.79 Tetrahydrobiopterin is believed to produce its Total cholesterol 197.0±51.6 195.1±45.6 0.78 antihypertensive effect via its effect on NO synth- (mg per 100 ml) esis. NO is a potent endothelium-derived vasodila- LDL cholesterol 113.9±44.7 119.3±37.9 0.38 1–6 (mg per 100 ml) tor that regulates BP and regional blood flow. HDL cholesterol 54.6±21.6 51.5±16.8 0.12 A decrease in NO production or bioavailability (mg per 100 ml) reduces endothelium-dependent dilation and Triglycerides 142.2±92.4 121.1±48.4 0.28 increases vascular tone. In addition, NO mediates (mg per 100 ml) Glucose (mg per 100 ml) 95.9±27.3 91.2±21.1 0.39 many of the protective functions of the endothe- Insulin (mUmlÀ1) 0.015±0.010 0.019±0.014 0.36 lium, including inhibition of vascular smooth muscle proliferation, platelet aggregation and ex- Abbreviations: BUN, blood urea, ; LDL, low density lipopro- pression of proinflammatory cytokines.19,20 NO is tein; HDL, high density lipoprotein. generated from L-arginine in the vascular endothe- ± Values are mean s.d. lium by constitutive eNOS in the presence of reduced BH4 that is an essential cofactor of eNOS. was no change in baseline diameter with BH4 Insufficiency of BH4 or its oxidation to dihydro- therapy in either group. by or other oxidants leads to uncoupling of eNOS that promotes generation of superoxide instead of NO.21 Thus, a deficiency of

Adverse effects of BH4 BH4 produces two additive physiologic con- There were no significant changes in renal function, sequences: (a) uncoupling of the L-arginine NO path- lipid parameters, glucose and insulin 4 weeks after way resulting in increased formation of oxygen-free 22 23 BH4 therapy in Study 2 subjects except a slight radicals by eNOS and (b) loss of NO production. decrease in serum sodium (Table 2). In Study 1, one In a recent study, we found that deoxycorticoster- À1 subject receiving the 10 mg kg dose of BH4 com- one acetate salt hypertension caused BH4 oxidation plained of worsening heartburn and hot flashes and in aortas of mice, leading to a marked increase in withdrew after 7 weeks of therapy. These symptoms superoxide production and a significant reduction were present before the patient enrolled in the in NO production. Treatment of these mice with oral KÀ study. Another patient reported mild nausea. In BH4 reduced vascular O2 production, increased Study 2, no subject withdrew due to adverse effects. NO production and blunted the increase in BP due Three subjects complained of mild orthostasis, but it to deoxycorticosterone acetate salt hypertension.11 was transient. Two subjects reported mild fatigue, Similar findings have been reported in other two reported mild ankle oedema, and one reported models of hypertension. Aortae of spontaneously dysgeusia. hypertensive rats had higher production of super- oxide and reduced release of NO compared with normotensive rats, an effect that was blunted in the 19,24 Discussion presence of exogenous BH4. Our present study confirms these experimental Our study demonstrates that oral BH4 is effective in observations in human subjects with essential lowering arterial BP in subjects with either poorly hypertension in whom oral BH4 reduced BP, likely controlled hypertension or in those who were newly by reversing the uncoupling of NO synthase and diagnosed. This effect was observed at a dose of improving NO bioavailability. A previous study by 400 mg or higher and was free of any changes in Higashi et al.25 showed that parenteral infusion of heart rate. Reduction in BP was evident within 1 BH4 improved endothelial function in hypertensive week of therapy and was sustained for up to 8 weeks patients, but this is the first study demonstrating the of continuous therapy without . BP effects of oral BH4 on endothelial function and BP. remained lower for at least a week after discontinua- BH4 has other potential actions such as and tion of therapy, but returned towards baseline after 4 hydroxylation that may also have con- weeks. Oral BH4 appeared to be well-tolerated tributed to these effects independent of the actions without any serious adverse effects. discussed above. We found that subjects had significant improve- ment in after BH4 therapy at the doses that also reduced BP. On the basis of our Study limitations previous experimental data, we speculate that this In the present study, the subjects remained on their improved vascular NO bioavailability and resultant previous medications during BH4 therapy. Further vascular relaxation contributed to the reduction in studies need to be performed to determine if BH4

Journal of Human Hypertension Tetrahydrobiopterin for hypertension M Porkert et al 406 will be equally effective in lowering BP when used endothelium-dependent responses by L-arginine. Circ as monotherapy. Our study was not blinded and did Res 1992; 70: 465–476. not have a placebo group, but the BP and endothelial 6 Ross R. The pathogenesis of atherosclerosis: a perspec- function measurements were performed without tive for the 1990s. Nature 1993; 362: 801–809. knowledge of treatment stage. Moreover, withdrawal 7 Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation 2004; 109: III27–III32. of therapy resulted in a return of BP and endothelial 8 Bolad I, Delafontaine P. Endothelial dysfunction: its function to pre-study levels. Vitamin C at higher role in hypertensive coronary disease. Curr Opin doses than those used to compound BH4 has been Cardiol 2005; 20: 270–274. demonstrated in some studies to have a mild 9 Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman antihypertensive effect26 and to improve endothelial BA, Harrison DG. Role of superoxide in angiotensin II- function, although this remains a subject of con- induced but not -induced hypertension. troversy.27–30 To overcome this possibility, we made Circulation 1997; 95: 588–593. baseline measurements after a 2-week run-in period 10 Somers MJ, Mavromatis K, Galis ZS, Harrison DG. with vitamin C alone in Study 2 and found no Vascular superoxide production and vasomotor func- change in BP. However, a synergistic effect of tion in hypertension induced by deoxycorticosterone acetate-salt. Circulation 2000; 101: 1722–1728. vitamin C with BH4 cannot be excluded. In fact, 11 Landmesser U, Dikalov S, Price SR, McCann L, Fukai recent studies have shown that the immediate T, Holland SM et al. Oxidation of tetrahydrobiopterin K product of BH4 oxidation is the BH3 radical and leads to uncoupling of endothelial cell nitric oxide K that vitamin C reacts with the BH3 radical and synthase in hypertension. J Clin Invest 2003; 111: 31 converts it back to BH4. 1201–1209. 12 Tiefenbacher CP, Bleeke T, Vahl C, Amann K, Vogt A, Kubler W. Endothelial dysfunction of coronary resis- What is known about this topic tance arteries is improved by tetrahydrobiopterin in K Nitric oxide bioavailability is reduced in hypertension and atherosclerosis. Circulation 2000; 102: 2172–2179. is associated with endothelial dysfunction. 13 Maier W, Cosentino F, Lutolf RB, Fleisch M, Seiler C, K This is partly secondary to uncoupling of nitric oxide Hess OM et al. Tetrahydrobiopterin improves endothe- synthase, which is secondary to reduced availability of lial function in patients with coronary artery disease. tetrahydrobiopterin, a cofactor for this enzyme. J Cardiovasc Pharmacol 2000; 35: 173–178. What the study adds 14 Fukuda Y, Teragawa H, Matsuda K, Yamagata T, K Oral therapy with tetrahydrobiopterin can safely lower Matsuura H, Chayama K. Tetrahydrobiopterin restores blood pressure in subjects with poorly controlled endothelial function of coronary arteries in patients hypertension. with hypercholesterolaemia. Heart 2002; 87: 264–269. K This is associated with improvement of endothelial 15 Heitzer T, Krohn K, Albers S, Meinertz T. Tetrahydro- dysfunction. biopterin improves endothelium-dependent vasodila- tion by increasing nitric oxide activity in patients with type II diabetes mellitus. Diabetologia 2000; 43: 1435–1438. Acknowledgements 16 Heitzer T, Brockhoff C, Mayer B, Warnholtz A, Mollnau H, Henne S et al. Tetrahydrobiopterin Markus Porkert was supported by an American improves endothelium-dependent vasodilation in Heart Association training Grant Award Number: chronic smokers: evidence for a dysfunctional nitric 0425402B. Other funding came from a research grant oxide synthase. Circ Res 2000; 86: E36–E41. from the Emory University General Clinical Re- 17 Ueda S, Matsuoka H, Miyazaki H, Usui M, Okuda S, search Center (Grant no. MO1-RR00039). Imaizumi T. Tetrahydrobiopterin restores endothelial function in long-term smokers. J Am Coll Cardiol 2000; 35: 71–75. References 18 Prasad A, Tupas-Habib T, Schenke WH, Mincemoyer R, Panza JA, Waclawin MA et al. Acute and chronic 1 Furchgott RF, Zawadzki JV. The obligatory role of angiotensin-1 receptor antagonism reverses endothe- endothelial cells in the relaxation of arterial smooth lial dysfunction in atherosclerosis. Circulation 2000; muscle by acetylcholine. Nature 1980; 288: 373–376. 101: 2349–2354. 2 Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri 19 Hong HJ, Hsiao G, Cheng TH, Yen MH. Supplemention G. Endothelium-derived relaxing factor produced and with tetrahydrobiopterin suppresses the development released from artery and vein is nitric oxide. Proc Natl of hypertension in spontaneously hypertensive rats. Acad Sci USA 1987; 84: 9265–9269. Hypertension 2001; 38: 1044–1048. 3 Rees DD, Palmer RM, Moncada S. Role of endothelium- 20 Angeja BG, Grossman W. Evaluation and management derived nitric oxide in the regulation of blood of diastolic . Circulation 2003; 107: pressure. Proc Natl Acad Sci USA 1989; 86: 659–663. 3375–3378. 21 Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, 4 Cosentino F, Katusic ZS. Tetrahydrobiopterin and Masters BS, Karoui H et al. Superoxide generation by dysfunction of endothelial in endothelial nitric oxide synthase: the influence of coronary arteries. Circulation 1995; 91: 139–144. cofactors. Proc Natl Acad Sci USA 1998; 95: 5 Kuo L, Davis MJ, Cannon MS, Chilian WM. Pathophy- 9220–9225. siological consequences of atherosclerosis extend 22 Stroes E, Kastelein J, Cosentino F, Erkelens W, into the coronary microcirculation. Restoration of Wever R, Koomans H et al. Tetrahydrobiopterin

Journal of Human Hypertension Tetrahydrobiopterin for hypertension M Porkert et al 407 restores endothelial function in hypercholesterolemia. diabetes and low vitamin C levels but does not J Clin Invest 1997; 99: 41–46. improve endothelial dysfunction or insulin resistance. 23 Schmidt K, Werner ER, Mayer B, Wachter H, Kukovetz Am J Physiol Heart Circ Physiol 2006; 290: WR. Tetrahydrobiopterin-dependent formation of H137–H145. endothelium-derived relaxing factor (nitric oxide) 28 Darko D, Dornhorst A, Kelly FJ, Ritter JM, Chowienc- in aortic endothelial cells. Biochem J 1992; 281: zyk PJ. Lack of effect of oral vitamin C on blood 297–300. pressure, oxidative stress and endothelial function in 24 Cosentino F, Patton S, d’Uscio LV, Werner ER, Werner- type II diabetes. Clin Sci (London) 2002; 103: 339–344. Felmayer G, Moreau P et al. Tetrahydrobiopterin alters 29 Kim MK, Sasaki S, Sasazuki S, Okubo S, Hayashi M, superoxide and nitric oxide release in prehypertensive Tsugane S. Lack of long-term effect of vitamin C rats. J Clin Invest 1998; 101: 1530–1537. supplementation on blood pressure. Hypertension 25 Higashi Y, Sasaki S, Nakagawa K, Fukuda Y, Matsuura 2002; 40: 797–803. H, Oshima T et al. Tetrahydrobiopterin enhances 30 Pellegrini MP, Newby DE, Johnston NR, Maxwell S, forearm vascular response to acetylcholine in both Webb DJ. Vitamin C has no effect on endothelium- normotensive and hypertensive individuals. Am J dependent vasomotion and acute endogenous fibrino- Hypertens 2002; 15: 326–332. lysis in healthy smokers. J Cardiovasc Pharmacol 26 Mullan BA, Young IS, Fee H, McCance DR. Ascorbic 2004; 44: 117–124. acid reduces blood pressure and arterial stiffness in 31 Kuzkaya N, Weissmann N, Harrison DG, Dikalov S. type 2 diabetes. Hypertension 2002; 40: 804–809. Interactions of peroxynitrite, tetrahydrobiopterin, as- 27 Chen H, Karne RJ, Hall G, Campia U, Panza JA, Cannon corbic acid, and thiols: implications for uncoupling III RO et al. High-dose oral vitamin C partially endothelial nitric-oxide synthase. J Biol Chem 2003; replenishes vitamin C levels in patients with Type 2 278: 22546–22554.

Journal of Human Hypertension