Tetrahydrobiopterin Supplementation Enhances Carotid Artery Compliance in Healthy Older Men: A Pilot Study

Abstract

Background

We performed a pilot study to test the hypothesis that acute oral ingestion of tetrahydrobiopterin (BH₄), a key cofactor modulating vascular nitric oxide (NO) synthase activity, improves large elastic artery stiffness with aging in men.

Methods

Healthy older (63 ± 2 years; n = 8) and young (age 25 ± 1 years; n = 6) men were studied 3 h after ingestion of BH₄ (10 mg·kg⁻¹ body weight) or placebo on separate days in a randomized, placebo-controlled, double-blind study.

Results

Baseline carotid artery compliance was 37% lower (0.17 ± 0.02 vs. 0.22 ± 0.02 mm/mm Hg·10⁻¹) and β-stiffness was 42% higher (7.3 ± 1.1 vs. 4.2 ± 0.5 AU) in the older men (both P < 0.05). BH₄ ingestion markedly increased circulating BH₄ concentrations in both groups (17–19-fold, P < 0.05), but increased compliance (+39% to 0.23 ± 0.02 mm/mm Hg^.10⁻¹, P < 0.01) and decreased β-stiffness index (–27% to 5.3 ± 0.7 AU, P < 0.01) only in the older men. BH₄ also reduced carotid systolic blood pressure (SBP) in the older men (P < 0.05).

Conclusions

These preliminary results support the possibility that limited BH₄ bioavailability contributes to impaired carotid artery compliance in healthy older men. Further studies are needed to determine if increasing BH₄ bioavailability though oral BH₄ supplementation may have therapeutic efficacy for improving large elastic artery compliance and reducing central SBP with aging.

American Journal of Hypertension, advance online publication 7 June 2012; doi:10.1038/ajh.2012.70

Aging is associated with progressive stiffening of the large elastic arteries of the cardiothoracic region (aorta, carotids) in humans, even in the absence of conventional risk factors for cardiovascular diseases (CVD).¹ Decreased compliance of the carotid artery and increased stiffness of the aorta act to increase central and peripheral systolic blood pressure (SBP) and pulse pressure (PP), as well as risk of CVD-related events such as myocardial infarction, stroke, and heart failure.^2,3 As such, insight into the mechanisms involved in reduced large elastic artery compliance with aging may be clinically valuable for identifying possible therapeutic targets.

The mechanisms in question are thought to include so-called “functional influences” that increase smooth muscle vascular tone as a result of decreased nitric oxide (NO)-mediated vasodilation^4,5 and/or increased endothelin-1 vasoconstrictor tone.⁶ Reduced vascular NO bioavailability with aging is mediated in part by reductions in the concentration or bioactivity of tetrahydrobiopterin (BH₄), the essential cofactor for NO synthase.⁷ Therefore, reduced vascular BH₄ bioavailability may be one mechanism contributing to decreased large elastic artery compliance with aging.

We performed a pilot study to test the hypothesis that acute oral ingestion of BH₄ improves large elastic artery stiffness with aging in men. In a randomized, placebo-controlled, double-blinded cross-over pilot study, we assessed carotid artery compliance and β-stiffness index, a less BP-dependent expression of compliance, in groups of healthy older and young men after administering a single therapeutic dose of BH₄ shown previously to restore peripheral vascular endothelial function in this group.⁸ Central (carotid) and peripheral (brachial) SBP and PP also were assessed.

Methods

Subjects. Eight older (age 55–75 years) and six young (age 19–30 years) healthy men participated in the protocol. Subjects were nonsmokers, nonobese (body mass index <30 kg^.m⁻²), had brachial artery BP <150/ <90 mm Hg and circulating total (<240 mg^.dl⁻¹) and low-density lipoprotein (<160 mg^.dl⁻¹) cholesterol in the clinically normal range. All study procedures complied with the Declaration of Helsinki and the informed consent and study documents were approved by the Human Research Committee of the University of Colorado at Boulder. The nature, benefits, and risks of the study were explained to the volunteers, and their written informed consent was obtained before participation. See Supplemental Methods ^{(32KB, doc)} online for additional information.

General experimental procedures. All BP measurements were performed at the University of Colorado at Boulder Clinical and Translational Research Center (CTRC) after a 12-h overnight fast and abstention from caffeine, alcohol and exercise for at least 24 h. See Supplemental Methods ^{(32KB, doc)} online for additional information.

Study design. Subjects were randomly assigned to ingest BH₄ (Schircks Laboratories, Jona, Switzerland) pills (single oral dose of 10 mg·kg⁻¹ body weight) or placebo (double-blinded, cross-over study design) on separate days (4–5 days apart) between 7–8 AM at the CTRC and then returned to the CTRC for experimental measurements 3–3½ h after ingestion. The placebo and BH₄ pills were encapsulated to look identical to each other. Pharmacokinetic studies in humans demonstrate that plasma BH₄ concentrations peak between 3 and 5 h after oral ingestion and that 4–5 days is an adequate washout period.⁹ All BH₄ tablets were stored at −20 °C until immediately before ingestion by the subject. This dose increases plasma biopterin concentrations ~25-fold,⁹ improves endothelium-dependent dilation in healthy middle-aged/older adults,⁸ and is within the dosing range used therapeutically to treat adults with phenylketonuria.¹⁰

BP and carotid artery measurements. BP was measured by oscillometry over the brachial artery according to established procedures as previously described our laboratory. Carotid arterial compliance and β-stiffness index were assessed noninvasively as previously described in detail by our laboratory^11,12 and others.^13,14 See Supplemental Methods ^{(32KB, doc)} online for details.

Measurement of circulating factors. See Supplemental Methods ^{(32KB, doc)} online for details.

Statistical analysis. All analyses were performed using IBM SPSS Statistics 19.0 (IBM, Armonk, NY). All data are presented as mean ± s.e. An independent t-test was used to compare older men (i.e., during placebo) to young men at baseline with statistical significance was set at P < 0.05. A 2 × 2 repeated-measures analysis of variance was used for between-group (older men, younger men) and within-group (placebo condition, BH₄ condition) comparisons. When a significant condition × group interaction was revealed (P < 0.05) within-group paired t-tests with Bonferonni correction for multiple comparisons were performed with statistical significance set at P < 0.025. Bivariate pearson correlation analyses were performed to examine relations between variables of interest.

Results

Baseline values. Clinical characteristics and baseline function are shown in Table 1. Clinical characteristics were within normal ranges for both groups. Older men had greater waist circumference, waist:hip ratio, and plasma glucose (all P < 0.05), but did not differ in body mass index compared with young men (P = 0.46). Brachial artery diastolic BP (DBP) (P = 0.02), carotid end-diastolic diameter (P = 0.05), carotid end-systolic diameter (P = 0.03), carotid distension (P = 0.06) and carotid intimal-medial thickness (P < 0.01) were greater (or trended greater) in the older men, although mean values for BP were well within the normotensive range (Table 1). Older and young men did not differ in leisure time physical activity levels (P = 0.59). Carotid artery compliance and β-stiffness were 37% lower and 42% higher in the older men, respectively (P < 0.05, Figure 1).

Table 1.

Subject characteristics, hemodynamic, and circulating factors after oral ingestion of placebo and 10 mg^.kg⁻¹ tetrahydrobiopterin (BH₄) in older and young men

Figure 1. Carotid artery compliance and β-stiffness index after acute placebo and tetrahydrobiopterin (BH₄) supplementation. (a) Carotid artery compliance and (b) β-stiffness index in older and young men 3 h after oral ingestion of placebo or 10 mg^.kg⁻¹ tetrahydrobiopterin (BH₄). Individual data of older men on (c) carotid artery compliance and (d) β-stiffness index 3 h after oral ingestion of placebo or 10 mg·kg⁻¹ tetrahydrobiopterin (BH₄). *P < 0.05 vs. Placebo within group. ^†P < 0.05 vs. older placebo.

Circulating BH₄ concentrations. BH₄ ingestion increased circulating BH₄ concentrations by 19-fold (30.0 ± 10.4–597 ± 192 nmol^.l⁻¹, P = 0.02) in the older men and ~17½-fold (5.4 ± 1.5–99.5 ± 27 nmol^.l⁻¹, P = 0.049) in the younger men (the group × condition interaction P = 0.06).

Effects of BH₄ administration in older men. After BH₄ there was no change in brachial artery SBP, DBP, PP, heart rate, or circulating factors compared with after placebo (Table 1). In contrast, there was a significant group × condition interaction for carotid compliance (P < 0.01) and β-stiffness index (P = 0.01) where BH₄ increased carotid artery compliance by 39% (P = 0.02) and reduced β-stiffness index by 27% (P < 0.01, Figure 1). There was also a group × condition interaction for carotid artery SBP (P < 0.01) and a trend for carotid PP (P = 0.09), where BH₄ decrease carotid SBP by ~6 mm Hg (P < 0.01, Table 1) compared with placebo. The effects of BH₄ were consistent, with seven of the eight older men showing improvements in both carotid compliance and β-stiffness (Figure 1; bottom panels).

Effects of BH₄ administration in young men. There was no effect of BH₄ on brachial or carotid SBP or DBP, heart rate or circulating factors compared with placebo in the young men. In contrast, there was a significant group × condition interaction for brachial PP (P < 0.01) where brachial PP was decreased in the younger men after BH₄ compared with placebo (P < 0.025, Table 1). BH₄ had no effect on carotid artery compliance or β-stiffness index compared with placebo in the young men (Figure 1; top panels; Supplementary Figure S1 ^{(63.5KB, doc)} online).

Discussion

The results of this pilot study provide preliminary evidence that reduced vascular BH₄ bioavailability could be an important mechanism contributing to age-associated reductions in large elastic artery compliance (increases in stiffness) in men. Specifically, we found that a single therapeutic dose of BH₄ improved carotid artery compliance by ~40% and reduced β-stiffness index by almost 30% in a small group of healthy older men, such that values attained (compliance) or approached (β-stiffness index) those of young controls. In the older men, BH₄ also reduced carotid (i.e., central) artery SBP in the absence of any changes in brachial (i.e., peripheral) artery SBP or PP. The latter observations suggest that reduced BH₄ bioavailability may selectively contribute to increases in central SBP in older men, perhaps via effects on large elastic artery compliance. However, we cannot discount the possibility that BH₄ induced a central BP-lowering effect, which, in turn, influenced carotid artery properties.

The acute BH₄ administration used in the present study presumably exerted its carotid artery and central BP effects by affecting “functional” influences on compliance, perhaps related to NO modulation of vascular smooth muscle tone, rather than affecting structural components of the artery. Indeed, there is direct evidence that at least some portion of increased arterial stiffness is regulated by reduced endothelial NO bioavailability leading to enhanced vascular smooth muscle tone.^4,5,15 Therefore, given the obligatory role of BH₄ in maintaining vascular NO production we speculate that the improvement in carotid compliance in this case is a result of improvement in vascular tone in part from an increase in NO rather than alterations in vascular wall structural proteins elastin or collagen.

It also is possible that BH₄ acted as an antioxidant and scavenged reactive oxygen species such as superoxide anion.¹⁶ However, paramagnetic resonance studies suggest that superoxide scavenging by BH₄ is not a major reaction in vivo.¹⁷ Additionally, intra-brachial artery infusion of BH₄ improves endothelium-dependent, NO-mediated dilation in smokers, whereas tetrahydroneopterin (NH₄), a biopterin with similar antioxidant properties as BH₄ but without the endothelial NO synthase coupling ability, has no effect.¹⁸ Moreover, although the formulation of BH₄ used includes 50 mg of vitamin C, we have shown previously that a much higher oral dose of vitamin C (500 mg·day⁻¹) administered for one month¹² or acute intravenous infusion of supraphysiological dose of vitamin C¹² does not alter carotid artery compliance or β-stiffness in older men.

The reasons for the difference in basal circulating BH₄ concentrations between the older and young men is unclear. One possibility to explain this is the small sample size. With a larger sample size you might expect to see a regression to the mean resulting in circulating values that are not different. Another possibility is that basal circulating concentrations are inversely related with vascular BH₄ levels,¹⁹ suggesting that vascular BH₄ levels are lower in our cohort of older adults. However, both groups demonstrated an approximately 17–19-fold increase in circulating concentrations 3 h after oral BH₄ ingestion that resulted in improvements in carotid compliance and β-stiffness only in the older group. Furthermore, baseline or the change in BH₄ concentrations did not correlate with the change in carotid compliance or β-stiffness index, confirming that circulating BH₄ concentrations may not be a good index of vascular BH₄ concentrations.

There are several limitations to our pilot study that we wish to emphasize. First, we did not assess NO bioavailability or its effects on vascular smooth muscle relaxation. Second, because of this study's pilot nature, our sample size was, by definition, small and consisted only of healthy older and young men without major CVD risk factors or with known CVD which may limit generalizability. Despite this, the responses to BH₄ were consistent among our healthy older men suggesting that similar results could be observed in a larger sample, including adults with more CVD risk factors. Finally, the reductions in carotid SBP (and trend for PP) in the absence of alterations in brachial SBP and PP makes it tempting to speculate that this was a direct result of a reduced amplitude or delayed return of the reflected wave to central arteries. However, we did not measure carotid-femoral pulse wave velocity or central augmentation index in the present study so it cannot be determined whether aortic stiffness or reflected wave properties were altered. Interestingly, a recent study in older adults with severe coronary artery disease demonstrated that 2–6 weeks of low- or high-dose BH₄ treatment had no effect on aortic pulse velocity or aortic and carotid distensibility measured by magnetic resonance imaging.²⁰ Future studies will be necessary to directly test whether chronic BH₄ supplementation favorably modulates aortic pulse wave velocity and augmentation index in healthy older men and women.

In conclusion, the results of the present pilot study support the hypothesis that reduced BH₄ bioavailability may be an important mechanism contributing to reductions in carotid artery compliance and increases in central SBP with aging in healthy men. These preliminary results provide the basis for larger investigations aimed at determining if increasing BH₄ bioavailability through oral supplementation can improve large elastic artery compliance, lower central SBP and perhaps reduce the risk of age-associated CVD.

Supplementary material is linked to the online version of the paper at http://www.nature.com/ajh

Diclosure

The authors declared no conflict of interest.