MacAllister and Aroon D. Hingorani Lila Mayahi, Simon Heales ...

(6R)-5,6,7,8-Tetrahydro-L-Biopterin and Its Stereoisomer Prevent Ischemia Reperfusion 

Injury in Human Forearm 

Lila Mayahi, Simon Heales, David Owen, Juan P. Casas, Joanne Harris, Raymond J. 

MacAllister and Aroon D. Hingorani 

Arterioscler Thromb Vasc Biol. 2007;27:1334-1339; originally published online April 5, 2007; 

doi: 10.1161/ATVBAHA.107.142257 

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(6R)-5,6,7,8-Tetrahydro-L-Biopterin and Its Stereoisomer 

Prevent Ischemia Reperfusion Injury in Human Forearm 

Lila Mayahi, Simon Heales, David Owen, Juan P. Casas, Joanne Harris, 

Raymond J. MacAllister, Aroon D. Hingorani 

Objective—6R-5,6,7,8-tetrahydro-L-biopterin (6R-BH4) is a cofactor for endothelial nitric oxide synthase but also has 

antioxidant properties. Its stereo-isomer 6S-5,6,7,8-tetrahydro-L-biopterin (6S-BH4) and structurally similar pterin 

6R,S-5,6,7,8-tetrahydro-D-neopterin (NH4) are also antioxidants but have no cofactor function. When endothelial nitric 

oxide synthase is 6R-BH4–deplete, it synthesizes superoxide rather than nitric oxide. Reduced nitric oxide 

bioavailability by interaction with reactive oxygen species is implicated in endothelial dysfunction (ED). 6R-BH4 

corrects ED in animal models of ischemia reperfusion injury (IRI) and in patients with cardiovascular risks. It is 

uncertain whether the effect of exogenous 6R-BH4 on ED is through its cofactor or antioxidant action. 

Methods and Results—In healthy volunteers, forearm blood flow was measured by venous occlusion plethysmography 

during intra-arterial infusion of the endothelium-dependent vasodilator acetylcholine, or the endothelium-independent 

vasodilator glyceryl trinitrate, before and after IRI. IRI reduced plasma total antioxidant status (P0.03) and impaired 

vasodilatation to acetylcholine (P0.01), but not to glyceryl trinitrate (P0.3). Intra-arterial infusion of 6R-BH4, 

6S-BH4 and NH4 at approximately equimolar concentrations prevented IRI. 

Conclusion—IRI causes ED associated with increased oxidative stress that is prevented by 6R-BH4, 6S-BH4, and NH4, 

an effect mediated perhaps by an antioxidant rather than cofactor function. Regardless of mechanism, 6R-BH4, 6S-BH4, 

or NH4 may reduce tissue injury during clinical IRI syndromes. (Arterioscler Thromb Vasc Biol. 2007;27:1334-1339.) 

Key Words: 6R-5,6,7,8-tetrahydro-L-biopterin antioxidant endothelial dysfunction ischemia reperfusion 

6R-5,6,7,8-terahydro-L-biopterin (6R-BH4) is an essential 

cofactor for endothelial nitric oxide synthase (NOS) that 

catalyzes production of the vasodilator and atheroprotective 

mediator NO. 1,2 Biosynthesis of 6R-BH4 occurs in the 

endothelium, promoting NO synthesis by facilitating electron 

transfer from the reductase domain of NOS to arginine. 

6R-BH4 also stabilizes the NOS dimer and exerts an allosteric 

effect, enhancing substrate binding. 3 Changes in 6R-BH4 

availability influence NO production. A reduction in 6R-BH4 

leads not only to a diminished NO synthesis but also to the 

generation of NOS-derived superoxide (O2 ) that has been 

implicated in the development of endothelial dysfunction. 4 

Ischemia reperfusion injury (IRI), such as occurs after 

thrombolysis or balloon angioplasty, is associated with endothelial 

dysfunction, which may contribute to cellular damage. 

The mechanism underlying endothelial dysfunction in IRI is 

incompletely understood but adhesion of activated neutrophils 

to endothelial cells, 5 an increase in oxygen radical 

generation, 6,7 the elaboration of inflammatory cytokines, and 

a reduction in NO production8,9 are all believed to play a role. 

In humans, local delivery of 6R-BH4 by intra-arterial 

infusion improves endothelium dependent vasodilation in 

patients with coronary artery disease, 10 type II diabetes 

mellitus, 11 elevated cholesterol, 12 raised blood pressure, 13 and 

in smokers, 14 leading to the proposal that an acquired deficiency 

in 6R-BH4 and defective NOS catalysis underlies 

endothelial dysfunction in these states. Supplementation with 

6R-BH4 also reduces IRI in the rat heart 15 and kidney 16 and 

in the pig heart. 17 In animals, the administration of antioxidants 

such as ascorbic acid or N-acetylcysteine reduces 

endothelial dysfunction induced by IRI. 18–20 

Therefore, the effect of 6R-BH4 on endothelial function in 

IRI could also be mediated by an antioxidant action, 21 a 

property shared with its stereoisomer 6S-5,6,7,8-tetrahydro- 

L-biopterin (6S-BH4), 22 as well as with 6R,S-5,6,7,8tetrahydro-D-neopterin 

(NH4), 14 rather than by correction of 

defective NOS catalysis. Although approximately equipotent 

as antioxidants, 14,22 only 6R-BH4 has substantial capacity to 

support NOS catalysis. 23,24 

We therefore examined, for the first time, the effect of 

6R-BH4 on endothelial dysfunction during IRI in humans and 

tested whether this was mediated through correction of 

6R-BH4 deficiency and defective NOS catalysis or through 

an antioxidant action. 

Original received November 17, 2006; final version accepted March 15, 2007. 

From Centre for Clinical Pharmacology (L.M., D.O., J.P.C., J.H., R.J.M., A.D.H.), University College London, London, UK; Neurometabolic Unit 

(S.H.), Hospital for Neurology and Neurosurgery (UCLH Foundation Trust), Queen Square, London, UK. 

Correspondence to Dr Lila Mayahi, Centre for Clinical Pharmacology, BHF laboratories, Department of Medicine, UCL, 5 University Street, London, 

UK, WC1E 6JJ. E-mail l.mayahi@ucl.ac.uk 

© 2007 American Heart Association, Inc. 

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/ATVBAHA.107.142257 


http://atvb.ahajournals.org/ 1334 by guest on June 29, 2013

Materials and Methods 

Forty-eight nonsmoking healthy volunteers, 34 males, 14 females, 

aged 19 to 42, with mean BMI (SD) of 23.4(3.1), blood pressure 

127/77 (15/10) mm Hg, heart rate 71 bpm, random glucose 

4.8(0.6) mmol/L, and random cholesterol 3.8(1.1) mmol/L, were 

recruited after written informed consent was obtained. All females 

were studied during their menstrual period to take into account the 

variation in endothelial function during the menstrual cycle. 25 The 

studies were approved by the UCLH research ethics committee. 

Assessment of Flow in Forearm 

Resistance Arteries 

Studies were performed in a temperature-controlled laboratory. 

Baseline blood pressure and heart rate was recorded. We used 

venous occlusion plethysmography using mercury-in-silastic strain 

gauges to measure forearm blood flow in the resistance vessels of 

both arms as described previously. 26 Drugs were administered in 

saline (0.9% sodium chloride) and infused at a rate of 0.5 mL/min 

through a 27-gauge needle (Cooper’s Needle Works Ltd, Birmingham 

UK) inserted into the nondominant brachial artery under local 

anesthesia using 1 to 2 mL of 2% lidocaine. Pterin compounds were 

prepared immediately before infusion in deoxygenated 0.9% sodium 

chloride solution. During the recording of forearm blood flow, the 

hands were excluded from circulation by inflation of the wrist cuffs 

to 200 mm Hg. 

To induce IRI, the nondominant forearm was made ischemic by 

inflation of the upper arm cuff to 200 mm Hg for 20 minutes 

followed by reperfusion by deflating the cuff for 15 minutes, as 

described previously. 26 

Venous Blood Sampling 

Venous plasma was obtained in EDTA tubes from an antecubital 

vein for measurement of total biopterin, total antioxidant status 

Mayahi et al 6R-BH4 and Ischemia Reperfusion Injury 1335 

Figure 1. Effect of 6R-BH4 on venous biopterin 

concentrations (a) and the baseline forearm 

blood flow (b). Dotted line indicates 6R-BH4 

Km for endothelial nitric oxide synthase in vitro. 

(TAOS), nitrate and nitrite concentration (NOx), and stored at 

80°C until analysis. 

TAOS 

The TAOS of plasma was determined by its capacity to inhibit 

the peroxidase-mediated formation of the 2,2-Azino-bis-3ethylbenzothiazioline-6-sulfonic 

acid (ABTS ) radical. 27,28 In this 

assay, the relative inhibition of ABTS formation in the presence of 

plasma is proportional to the antioxidant capacity of the sample. 

Briefly, 2.5 L of plasma was incubated for 3 minutes at 37°C in a 

96-well plate with a reaction mixture comprising (final concentrations): 

20 L ABTS (20 mmol/L), 20 L horseradish peroxidase (30 

mU/mL), and 37.5 L phosphate-buffered saline, pH 7.4. The 

reaction was initiated by the addition of 20 L hydrogen peroxide 

(0.1 mmol/L), and the increase in absorbance over 6 minutes was 

monitored at 405 nm, using a microplate reader. At 6 minutes, 

sample absorbance was read together with a control (containing 2.5 

L phosphate-buffered saline instead of plasma). The proportional 

difference in absorbance between the control and test samples was 

used to represent percentage inhibition of the reaction. 

Plasma Total Biopterin 

Plasma total biopterin concentration was measured using the method 

of Fukushima and Nixon. 29 Briefly, reduced pterins were oxidized 

using 3% iodide solution (2% iodine in 6% KI) after removal of 

proteins with 60% perchloric acid. Total biopterin was detected by 

fluorescence set at 360 nm excitation and 440 nm emission after 

using a high-performance liquid chromatography Spherisorb 

ODS-1 column with a 5% methanol mobile phase run at a flow 

rate of 1 mL/min. 



1336 Arterioscler Thromb Vasc Biol. June 2007 

Measurement of Nitrite and Nitrate Concentration 

Plasma NO 2 and NO3 (NOx) were measured using the Griess 

assay. Nitrate was converted to nitrite with nitrate reductase before 

the addition of the Griess reagent as described previously. 30 

Drugs and Infusates 

Acetylcholine chloride (ACh) was obtained from Merck Biosciences, 

and glyceryl trinitrate (GTN), lidocaine hydrochloride, and 

normal saline (0.9% sodium chloride) solution were from East 

Anglia Pharmaceutical, Phoenix, and Baxter, respectively. 6R-BH4, 

6S-BH4, and NH4 were purchased from Schircks Laboratories. 

Reagents for Assays 

For measurements of total plasma antioxidant capacity, Dulbecco 

phosphate-buffered saline was purchased from Invitrogen, horseradish 

peroxidase was from Merck Biosciences, and hydrogen peroxide 

and 2,2-Azino-bis-3-ethylbensthiazoline-6-sulfonic acid (ABTS) 

were from Sigma-Aldrich. For plasma total biopterin and measurement 

of nitrate (NO 3 ) and nitrite (NO2 ), perchloric acid 60%, 

sodium ascorbate, iodine and potassium iodide solution, glucose-6phosphate 

dehydrogenase, nitrate reductase, glucose-6-phosphate, 

and reduced form of nitrate reductase were purchased from 

Sigma-Aldrich. 

Experimental Protocols 

Endothelial and smooth muscle function were assessed by measuring 

the forearm blood flow response to intra-arterial infusion of ACh 

(25, 50, and 100 nmol/min) and GTN (8, 16, and 32 nmol/min), 

respectively. Each dose was infused for 3 minutes; 0.9% sodium 

chloride was infused between study drugs. The effect of infusion of 

pterin compounds on endothelial and smooth muscle dilator function 

to IRI was assessed in separate studies as follows. 

Protocol 1: Effect of 6R-BH4 on Baseline Forearm 

Blood Flow 

Forearm blood flow was measured during intra-arterial infusion of 

50 or 500 g/min 6R-BH4, each dose for 30 minutes. Venous blood 

was drawn from the same arm at 10-minute intervals during and after 

the infusions. 

Protocol 2: Effect of Pterins on Vasodilation to ACh 

Forearm blood flow was measured in response to ACh before and 

after infusion of 500 g/min 6R-BH4 or NH4. 

Protocol 3: Effect of IRI on Plasma TAOS, Biopterin, 

and NOx 

In 8 subjects an antecubital vein was cannulated in both arms for 

blood sampling. The nondominant arm was then subjected to 20 

minutes of ischemia and 15 minutes of reperfusion as described 

previously. Venous samples were obtained simultaneously from both 

arms before initiation of ischemia, at the end of ischemia, 15 minutes 

after reperfusion, and then in recovery 4 hours later. 

Protocol 4: Effect of IRI on Endothelial Function 

Forearm blood flow responses to ACh or GTN were assessed before 

and immediately after IRI. 

Protocol 5: Effect of Pterins on IRI Induced 

Endothelial Dysfunction 

In separate experiments, to assess the effect of pterin compounds on 

endothelial function after IRI, vasodilation to ACh and GTN was 

measured before and immediately after IRI. 6R-BH4, 6-SBH4, or 

NH4 (500 g/min) were infused intra-arterially in the study arm 

during both ischemia and reperfusion. 

For all the protocols, please see supplemental Figure I (available 

online at http://atvb.ahajournals.org). 

Statistical Analysis 

Forearm blood flow was measured as mL/100 mL forearm volume/ 

min. The average ratio of flow in the infused (active)/noninfused 

Figure 2. Effect of IRI on venous plasma TAOS (a), biopterin (b), 

and NOx (c) (probability values refer to the difference between 

control and active arms at IRI. 

(control) arm was calculated over the last minute of infusion for each 

dose of drug and comparisons were made by 2-way ANOVA. A 

value of P0.05 was considered statistically significant. Where an 

alteration in baseline flow was observed between study periods, 

responses were evaluated after adjustment for these differences using 

logistic regression analysis. Within-group comparisons for other 

parameters were analyzed by paired Student t test. All analyses were 

performed using Graph Pad Prism or Stata 8.2. 



Results 

A total of 109 forearm studies were conducted among 48 

healthy volunteers. 

Effect of 6R-BH4 and NH4 on Baseline Forearm 

Blood Flow and Venous Biopterin Concentrations 

6R-BH4 (50 g/min intra-arterial for 30 minutes) increased 

forearm venous total biopterin concentrations to approximately 

61 mol/L. At a rate of 500 g/min for 30 minutes 

the forearm venous concentration of total biopterin increased 

to 10840 mol/L (Figure 1a). There was no alteration in 

forearm blood flow at either dose of 6R-BH4 (Figure 1b). 

Forearm blood flow was also unchanged during infusion of 

NH4 (500 g/min for 30 minutes; data not shown). 

Effect of Pterins on Vasodilation to ACh 

Neither 6R-BH4 nor NH4 altered the dilator response to ACh 

in the absence of IRI (supplemental Figure II). 

Effect of IRI on TAOS, Biopterin, and NOx 

There was a significant reduction of forearm venous plasma 

TAOS (Figure 2a) immediately after IRI with no detectable 

change in plasma total biopterin or NOx (Figure 2b, 2c). 

Effect of IRI on Endothelial Function and the 

Effect of Pterins on Endothelial Function 

Following IRI 

As reported previously, 26 IRI attenuated endotheliumdependent 

vasodilation to ACh (P0.01) but did not affect 

a 

forearm flow ratio 

6 pre 

IRI 

post IRI 

5 

4 

3 

2 

1 

0 

6R-BH4 

n= 

14 

p= 

0. 

81 

25 50 100 

ACh 

nmol/ 

min 

b 



3 

2 

1 

0 

6S-BH4 

Figure 3. Effect of IRI on endothelial function. 

Forearm blood flow responses to endothelium 

dependent vasodilator ACh (a) and to endothelium 

independent vasodilator GTN (b). 

the response to endothelium-independent dilator GTN 

(P0.3; Figure 3a , 3b). Continuous infusion of 6R-BH4, 

6S-BH4, or NH4 (all at 500 g/min) during ischemia and 

reperfusion preserved the response to ACh dilation, in keeping 

with protection from reperfusion injury (Figure 4a, 4b, 

4c). The response to GTN was unaltered (data not shown). 

Discussion 

In this study 6R-BH4, an essential endogenous NOS cofactor, 

prevented endothelial dysfunction caused by IRI, consistent 

with observations in animal models. The protection from 

endothelial dysfunction observed here with 6R-BH4 is similar 

to that seen in humans after other acute endothelial insults 

such as cigarette smoking or an oral glucose load, and after 

chronic exposure to cardiovascular risk factors. However, in 

our studies, a similar protective effect on endothelial IRI was 

also observed with 6S-BH4, and with NH4, at doses that 

would have resulted in approximately equimolar intra-arterial 

concentrations. 6S-BH4 is 60-times less potent as a NOS 

cofactor than 6R-BH4, 23,31–33 and NH4 has no cofactor 

activity. 24 Therefore, it is unlikely that this protective action 

is the result of increased NOS catalysis. However, all 3 

pterins are equally efficacious as scavengers of the superoxide 

radical. 21,22,34 Because superoxide may quench the effect 

of NO by a rapid chemical interaction altering its vasodilator 

and other actions, 35–37 this might provide a mechanism for the 

observed protection from endothelial IRI produced by all 3 

pterins. 

5 pre 

IRI 

post IRI 

4 

25 50 100 

AChnmol/ min 

n= 

15 

p= 

0. 

75 

c 


5 

4 

3 

2 

1 

0 

NH4 

n= 

11 

p= 

0. 

61 

25 50 100 

AChnmol/ min 

pre 

IRI 

post IRI 

Figure 4. Effect of pterins on IRI. Forearm blood flow responses to cumulative doses of ACh before and after IRI, in the presence of 

6R-BH4 (a), 6S-BH4 (b), and NH4 (c). 



1338 Arterioscler Thromb Vasc Biol. June 2007 

IRI is associated with increased generation of O 2 and 

related radicals during the early phase of reperfusion, 7,38 

although evidence for this is indirect and based on experiments 

where there has been reduction in injury with use of 

antioxidants such as ascorbic acid or N-acetylcysteine 18–20 or 

superoxide dismutase. 39 Potential sources of O 2 include 

cyclo-oxygenases, 40 xanthine oxidase, NADPH oxidase, uncoupled 

NOS, and the mitochondrial electron transport system, 

all of which have been implicated in IRI. 41 In this study, 

consistent with increased O 2 production, there was a marked 

reduction in TAOS of venous plasma after IRI. Therefore, the 

simplest explanation of our findings is that all 3 pterins exert 

their protective effects in this model through free-radical 

scavenging. 

Intrabrachial infusion of 6R-BH4 at a dose of 500 g/min 

also markedly improves endothelium-dependent vasodilation 

in patients with cardiovascular risk factors. 11–14 This observation 

has been taken as evidence that these risk factors lead 

to a depletion of endogenous 6R-BH4. Because all of these 

exposures have also been associated with the increased 

generation of oxygen radicals by the vascular wall, 42–44 and 

because 6R-BH4 is itself susceptible to oxidative inactivation, 

4,45,46 this model has good biological plausibility. However, 

as we demonstrate in this study, the dose of 6R-BH4 

commonly used (500 g/min) raises its intravascular concentration 

to 100 mol/L, 1000-times the Michaelis constant 

(Km; 100 nmol/L) of the isolated NOS enzyme for 6R-BH4. 47 

Though it is uncertain whether intracellular concentrations 

would be quite as high, the free radical scavenging action of 

6R-BH4 at these concentrations could outweigh its specific 

“physiological” role in NOS catalysis at this dose. Few of the 

prior studies in humans have compared the effects of 6R-BH4 

on endothelial function with those of 6S-BH4 or NH4, which 

are equipotent as antioxidants but have little or no NOS 

cofactor actions. The beneficial effect of 6R-BH4 on endothelial 

function in smokers 14 was not shared by NH4, nor by 

6S-BH4, after an oral glucose load in healthy subjects. 22 

These observations suggest that, in some situations, the 

effects of 6R-BH4 could be mediated by direct improvement 

of NOS catalysis, possibly by restoration of 6R-BH4 deficiency, 

while in others by an antioxidant effect. Further work 

will be required to understand which of the 2 actions of 

6R-BH4 is important in the endothelial dysfunction induced 

by diabetes, elevated levels of cholesterol, or high blood 

pressure. 

Although the evidence suggests that most of the injury in 

IRI occurs early after reperfusion, 38 we chose to infuse 

6R-BH4 throughout the IRI period to maximize protection 

against injury. To further understand the mechanism of action 

of BH4 in IRI, it will be important to establish the critical 

period during which pterins exert their protective actions, 

particularly if these agents are to be considered as potential 

therapies for IRI in clinical situations. 

Understanding the role of 6R-BH4 in endothelial dysfunction 

has been limited by the lack of a simple specific assay in 

plasma. In our experiments we measured total plasma biopterin 

after acid oxidation of plasma. The total biopterin thus 

measured derives from both 6R-BH4 and its oxidized product 

7,8-dihydrobiopterin that are thought to circulate in plasma in 

the approximate ratio of 3 to 2. 48 Measuring each species 

separately in human plasma has proved challenging, although 

other workers have developed assays that are reliable in 

animals. However if a simple method was developed for 

human plasma, this might enhance understanding of whether 

6R-BH4 concentrations are reduced in situations in which 

endothelial dysfunction occurs. 

In summary we have shown, for the first time in humans, 

that reduced pterins 6R-BH4, 6S-BH4, and NH4 prevent 

endothelial dysfunction induced by IRI in the human forearm. 

Regardless of mechanism, this may represent a new therapeutic 

approach in clinical settings where ischemiareperfusion 

injury occurs. 

Acknowledgments 

The authors thank Dr Jeffery Stephens for helping with the 

TAOS assay. 

Sources of Funding 

L.M. is supported by a BHF PhD studentship FS/02/070/14413. 

A.D.H. is supported by BHF Senior Research Fellowship FS/05125. 

None. 

Disclosures 

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Figure I 

a. Protocol 1. Effect of 6R-BH4 on forearm blood flow. Forearm blood flow 

(FBF) was measured at 10 minute intervals during i.a infusion of 50 and 

500mcg/min 6R-BH4 and 0.9% saline. Venous blood samples (BS) were also 

taken at these time points. 

b. Protocol 2. Effect of pterins on vasodilation to ACh. Cumulative dose 

responses to ACh were measured at baseline and after 30 minutes of i.a 6R- 

BH4 or NH4. 

c. Protocol 3. Effect of IRI on plasma TAOS, biopterin and NOx. BS were 

taken from active (IRI) and control arms. The active arm was subjected to the 

IRI and the control arm remained at rest with no intervention (the time line is 

not to scale). 

d. Protocol 4. Effect of pterins on endothelial dysfunction following IRI. FBF 

responses to cumulative doses of ACh and GTN were measured before and 

after IRI. This was repeated with i.a infusion of 6R-BH4, 6S-BH4 or NH4 

(500mcg/min) during IRI. 

Figure II. Effect of pterins on vasodilation to ACh. Forearm blood flow 

responses to cumulative doses of ACh before and after 500mcg/min i.a 6R- 

BH4 (a) and NH4 (b) show no significant differences 



Figure I 

Time (min) 

BS & FBF 

b 

c 

0.9%NaCl ACh 6R-BH4 or NH4 

0.9%NaCl ACh 

Ischemia Reperfusion 

Intervention Active Active (IRI) (IRI) arm arm 

arm 

Time (mins) 

BS 0 20 35 275 

Intervention Nil 

Control Control Control Control arm arm arm 

arm 

d 

a 

FBF 

FBF 

0.9%NaCl 

6R-BH4 50mcg/min 6R-BH4 500 mcg/min 

0 15 25 35 45 55 65 75 85 95 105 115 125 135 

Ischemia 

0.9%NaCl 0.9%NaCl 

Reperfusion 

0.9%NaCl ACh or GTN 6R-BH4 or 6S-BH4 or NH4 or N saline 0.9%NaCl ACh or GTN 



Figure II 

a 


b 


5 pre BH4 

4 

3 

2 

1 

0 

5 pre NH4 

4 

3 

2 

1 

0 

n=10 

p=0.12 

25 50 100 

ACh nmol/min 

n=8 

p=0.6 

25 50 100 

ACh nmol/min 

post BH4 

post NH4

MacAllister and Aroon D. Hingorani Lila Mayahi, Simon Heales ...

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