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European Journal of Heart Failure 2007 9(3):287-291; doi:10.1016/j.ejheart.2006.06.006
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© 2007 European Society of Cardiology

Chronic oral ascorbic acid therapy worsens skeletal muscle metabolism in patients with chronic heart failure

Angus K. Nightingalea,b,*, Jenifer G. Crilleyc, Nicholas C. Peggeb, Ernie A. Boehmc,d, Catherine Mumfordb, Doris J. Taylorc,d, Peter Stylesc,d, Kieran Clarkec and Michael P. Frenneauxb,e

a Bristol Heart Institute Bristol University, Bristol, UK
b Wales Heart Research Institute University of Wales College of Medicine, UK
c Department of Physiology, Anatomy and Genetics University of Oxford, UK
d MRC Biochemical and Clinical Magnetic Resonance Unit Oxford Radcliffe Hospital, Oxford, UK
e Department of Cardiovascular Medicine Birmingham University, UK

* Corresponding author. Department of Cardiology, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom. Tel.: +44 117 342 0492; fax: +44 117 342 0496. E-mail address: drangus{at}doctors.org.uk


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Chronic heart failure (CHF) is associated with abnormalities of skeletal muscle metabolism. This may be due to impaired oxygen delivery as a result of endothelial dysfunction.

Aims: We postulated that ascorbic acid would improve oxygen delivery to exercising muscle and improve skeletal muscle metabolism.

Methods: We studied skeletal muscle metabolism using 31P magnetic resonance spectroscopy in 39 CHF patients. Endothelial function was assessed by changes in pulse wave velocity. Subjects were randomised to receive 4g ascorbic acid daily for 4weeks in a placebo-controlled double-blind study.

Results: Ascorbic acid significantly increased phosphocreatine utilization during exercise. In addition, glycolytic ATP synthesis increased in the ascorbic acid group (change in rate of ATP synthesis at 1min –0.21±0.76 with placebo, 2.06±0.60 following ascorbic acid; p<0.05). Phosphocreatine and ADP recovery after exercise were not changed. The fall in pulse wave velocity during reactive hyperaemia was increased by ascorbic acid from –6.3±2.6% to –12.1±2.0% (p<0.05).

Conclusions: These findings suggest that ascorbic acid increased both phosphocreatine utilization and glycolytic ATP synthesis during exercise in patients with CHF implying worsened skeletal muscle metabolism despite improvements in endothelial function.

Key Words: Magnetic resonance spectroscopy • Ascorbic acid • Chronic heart failure • Endothelial function

Received November 23, 2005; Revised May 10, 2006; Accepted June 22, 2006


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Chronic heart failure (CHF) is characterized by exercise limitation. There has been considerable research interest in understanding the mechanisms of exercise limitation in CHF. Endothelial dysfunction has been well recognized in CHF [1] and is an important prognostic factor in several conditions [2]. Recent studies on the role of exercise training have identified that this can improve both endothelial function and skeletal muscle metabolism [3]. Whether there is a direct link between these two factors is less clear. The finding that L-arginine supplementation improves muscle metabolism to a similar extent as exercise have suggested that there may be a mechanistic link [4]. Other studies, in contrast, have suggested that changes in muscle metabolism are unrelated to changes in muscle blood flow [5,6].

Skeletal muscle blood flow and endothelial function might be important in several ways. Most importantly, increased oxygen delivery to muscle might improve muscle metabolism. Whilst some studies have suggested that muscle blood flow is impaired in CHF during exercise [7], other studies have not shown this [5,6]. Secondly, endothelial function might be important in determining whether blood flow is directed to active or resting muscle, termed nutritive or non-nutritive flow [8]. Finally, nitric oxide has direct effects on mitochondrial function [9]. Therefore, altering the levels of local nitric oxide within active skeletal muscle might be an important therapeutic target.

Several studies have shown that intravenous ascorbic acid improves endothelial function in CHF although it may not restore it completely to normal [10,11]. We have shown that chronic oral ascorbic acid improves endothelial function in CHF [12]. In this study we investigated whether short-term oral ascorbic acid improves skeletal muscle metabolism in subjects with CHF. In addition, we investigated whether changes in metabolism are related to changes in large artery endothelial function.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Patients with clinical evidence of CHF and left ventricular systolic dysfunction (ejection fraction <35%) were recruited from heart failure clinics at two university hospitals in the UK. All subjects had been stable on medical treatment for the preceding 2 months; none of the subjects were taking vitamin supplements. The respective Research Ethics Committees approved the study and all subjects gave written informed consent. The investigation conforms with the principles outlined in the Declaration of Helsinki. Subjects were randomised to 1 month of oral vitamin C (4 g per day) or placebo in a double-blind placebo controlled parallel group study. We chose this dose and duration of therapy because we have previously shown that it significantly improved brachial artery flow mediated dilatation in a similar group of patients [12]. Skeletal muscle metabolism was assessed using 31P magnetic resonance spectroscopy (31P MRS) and large artery endothelial function was measured by pulse wave velocity (PWV). Following 1 month of treatment, the assessments were repeated in the same way. Drug therapy was continued throughout the studies. Subjects who were fluid overloaded or with evidence of peripheral oedema were excluded.

2.1. Plasma ascorbic acid measurement
Venous blood was obtained at baseline and on completion of the intervention limb, and immediately centrifuged. Plasma (100 µl) was added to 5% metaphosphoric acid (900 µl) and frozen at –70 °C for later estimation of ascorbic acid concentrations as described by Vuillemier and Keck [13].

2.2. Skeletal muscle metabolism
The technique of 31P MRS allows changes in skeletal muscle phosphocreatine (PCr), inorganic phosphate (Pi) and pH to be assessed non-invasively during exercise and recovery. We have described this technique in detail previously and give a brief overview here [14]. Subjects lay in a 2.0 T superconducting magnet (Oxford Magnet Technology, Eynsham, Oxford, UK) that was interfaced to a spectrometer (Brucker, Coventry, UK) with the right calf overlying a 6 cm diameter surface coil and fixed in place. 31P MR spectra were collected during a rest-exercise-recovery protocol using a 2 s interpulse delay. Exercise consisted of plantar flexion performed at 0.5 Hz lifting 10% of lean body mass (LBM, calculated from body weight and skin fold thickness), through a distance of 7 cm. After 4 min, the workload was incremented by 2% of LBM for each further minute until PCr consumption reached approximately 55% of the pre-exercise level or until the subject was unable to continue (e.g., due to fatigue). Spectra were acquired at rest (64 scans) during exercise (16 scans) and in recovery.

Measurements of changes in pH and [PCr] from rest to the first exercise spectrum were used to calculate the initial rates of glycolytic ATP synthesis and PCr depletion, whose sum is a reasonable estimate of the initial rate of ATP synthesis.

The rate of PCr consumption during exercise was plotted as a function of time.

During recovery from exercise, the initial rate of PCr re-synthesis is believed to be a good estimate of the end-exercise rate of oxidative ATP synthesis. Initial rates of PCr re-synthesis after exercise (V, in mM/min) were determined from the exponential rate constant of PCr recovery (k=ln(2)/t1/2) and the net decrease in [PCr] during exercise.

End-exercise [ADP] and V were used to calculate the maximum rate of oxidative ATP synthesis (a measure which is independent of muscle mass). Estimates of PCr and ADP recovery half times (t1/2) were calculated from the slope of semi-logarithmic plots; these provide another index of mitochondrial function.

2.3. Pulse wave velocity
We used a recently described technique to assess changes in arterial pulse wave velocity in response to reactive hyperaemia [15]. These changes are endothelial dependent as demonstrated by blockade with N-monomethyl-L-arginine (L-NMMA, an inhibitor of nitric oxide (NO) synthase).

A commercially available peripheral pulse waveform recorder (QVLP84, SciMed, Bristol, UK) was programmed to record PWV between standard sphygmomanometer cuffs attached to ipsilateral thigh and ankle. Following 5 min acclimatisation, transit times between the arrival of 10 consecutive pulse waves at proximal then distal cuff were averaged to yield a basal PWV. Inflation of the ankle cuff to 250 mm Hg for 5 min delivered foot ischaemia. After abrupt cuff deflation, transit times were recorded during subsequent reactive hyperaemia. PWV was calculated as cuff separation divided by transit time. Subjects in atrial fibrillation were excluded from measurement.

2.4. Statistical analysis
Results are presented as mean±S.E. Data were analyzed using SPSS version 10.07. Statistical significance of differences was determined by three-way ANOVA with subject, time and treatment as factors. To analyze the time course of PCr depletion during exercise we calculated the area under the PCr curve (AUC) for each group. The AUC were compared using MANOVA and the results were adjusted to take account of baseline values. Continuous data were compared using Student's paired and unpaired t-test as appropriate and proportions (age and medication) were compared with Chi-square tests. Statistical significance was accepted at a level of p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
The groups receiving ascorbic acid and placebo were well matched for age, sex and NYHA class (see Table 1). Drug treatment was similar in both groups (Table 1) apart from β blocker therapy, which was higher in the placebo group (10 versus 4; p=0.1). All patients were treated with an ACE inhibitor or an angiotensin receptor blocker. Smoking status, which can potentially alter antioxidant capacity, was similar in both groups with only two current smokers in each group. Ascorbic acid had no effect on resting blood pressure or heart rate. Heart rate and blood pressure were similar in both groups and did not change following ascorbic acid as shown in Table 3.


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  		Table 1 Clinical characteristics of the CHF patients

 
3.1. 31P MRS
Metabolite concentrations at rest were similar in the ascorbic acid and placebo groups and did not change significantly between studies (Table 2). There was a greater initial fall in absolute PCr concentration during vitamin C compared to placebo. The AUC for the whole time course was 151.0±8.6 pre-placebo, 156.1±7.1 post-placebo, 147.0±7.1 pre-ascorbic acid and 135.0±7.1 post-ascorbic acid (p<0.05; by MANOVA using baseline values as covariates) as shown in Fig. 1. In addition, glycolytic ATP synthesis increased in the ascorbic acid group (change in rate of ATP synthesis at 1 min –0.21±0.76 with placebo, 2.06±0.60 following ascorbic acid; p<0.05). There was no improvement in PCr or ADP recovery half time following ascorbic acid (Table 2).


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  		Table 2 Effects of vitamin C on skeletal muscle energetics in CHF patients

 


Figure 01
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  		Fig. 1 Effect of vitamin C on phosphocreatine concentration during exercise. *Significant fall in phosphocreatine (PCr) during exercise in the group treated with ascorbic acid (Vit C) compared to placebo (p<0.05 for area under curve).

 
3.2. Large artery function
Resting PWV in the leg was similar in both groups and did not change with vitamin C (Table 3). PWV reactive hyperaemia was improved by ascorbic acid from –6.3±2.6% to –12.1±1.97% but did not change with placebo (p<0.02 by ANOVA) (Table 3).


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  		Table 3 Effects of ascorbic acid on haemodynamic parameters

 
3.3. Vitamin C levels
Plasma levels of ascorbic acid increased significantly following 1 month of treatment from 39.1±7.3 µmol/l to 115.6±17.5 µmol/l (p<0.01), whereas no change was seen following placebo (39.0±5.6 µmol/l to 33.5±6.2 µmol/l; p=NS). There was no correlation between renal function at baseline or following treatment and ascorbic acid concentration.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The first important finding of this study is that there was no improvement in skeletal muscle metabolism following 1 month of treatment with ascorbic acid in subjects with mild to moderate chronic heart failure despite an improvement in large artery endothelial function. In fact, whilst recovery of high-energy phosphates was unaffected, PCr hydrolysis was more rapid after ascorbic acid therapy.

CHF is characterized by increased PCr depletion during exercise, and impaired skeletal muscle perfusion has been proposed as a mechanism [16]. One animal study has shown that limb blood flow is dependent on nitric oxide bioavailability [17] and endothelial function is impaired in most studies on patients with chronic heart failure [1]. We have previously shown that impaired endothelial function can be improved by chronic oral administration of ascorbic acid [12] and other groups have found similar effects of ascorbic acid [10]. A study demonstrating that the improvement in muscle energetics by exercise training correlated with improvement in endothelial function, supported a role for endothelial dysfunction in limiting skeletal muscle blood flow during exercise, although reversal of endothelial dysfunction did not return exercise capacity to normal [18]. This implies that whilst having an important role, other factors, such as muscle changes, are equally or more important.

Our observations are consistent with those of Hanada et al., who showed that PCr recovery was dissociated from oxygen kinetics suggesting that oxygen delivery was not the limiting step [19]. Our observation of an increase in PCr utilization during exercise with ascorbic acid as well as increased ATP synthesis from glycolysis is surprising. The lack of effect on PCr re-synthesis after exercise suggests that mitochondrial oxidative metabolism was not affected. There was a trend towards an improvement in PCr recovery following exercise with ascorbic acid although this was non-significant. There are several possible explanations for these findings. Local effects of nitric oxide on muscle metabolism are complex and high levels of iNOS are associated with a reduced peak VO2, probably via inhibition of mitochondrial creatinine kinase expression [20,21]. Thus, increased nitric oxide bioavailability might be associated with impaired skeletal muscle energetics as seen in our study. The effects of ascorbic acid on iNOS are complex. One study has suggested that ascorbic acid reduces iNOS expression [22] but other studies have shown that there is a dose-dependent effect with low doses of ascorbic acid worsening the effects of reactive oxygen species in L6 muscle cells while high dose ascorbic acid has the opposite effects [23].

4.1. Study limitations
Whilst we have previously shown improvements in endothelial function in conduit arteries of patients with heart failure, little is known of the effect of ascorbic acid on resistance vessels which are likely to be a more important determinant of local oxygen delivery to skeletal muscle. We are not aware of any studies examining the effect of chronic oral ascorbic acid on resistance vessel endothelial function in patients with CHF although intra-arterial ascorbic acid (24 mg/min) improved resistance vessel endothelial function in subjects with hypertension [24].

It is possible that a beneficial effect of ascorbic acid might have been seen if therapy had been maintained for a period of longer than 1 month. We used this time scale because we [12] and others [10] have reported improvements in endothelial function within 1 month of commencing ascorbic acid and because endothelial function and exercise capacity improved within 1 month following commencement of ACE inhibitor therapy in patients with CHF [25]. We did see a significant effect of vitamin C on skeletal muscle metabolism in this study period, albeit a negative one. As we did not measure nitrate/nitrite so we can only speculate on changes in NO in the muscle.

In summary, ascorbic did not improve skeletal muscle metabolism despite improving endothelial function. In fact, ascorbic acid increased phosphocreatine utilization during exercise, possibly because of negative effects of NO derived from iNOS on muscle metabolism. Exercise capacity is closely correlated with mortality, and is an important treatment goal in its own right, but these findings suggest that high-dose ascorbic acid may be deleterious rather than beneficial in CHF.


   Acknowledgements
 
This study was supported by funding from the British Heart Foundation.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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