Chronic Pantothenic Acid Supplementation Does Not Affect Muscle Coenzyme a Content Or Cycling Performance

Applied Physiology, Nutrition, and Metabolism

Chronic pantothenic acid supplementation does not affect muscle coenzyme A content or cycling performance

Journal: Applied Physiology, Nutrition, and Metabolism

Manuscript ID apnm-2020-0692.R2

Manuscript Type: Brief communication

Date Submitted by the 09-Oct-2020 Author:

Complete List of Authors: Whitfield, Jamie; Australian Catholic University, Mary MacKillop Institute for Health Research Harris, Roger; Junipa Ltd Broad, Elizabeth; Australian Institute of Sport Patterson, DraftAlison; Australian Institute of Sport Ross, Megan; Australian Catholic University, Mary MacKillop Institute for Health Research; Australian Institute of Sport Shaw, Gregory; Australian Institute of Sport Spriet, Lawrence; University of Guelph, Human Health and Nutritional Sciences Burke, Louise; Australian Catholic University, Mary MacKillop Institute for Health Research; Australian Institute of Sport

Novelty bullets: points that Supplementation with pantothenic acid for 16-wk had no effect on summarize the key findings in skeletal muscle CoASH or acetyl-CoA content, As a result, exercise the work: performance in trained male cyclists was unaltered

exercise performance < exercise, skeletal muscle < muscle, coenzyme Keyword: A, acetyl-CoA, skeletal muscle metabolism < metabolism, ergogenic aids < athlete performance

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

Chronic pantothenic acid supplementation does not affect muscle coenzyme A content or cycling performance

Jamie Whitfield1, Roger C. Harris2, Elizabeth M. Broad3, Alison K. Patterson3, Megan L. R. Ross1,3, Gregory Shaw3, Lawrence L. Spriet4, and Louise M. Burke1,3

1Exercise and Nutrition Research Program, Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, Australia. 2Junipa Ltd, Newmarket, Suffolk, United Kingdom 3Australian Institute of Sport, Belconnen, Australia 4Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada

Corresponding Author:

Jamie Whitfield, PhD Exercise and Nutrition Research ProgramDraft Mary MacKillop Institute for Health Research Australian Catholic University Melbourne, Australia

Phone: +61 3 9230 8252 Fax: +61 3 9663 5726 E-mail: [email protected]

Abstract: This study determined if supplementation with pantothenic acid (PA) for

16-wk could increase skeletal muscle coenzyme A (CoASH) content and exercise performance. Trained male cyclists (n=14) were matched into control or PA (6 g·d-

1) groups. At 0, 4, 8 and 16-wk, subjects performed an incremental time to exhaustion cycle with muscle biopsies taken prior to and following exercise.

Prolonged PA supplementation did not change skeletal muscle CoASH and acetyl-

CoA contents or exercise performance.

 Supplementation with pantothenic acid for 16-wk had no effect on skeletal muscle CoASH and acetyl-CoA content or exercise performance in trained male cyclists.

Keywords: exercise performance;Draft skeletal muscle; coenzyme A, acetyl-CoA

Introduction

Coenzyme A (CoASH) is an indispensable cofactor in living organisms and is

involved in over 100 different reactions in intermediary metabolism (Leonardi et

al. 2005). CoASH is required in the cytoplasm of skeletal muscle for the activation

of fatty acids (FA) for subsequent storage as intramuscular fat or transport into the

mitochondria. Within the mitochondria, CoASH is required as a substrate for the

activation of FA via the carnitine palmitoyl II reaction, the production of acetyl-

CoA via both -oxidation and pyruvate dehydrogenase (PDH) and succinyl-CoA

via -ketoglutarate dehydrogenase in the tricarboxylic acid (TCA) cycle, and as a Draft substrate for the reversal of acetylcarnitine to acetyl-CoA when exercise ceases or

acetyl-CoA provision wanes late in exercise.

Despite the central role this cofactor plays in both carbohydrate (CHO) and FA

metabolism, total skeletal muscle concentrations of CoASH are <100 µmol·kg-1 dry

weight (dw) (Putman et al. 1998). In contrast carnitine, which serves to buffer

acetyl groups and maintain a viable pool of free CoASH at the onset of and

throughout exercise (Stephens et al. 2007), has an intracellular concentration of

~20,000 µmol·kg-1 dw (Putman et al. 1998). Estimates suggest that if supported

solely by free CoASH, flux through PDH during the onset of high-intensity

exercise would see the entire pool acetylated within ~1 s (Constantin-Teodosiu et

al. 1991). It has also been demonstrated that muscle [acetyl-CoA] increases and

[free CoASH] decreases (while [combined CoASH] is constant) during exercise at

4 varying intensities (Howlett et al. 1998). Accordingly, it has been proposed that

CoASH may limit both fat and CHO oxidation, and therefore exercise capacity and performance.

Research suggests that the 5-step biosynthesis of CoASH from pantothenic acid

(PA, also known as pantothenate or vitamin B5) and cysteine may be limited by PA availability (Robishaw and Neely 1985). However, studies of PA supplementation have failed to demonstrate increased resting skeletal muscle [CoASH] or improve metabolic control as determined by reductions in phosphocreatine utilization, glycogen depletion and lactate accumulation in blood and muscle in PA vs. Draft placebo groups (Webster 1998; Wall et al. 2012). Notably, these studies have involved low (< 2 g·d-1) PA doses and a short 7-d supplementation period.

Given the lack of investigations of the regulation of PA transport into skeletal muscle, it is possible that supplementation for longer periods and/or higher doses is required to increase CoASH content. We therefore examined whether chronic supplementation (16-wk) with a high daily PA dose could increase muscle free

CoASH and acetyl-CoA contents and exercise capacity in well trained cyclists.

Methods

Subjects

Fourteen healthy male competitive cyclists (30.7 ± 7.5 y; 78.0 ± 15.8 kg; 푽 O2peak

58.9 ± 8.1 ml·min-1·kg-1) currently undertaking > 10 h cycling per week completed

this study. Ethical clearance was obtained from the Australian Institute of Sport

(ACT, Australia) and the University of Guelph (ON, Canada) human research

ethics committees and all procedures conformed to the Declaration of Helsinki.

Subjects were informed of the nature and risks of the study before written and oral

consent was obtained. Prior to the study, subjects were screened with a medical

questionnaire to confirm the absence of injury or illness.

Study Overview

We used a matched group design to investigate the effects of 16-wk of supplementation with large (6 g·dDraft-1) doses of PA on muscle and exercise capacity. Given the large participant commitment (16-wk of training and 4 testing protocols

involving muscle biopsies) and the focus on exercise muscle measurements, we

compared outcomes to a control (CON) group matched for training.

Prior to the first experimental trial, subjects performed a 60 s/stage progressive

maximal exercise test on a cycle ergometer (Lode Excalibur Sport, Groningen, The

Netherlands) to determine 푽 O2peak. Following a 5 min warm-up at 150 W, the test

protocol started at 175 W and increased 25 W every 60 s until volitional exhaustion.

The results of this test and information on current training-loads were used to pair-

match subjects prior to being randomly assigned to the CON or PA groups.

Subjects completed a 180 s/stage incremental cycling time to exhaustion (TTE) test

at baseline within 7 d to determine 푽 O2peak, maximal fat oxidation (MFO) and the

%O푽 2peak where MFO occurred (FATmax) (Achten et al. 2002). Muscle biopsies were

6 collected from the vastus lateralis 30 min before and immediately following the

TTE. All subjects then commenced 16-wk of normal training, during which only the PA group undertook daily supplementation. The TTE was repeated at the same time of day after 4, 8 and 16-wk of normal training.

Supplementation

Immediately following the completion of baseline testing, the PA group (n=7) commenced supplementation with 6g·d-1 of a commercial PA product

(Thompson’s B5 500 mg, Integra Health Care, QLD, Australia). Intakes of 4-6 mg·d- 1 are suggested as Adequate IntakeDraft in Australia for healthy adults (NHMRC, Australia), while intakes of 10 g·d-1 over several months were not associated with side-effects (Leung 1995). Supplements were consumed as 0.5 g·h-1 for 12 h each day to optimize PA availability, with compliance determined from data recorded by pill-taking mobile applications (MedHelper, Earth Flare, http://medhelperapp.com, and Dosecast, version 7.06, Montuno Software, http://montunosoftware.com), and sent via e-mail to one investigator.

Experimental Trial Overview

Subjects followed a standardized diet and training schedule for 24 h prior to each experimental trial. Individualized menus were prepared using FoodWorks

Professional Edition (Version 6.0, Xyris Software, Brisbane, Australia). Pre- packaged meals and snacks provided 7.9 ± 0.2 g·kg-1 body mass (BM) CHO; 1.5 ±

0.0 g·kg-1 BM protein; 1.5 ± 0.0 g·kg-1 BM fat. During this period, subjects refrained

from caffeine or alcohol and undertook light exercise which was repeated for

subsequent trials. Compliance to the diet and exercise protocol was determined

from a maintained checklist presented prior to each trial.

Subjects arrived at the laboratory following an overnight fast and consumed a

standardized ‘pre-race meal’ (2.0 ± 0.0 g·kg-1 BM CHO) and their final dose of PA 2

h prior to exercise. Local anesthesia (2-3 ml of 1% Xylocaine (Lignocaine)) was

administered to the skin, subcutaneous tissue and fascia of the vastus lateralis in

preparation for muscle biopsies. Two sites on the same leg (~3 cm apart) were

prepared for each trial, with the contralateral leg sampled during the subsequent Draft trial. Muscle biopsy samples were collected at 30 min prior to and immediately

following the TTE, using a 5-mm Bergstrom needle modified with suction (Evans

et al. 1982).

Subjects started cycling at 95 W with an increase in power of 20 W every 180 s

until volitional exhaustion to provide a measure of exercise capacity (TTE). Expired

gases were collected every 30 s and concentrations of O2 and CO2 (AEI

Technologies, Pittsburgh, PA) and the volume of air displaced were quantified for

푽O2 measurement. 푽 O2peak was determined towards the end of the TTE, when

subjects met specific criteria, including volitional exhaustion, respiratory exchange

-1 -1 ratio >1.10 and a plateau in O2 consumption (< 2 ml·kg ·min ). Substrate oxidation

was determined from respiratory gas collected over the last 90 s of every workload

8 and used to determine MFO and Fatmax. Experimental trials were replicated after 4,

8 and 16-wk of habitual exercise training and diet, and PA supplementation.

Muscle Analyses

Muscle samples were immediately snap frozen in liquid N2 and stored at

80°C, before being freeze-dried, dissected free of visible blood and connective tissue, and powdered for metabolic analyses. Freeze-dried muscle (10-12 mg) was extracted with 0.5 M perchloric acid, containing 1 mM EDTA, and neutralized with

2.2 M KHCO3. The supernatant was used to measure [CoASH] and [acetyl-CoA] for the calculation of [combined CoA]Draft with radioenzymatic methods as previously described (Cederblad et al. 1990).

Statistical Analysis

Data, expressed as mean ± SD, were analyzed using two-way repeated measures ANOVA. If significance was detected, a Bonferroni post hoc was applied.

Significance was set at P < 0.05 with confidence intervals ≥ 95%. All graphs and statistical analyses were performed using GraphPad Prism 8.4.3.

Results

All 14 participants completed the study with the PA group showing >98% compliance to their supplementation protocol. There were no changes in skeletal muscle concentrations of CoASH (Figure 1A-B), acetyl-CoA (Figure 1C-D) or

combined CoA (Figure 1E-F) at any time point throughout the 16-wk

supplementation period when compared in the pre- or post-exercise situation.

PA supplementation had no effect on 푽 O2peak, MFO, TTE over time or

compared to the CON group (Table 1). There was a main effect of treatment for

Fatmax in the PA group vs. CON, however, post hoc testing revealed this only

occurred at Week 4. 푽 O2peak did not change in either group over the 16-wk,

indicating a stable state of training.

Discussion

This study demonstrated that Draftchronic supplementation with a novel protocol

of high PA doses (6 g·kg-1) spread in multiple daily intakes did not increase

skeletal muscle CoASH availability and failed to improve fat oxidation or maximal

exercise capacity. These findings are in agreement with previous studies which

used short (7-d) supplementation periods with substantially lower doses (1.5-1.8

g·kg-1) (Webster 1998; Wall et al. 2012). While there were no statistically significant

changes detected in any of our skeletal muscle measurements, we did observe a

main effect of treatment for MFO in the PA group, which had a moderate effect

size. It therefore remains possible that we are underpowered to detect long-term

changes in muscle metabolite concentrations as well as the associated changes in

exercise capacity and fuel selection. However, the sample size used in this study

(n=7 per group) was based upon previous work performed on PA supplementation

(Webster 1998; Wall et al. 2012), as well as studies on other supplements (e.g.,

10 chronic L-carnitine) that have successfully altered muscle metabolite concentrations, fuel selection and exercise performance in humans (Wall et al.

2011). Given this, and the “hammer” approach of chronic supplementation with high daily dosages, it is currently unclear why skeletal muscle CoASH content was not increased, especially as similar strategies involving creatine and β-alanine have previously proved effective (Harris et al. 1992, 2006).

While the regulation of skeletal muscle PA uptake remains unexplored,

previous studies have demonstrated similar Km uptake values (7-17 µM) in a variety of tissues including heart (Beinlich et al. 1990), liver (Smith and Milner Draft 1985), and kidney (Karnitz et al. 1984). No plasma measurements were made in the current study, however previous work estimated that supplementing with 500 mg

3x·d-1 would result in an ~3-fold increase in plasma concentrations (Wall et al.

2012) above the normal PA baseline of ~7 µM (Eissenstat et al. 1986). Therefore,

assuming the Km for PA uptake in skeletal muscle is similar to other tissues, our consistently higher sustained dose should have been sufficient to load the muscle.

This suggests, much like taurine (Spriet and Whitfield 2015), that transport/utilization of PA in skeletal muscle is highly regulated and warrants further investigation.

Free CoASH concentrations only decreased by a small amount (~10 µmol·kg-

1·dw-1) following incremental exercise, suggesting that muscle has been designed to maintain sufficient free CoASH to handle the metabolic needs, even when

acetyl-CoA increased from ~7 to ~20 µmol·kg-1·dw-1. This suggests a feedback

system that limits further PA entering the muscle and being converted into free

CoASH.

Conclusion

This study demonstrated that 16-wk of high dose PA supplementation was

unable to increase skeletal muscle CoASH content. Consequently, exercise

metabolism and capacity were unaltered in endurance trained cyclists.

Funding: This study was funded Draftby a Big Ideas grant from the Australian Institute

of Sport’s High Performance Research Program to L.M. Burke.

Acknowledgments: The significant technical assistance of Dr. Kimberley Wells is

gratefully acknowledged.

Conflicts of Interest: none.

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Figure and Table Legends

Figure 1. Skeletal muscle coenzyme A (CoASH), acetyl-CoA and combined CoA contents at rest (pre-exercise) and following a cycling test (post-exercise) across 16-wk of training and pantothenic acid (PA) supplementation or control (CON). Data are means ± SD, n=7 per group.

Table 1. Metabolic and exercise responses to 16-wk of pantothenic acid (PA) supplementation or control (CON) in well trained cyclists.

Draft

Time Point PA CON

-1 -1 푉O2peak (ml·min ·kg ) 59.3 ± 6.2 55.9 ± 8.3

MFO (g·min-1) 0.36 ± 0.17 0.32 ± 0.11 Week 0 Fatmax (% 푉 O2peak) 61 ± 4 51.5 ± 8

TTE (mm:ss) 35:43 ± 08:57 35:47 ± 04:13

-1 -1 푉O2peak (ml·min ·kg ) 60.1 ± 5.8 57.2 ± 8.8

MFO (g·min-1) 0.33 ± 0.18 0.29 ± 0.13 Week 4 Fatmax (% 푉 O2peak) 60 ± 9* 48 ± 7

TTE (mm:ss) 35:56 ± 09:41 36:43 ± 03:50

-1 -1 푉O2peak (ml·min ·kg ) 60.0 ± 5.6 57.5 ± 9.5

MFO (g·min-1) 0.30 ± 0.11 0.28 ± 0.11 Week 8 Fatmax (% 푉 O2peak) Draft58 ± 10 48 ± 5 TTE (mm:ss) 35:06 ± 09:43 37:04 ± 04:01

-1 -1 푉O2peak (ml·min ·kg ) 61.6 ± 6.3 57.8 ± 8.9

MFO (g·min-1) 0.35 ± 0.20 0.32 ± 0.11 Week 16 Fatmax (% 푉 O2peak) 57.5 ± 9 50 ± 11

TTE (mm:ss) 34:16 ± 09:07 36:43 ± 04:48 Time: p= 0.994, ηp²=0.002 -1 -1 푉O2peak (ml·min ·kg ) Treatment: p= 0.558, ηp²=0.007 Interaction: p= 1.000, ηp²=0.0002 Time: p= 0.771 ηp²=0.07 MFO (g·min-1) Treatment: p= 0.956, ηp²=0.001 ANOVA Interaction: p= 0.466, ηp²=0.22 Results Time: p= 0.782, ηp²=0.06 Fatmax (% 푉 O2peak) Treatment: p= 0.011, ηp²=0.68 Interaction: p= 0.746, ηp²=0.11 Time: p= 0.606, ηp²=0.05 TTE (mm:ss) Treatment: p= 0.735, ηp²=0.06 Interaction: p= 0.271, ηp²=0.02

MFO, maximal rate of fat oxidation; Fatmax, %푉 O2peak at which MFO occurred; TTE, incremental time to exhaustion cycling test. Data are means ± SD, n=7 per group. Significant difference between groups at respective time-point, *(p<0.05).