Effect of Quercetin Complex Supplementation on Indirect Markers of Exercise-Induced Muscle Damage
Mc Auley, C.J. (2004)
Table of Contents / Abstract / Introduction / Literature Review / Methods
Results / Discussion / References
This study investigated the influence of Quercetin/Vitamin C complex supplementation on functional, biochemical,
anthropometrical and subjective indices of delayed onset muscle soreness (DOMS).
The test subjects comprised 4 volunteers whom were randomly assigned to either the supplement or placebo group
following a double blind protocol. Pre-supplementation, subjects were measured for upper leg circumference (CIR) on
their non-dominant leg, and a capillary blood sample was obtained to determine a baseline measure for creatine kinase
(CK) activity. Subjects then underwent a familiarisation session on an isokinetic dynamometer (Cybex Norm™), to
determine baseline measurements for peak torque (PT) in their non-dominant knee extensors, performing 8 repetitions -
3 sub-maximal and 5 maximal - using the CON/ECC test protocol at an angular velocity of 90ºsec-1
(1.57rad sec-1). Daily (Quercetin/Vit C 1000mg or placebo) supplementation commenced 14d pre-exercise and
continued 5d post-exercise. Subjects performed two bouts of exercise (downhill running) ~8w apart. Immediately
pre-exercise and post-exercise (1h, 24h, 48h & 96h), subjects were assessed for CIR, CK, PT, and muscle tenderness (MT).
The subjects were then reassigned to the opposite treatment group after a period of ~8 to 10 weeks, supplementation
followed the original time scale. Subjects were retested following a second bout of downhill running ~10-12w after the
first. The study found no significant difference (p>0.05) for any of the parameters tested, between the treatment and
the placebo groups. It can therefore be concluded that Quercetin Complex supplementation, daily at
1000mg, has no significant effect on the indirect markers of muscle damage tested for.
Introduction
Strenuous aerobic exercise utilises significantly greater quantities of oxygen intramuscularly than during rest (Keul & Doll, 1972), with a concomitant increases in the production of oxygen intermediate free radicals (Alessio et al., 1988). Secondary free radical production can also occur post-exercise, in response to neutrophil and phagocyte infiltration as a results of exercise-induced muscle damage (Zebra et al., 1990; Pizza et al., 2002), induced by the inflammatory response (Camus et al., 1993). These highly reactive oxygen species (ROS) are implicated in the destruction of cellular material (Kanter et al., 1993; Reznick et al., 1992; Ebbeling & Clarkson, 1989) if not neutralised by the cells inherent antioxidant defences that include enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidases, and nonenzymatic substance such as vitamins and glutathione (Sen, 1995). Many studies have detected and quantified the generation of free radicals by skeletal muscle (Balon & Nadler, 1994; Diaz et al., 1993; Reid et al., 1992).
It is generally accepted (Alessio & Goldfarb, 1988; Alessio, 1993; Kanter & Eddy, 1992; McBride et al., 1988) that certain vitamins within the body, particularly vitamin C and E, play important roles as antioxidants by quenching or neutralising free radicals. Vitamin E has been highlighted as being unique among antioxidant vitamins, due to its status as a membrane bound antioxidant and its mechanism of action (Bucci, 1995). Several studies have investigated the effect of supplementation with these vitamins on reducing the magnitude of free radical production and muscle damage in response to exercise (Goldfarb et al., 1989; Helgheim et al., 1979; Kanter & Eddy, 1992; Kanter et al., 1993; McBride et al., 1988). However, the results of these studies have proved inconclusive. Several studies have reported positive effects of vitamin supplementation on reducing the products of lipid peroxidation (Goldfarb et al., 1989; Kanter et al., 1993; McBride et al., 1998), whilst others have reported no effect (Helgheim et al., 1979; Kanter & Eddy, 1992). To date the author knows of only one other study that has examined the effect of a flavonoid containing vitamin mixture supplement on the markers of exercise-induced muscle damage (Phillips et al., 2003). Phillips et al. (2003) reported an attenuated inflammatory response accompanied by an increase in the molecular markers of muscle damage CK and lactate dehydrogenase (LDH). Further research with these natural antioxidant compounds is clearly required.
The purpose of this study was to determine whether supplementation with a Quercetin Complex (QC) - 14d pre-exercise and 5d post-exercise - would have a positive effect on reducing the functional, anthropometrical, biochemical and subjective indices of exercise-induced muscle damage. This will be achieved through the random assignment of the test subjects to either the supplement or placebo group following a double blind protocol. Subjects will then receive either the supplement or the placebo for 14 days, before performing a bout of damage-inducing eccentric exercise - a downhill running protocol that is performed at a decline of 10% and an exercise intensity of 85% of their age adjusted maximum heart rate (AAMHR) - corresponding to ~70VO2max. The protocol was designed to be simultaneously highly aerobic whilst utilising an exercise modality with a functional eccentric bias. This exercise protocol should ensure heightened free radical production during the exercise performance, and post-exercise as a result of the inflammatory response initiated by eccentric exercise-induced muscle damage.
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Literature Review
Delayed Onset Muscle Soreness
It is well established that delayed onset muscle soreness (DOMS) occurs as a results of strenuous, unaccustomed, or prolonged exercise (Armstrong, 1984; Schwane et al., 1983). This type of pain is experienced in response to palpation or motion and the tenderness is frequently localised in the region of the myotendinous junction, although it can also be generalised throughout the body of the muscle (Armstrong, 1984). The soreness initiates 8 or more hours post-exercise, reaches maximal level within 1 to 3 days, then gradually subsides (Bobbert et al., 1986).
DOMS has been shown to be correlated with temporary changes in muscle function, including decreased force production and reduced range of motion (Byrne & Easton, 2002a; Byrne & Easton, 2002b; Clarkson et al., 1992; Eston et al., 1996), as well as physiological changes such as altered muscle volume (Bobbert et al., 1986), increased concentrations of muscle enzymes (Komulainen et al., 1994; Pizza et al., 1999; Shwane et al., 1983), and by-products of lipid peroxidation being found in blood plasma (Alessio, 1993).
DOMS is routinely measured as an indirect marker of exercise-induced muscle damage (Friden et al., 1986; MacIntyre et al., 1996; Thomson et al., 1999 ). Muscle soreness has been quantified subjectively using various scales (Schwane et al., 1983), and the use of a myometer to determine pressure pain thresholds (Dannecker et al., 2002; Newham et al., 1987). Tenderness has been shown to be greatest in the gluteus maximus, rectus femoris, vastus medialis, vastus lateralis, tibialis anterior, gastrocnemius and biceps femoris when assessed 2 days after downhill running (Eston et al., 1994). Soreness has been shown to last several days then gradually diminish following a bout of eccentric exercise with the elbow flexors (Nosaka & Clarkson, 1995).
Eccentric exercise and muscle damage
Eccentric exercise has been highly implicated in the development of exercise-induced muscle damage (Armstrong et al., 1983; Miles & Clarkson, 1994). Eccentric exercise has a lower metabolic cost than concentric exercise, utilising fewer motor units for a given load (Bigland-Ritchie & Woods, 1976), which results in an elevated level of tension within the active skeletal muscle fibres (Davies & White, 1981). Downhill running has been demonstrated to accentuate the eccentric activity of the hip and knee extensors as well as the ankle dorsiflexors - particularly during the stance phase (Eston et al., 1994).Direct evidence of eccentrically induced muscle damage has been verified through histological and ultrastructural examination (Friden et al., 1986; Jones et al., 1986). The performance of a prior bout of eccentric exercise has been shown to illicit a protective effect on subsequent bouts of eccentric activity (Nosaka & Clarkson, 1995; Schwane & Armstrong, 1983; Westerlind et al., 1992). Further studies have indicated that the protective effect is not dependant on the initial bout causing appreciable damage (Brown, et al., 1997; Clarkson & Tremblay, 1988), and two studies have indicated that the performance of a repeated bout, prior to complete recovery, did not exacerbate existing symptoms (Mair et al., 1994; Nosaka & Clarkson, 1995).
Secondary muscle damage, as a result of neutrophil infiltration into previously damaged muscle tissue, and a further release of oxygen centred free radicals during phagocytosis may exacerbate existing muscle damage (Pizza et al., 2002).
Aerobic exercise and free radical production
Strenuous aerobic exercise has the capacity to increase whole body oxygen uptake 20-fold, and oxygen consumption within active muscle fibres by up to 200-fold over resting levels (Keul & Doll, 1972). A substantial increase in the production of oxygen free radicals is thought to result from mitochondrial metabolic leaks (Jenkins, 1993), with a predicted 2-5% of electron flux through the cytochrome chain resulting in the formation of the superoxide radical (Boveris et al., 1972).
Current literature on the relationship between exercise and free radical production is both limited and inconclusive. A study by Dillard et al. (1978) supports the relationship between exercise and free radical production. Whereas, Viinkka et al. (1984) found no relationship between exercise and free radical production - as indicated by altered plasma peroxide levels. Lovlin et al. (1987) suggested that maximal exhaustive exercise (~100%VO2max) induced significant quantities of free radical generation, with a concomitant increase in plasma MDA - indicative of lipid peroxidation and therefore muscle damage, whilst short periods of submaximal exercise ( less than 70% VO2max) may actually inhibit it. Although, these anomalies in findings may be due more to variations between protocols regarding exercise modality, intensity and time.
Many studies have analysed the protective role of antioxidant vitamin supplementation on the indices of exercise-induced muscle damage, with inconclusive results. Several studies have reported the positive effects of Vitamin E supplementation on reducing markers of lipid peroxidation (Kanter, 1994; Sen et al., 1997; Oostenburg et al., 1997), and protecting against oxidative stress (Sen et al., 1997; Sacheck et al., 2003). Itoh et al. (2000) reported a reduction in the leakage of the muscular enzymes CK and LDH following supplementation with vitamin E. However, the results of the study by Itoh et al. (2000) are equivocal, due to the small sample size (~7 in each group) and the large inherent variation commonly found between subjects in CK and LDH response. In contrast, Dawson et al (2002) investigated the effects of vitamin C and E supplementation, on the biochemical and ultrastructural indices of muscle damage following a 21 km run, and found no significant differences between the vitamin and placebo group. Similarly, Beaton et al. (2002) concluded that Vitamin E supplementation had no effect on the indices of eccentrically biased contraction-induced muscle damage. One of the limitations of the previous studies concerning vitamin supplementation on the markers of muscle damage is that most of these studies lack the evaluation of the antioxidant status of the participants. In participants with an antioxidant vitamin deficiency, supplementation could be expected to have a greater effect on reducing these markers of exercise-induced muscle damage.
A study by Ohshima et al. (1998) indicated that flavonoids have antioxidant properties, but in the presence of nitric oxide (NO) some act as pro-oxidants. Flavonoids are found in plants and plant products and their average daily consumption was estimated to be around 23mg per day in the Netherlands (Keli et al., 1996). Movileanu et al. (2000) proposed that the optimised radical scavenging activity of quercetin might be correlated with its translocation in the polar part of the lipid bilayer.
Creatine kinase activity
It is well documented that CK activity increases in response to muscle fibre disruption as a consequence of exercise-induced muscle damage (Blais et al., 1999; Itoh et al., 2000; Hortobagyi et al., 1998). This cytoplasmic muscular enzyme, that is not normally able to cross the cell membrane, enters the general circulation via the lymph fluid as a consequence of skeletal muscle fibre necrosis (Salminen, 1985).
Many studies have measured CK activity as an indirect molecular marker of exercise-induced muscle damage (Byrnes et al., 1985; Brown et al., 1997; Dawson et al., 2002; Nosaka et al., 1991; Hilbert et al., 2003; Hortobagyi et al., 1998; Totsuka et al; 2002). Following downhill running, CK activity has been shown to increase significantly 6h post-exercise (Maughan et al., 1989), peak between 24 (Schwane et al., 1983; Maughan et al., 1989; Sacheck et al., 2003) and 48 h (Eston et al., 1996), and return toward baseline values by 72h post-exercise (Schwane et al., 1983; Sacheck et al., 2003; Eston et al., 1996), although may remain elevated in high responders even after 72 h (Maughan et al., 1989).
Normal CK values in the resting condition have been reported at around
100 IU·l-1 (Schwane et al., 1983; Totsuka et al., 2002). Schwane et al.
(1983) demonstrated mean peak CK values of ~500 µ·l-1 at 24h post-exercise,
after performing 45 mins of downhill (-10 ) treadmill running at ~57% of
VO2max, in their study that utilised 7 moderately
aerobically active male volunteers (mean age (SEM) = 20 ± 1yr). Similar
findings were reported by Eston et al. (1996), their subjects demonstrated
mean peak CK values of ~600 µ·l-1 at 48h post-exercise
- CK values were not recorded at 24h. Although their protocol utilised a
40min intermittent bout of 5 x 8 min, it was performed at a higher relative
intensity, corresponding to a treadmill speed that elicited 80% of their
subject's age-predicted maximum heart rate during the downhill run. In contrast,
Maughan et al. (1989) reported mean peak CK values of ~300 µ·l-1
at 24h post-exercise, following a similar treadmill running protocol, with
16 healthy and normally physical active young males. The higher CK values
reported in some studies may have been due to differences in the protocols
used, although it seems just as likely due to the extensive intersubject
variability that is found in CK response, this has previously been demonstrated
by Maughan (1989) and several other studies (Newham et al., 1983a; Nosaka
& Clarkson, 1996a).
The inflammatory response and post-exercise swelling
Repetitive eccentric contractions can result in severe morphological damage to the active muscle fibres. This can result in significant numbers of muscle fibres becoming necrotic post-exercise, and the initiation of the inflammatory response (Jones et al, 1986). This is supported by the findings of other studies (Ebbeling & Clarkson, 1989; Friden & Leiber, 1992), which reported that the extent of the ultrastructural evidence to support muscle damage becomes greater well after the initial exercise-induced muscle damage. Swelling is a classic symptom of inflammation (Smith, 1991), and has been quantified as an enlargement in muscle circumference (Clarkson et al., 1992; Nosaka & Clarkson, 1996b). Bobbert et al. (1986) proposed that the formation of oedema was responsible for an increase in the experimental lower leg volume, following repeated plantar and dorsi flexion of the gastrocnemius during the raising and lowering of the body.
The extent of post-exercise muscle oedema is commonly determined by measuring the circumference of the affected limb (Eston et al., 1996; Chen, 2003; Beaton et al., 2002; Nosaka & Clarkson, 1996b). Several studies have measured changes in upper arm circumference as an indicator of post-exercise muscle damage and have reported significant differences in circumference in response to forced lengthening during percutaneous stimulation (Nosaka et al., 2002), and maximal voluntary eccentric contractions (Chen, 2003; Nosaka et al., 2001). In contrast, Beaton et al. (2002) reported no difference in mid-thigh circumference immediately post-exercise, or at 2, 4 or 7 days post-exercise, following a bout of 240 maximal eccentric contractions of the knee extensors. These conflicting findings may have been due to methodological differences in the exercise protocols, although all the studies utilised maximal eccentric contractions to induce muscle damage. Beaton et al. (2002) provided evidence of significant post-exercise ultrastructural damage via muscle biopsy examination, it appears probable that this measure of oedema - changes in limb circumference - is possibly not sensitive enough for detecting oedema in the quadriceps muscle group.
Strength decrements as a result of exercise-induced muscle damage
One of the most noticeable effects of exercise-induced muscle damage as a consequence of downhill running is the acute strength loss that can last for several days post-exercise (Clarkson et al., 1992; Clarkson & Tremblay, 1988; Sargeant & Dolan, 1987; Eston et al, 1994). Strength is typically reduced immediately post-exercise, and thereafter follows a linear recovery over hours (Davies & White, 1981; Newham et al., 1983b), days (Hortobagyi et al., 1998; Byrne & Eston, 2002) or in excess of a week (Clarkson et al., 1992).
It has been proposed that eccentric exercise selectively recruits or damages type II muscle fibres (Byrne & Eston, 2002; Jones et al., 1986 ) and a prolonged decrease in muscle glycogen is normally observed (Costill et al., 1990). The performance of maximal intensity exercise and the rapid decline in force and power output this produces, is thought to be linked to the activation and fatigue of type II muscle fibres, as a result of the increased demand and regeneration of Adenosine Triphosphate (Woledge et al., 1985). Newham et al. (1988) proposed that the decrements in knee extensor muscular strength - associated with downhill running - were related more to the working range of the muscle and the magnitude of work done at its longer length, and reported similar findings for the elbow flexors.
The rapid recovery of concentric strength is in contrast to the prolonged recuperation of eccentric strength (Clarkson & Tremblay, 1988). Peak torque has been measured in numerous studies post-exercise (downhill running) to quantify the loss in muscular function (Clarkson et al., 1992; Clarkson & Tremblay, 1988).
Overview of study
Aerobic exercise has been shown to increase the production of oxygen centred free radicals, which are implicated in the destruction of cellular material through oxidation. Many substances including vitamins and bioflavonoids have been demonstrated to have antioxidant properties (Halliwell & Gutteridge, 1999). The water-soluble vitamin C has been reported as reducing the parameters of exercise-induced oxidative stress following dietary supplementation (Dawson et al., 2002). Studies have indicated that quercetin has both antioxidant and anti radical properties, due to its molecular structure and its translocation into the lipid membrane (Movileanu et al., 2000). Major radical formation starts in the lipoidic layers of the cell membrane and is transferred to the aqueous compartments (Karlson, 1997). Therefore supplementation with a complex that includes both a hydrophilic and lipophilic antioxidant may act to optimise radical scavenging, by exploiting the lipid-soluble and water-soluble antioxidant capabilities of both systems. To date the author knows of only one other study that has examined the effect of supplementation with a bioflavonoid and vitamin mixture on reducing the markers of exercise-induced muscle damage.
The variables that will be assessed include functional strength, muscle tenderness, muscle oedema and CK activity - a molecular marker of muscle damage. Functional strength will be assessed by using a dynamometer to measure the concentric and eccentric peak torque of the leg extensors in the non-dominant thigh. Muscle tenderness was measured using a digital algometer, and recording the amount of pressure that was required to illicit the onset of pain, measurements were taken at three mid-thigh sites. Muscle oedema will be assessed by measuring the circumference of the mid-thigh in the non-dominant leg, pre and post-exercise (1,24,48 & 96h) at the same location. The concentration of CK - the blood borne skeletal muscle specific enzyme - will be measured by collecting a capillary sample of whole fresh blood. This blood will be centrifuged to isolate the serum that will be later assayed to allow the calculation of CK activity.
To overcome the random sources of error in the experimental design, care was taken to ensure that all measurements were performed on or with the same equipment, and around the same time of day. Where applicable, the equipment was calibrated pre-testing (e.g. the cybex). The sites for thigh girth and algometer measurement were marked with a semi-permanent marker to facilitate accurate re-measurement. Subjects received the same instruction, motivation (verbal encouragement) and feedback (real time computer feedback) during all cybex testing, and the same researcher administered the tests.
The research hypothesis is that supplementation with quercetin complex will reduce the functional, molecular, anthropometrical and subjective indirect markers of muscle damage being assessed.
Methods
Subjects
The subjects comprised 4 sports science students who volunteered to participate in the study. Their characteristics are summarised in Table 1. All subjects were moderately active, although none participated in eccentrically biased exercise - e.g. downhill or fell running. Subjects were requested to maintain their regular activity patterns and levels throughout the course of the study, and to refrain from initiating new activities and training programmes. Subjects were required to abstain from strenuous physical activity for several days pre-exercise, and throughout the course of the post-exercise testing period. None of the subjects took any supplements or medication throughout the study period, including anti-inflammatory agents. The project was approved by the Napier University Ethics Board (Appendix A). The subjects were issued with a study outline guide (Appendix B) and the study was fully explained to them. All subjects gave their written informed consent (Appendix C) before participation.
Table 1. Characteristics of subjects (n = 4)
Characteristic X +/- SD
Age (years) 27.7 +/- 7.9
Height (cm) 170.9 +/- 8.2
Weight (kg) 68.6 +/- 12.6
85%AAHRmax (bpm) 163.5 +/- 6.7
Treadmill Speed (kph) 12.0 +/- 1.7
Supplement and Placebo Details
The supplement was a Quercetin Complex (Solgar, USA), which primarily comprises Quercetin (500mg) and Vitamin C (500mg), it also contained small amounts of other substances (Appendix D). Subjects were required to take 2 capsules per day to achieve the proposed dosage of 1000mg·d-1. In accordance with the manufacturers recommendations, subjects were directed to take 2 capsules with their main meal. The placebo comprised a capsule that was identical in size and taste to that of the supplement (VegicapsTM single"0"; Solgar, USA), which was filled with sucrose (table sugar). As the capsules were different in appearance (Figure 1), subjects received their capsules in an opaque plastic container and were instructed to take them blind.
Adherence to supplementation schedule and compliance with study requirements
Subjects were issued with a guide that outlined the nature and potential risks associated with the study. It also indicated the requirements of the study in terms of the subjects physical activity patterns and use of dietary supplements and anti-inflammatory medication. The guide also contained a supplement/placebo adherence chart, which subjects were required to tick each time they took the supplement/placebo.Pilot study
The pilot study was conducted with a subject who fulfilled all the criteria for inclusion in the study, but was unable to take part. The physical characteristics of the subject were: 28 yr of age, 78.7 kg of body mass and 174 cm height. The subject performed a continuous treadmill protocol, once a pre-determined exercise intensity had been achieved on a level gradient - a treadmill speed corresponding to 85% of their age adjusted HR max. 30 min on a decline of -10 .
Post-exercise (1,6,24,48 & 96h) the subject was verbally interviewed as to the nature, location and intensity of any DOMS present. The subject reported sensations of pain in the quadriceps, hamstring and calf muscles, particularly ~24 & 48h post-exercise. Symptoms of knee pain were also reported in response to the downhill running bout (30min at -10%), although the severity was rated as varying between moderate to mild.
Anthropometrical data
Body mass and height were determined with the subjects wearing normal exercise kit and without shoes. The time of these measurements was standardised for all subjects (around noon), to account for the normal diurnal variation that subjects exhibit (Eston & Reilly, 1996). Body mass was measured using beam scales (Weylux, UK). Height was established using the Leicester height measure (SECA Ltd, UK). Subjects were instructed to stand with an erect posture, with their head in the Frankfurt horizontal plane, and to distribute their weight evenly over both feet.
Thigh girth was measured at the thickest mid-thigh section of the non-dominant leg, with a flexible non-stretchable fibreglass tape (Bodycare, UK). The tape was applied to the appropriate site, and care was taken to ensure that the tape remained in physical contact with the skin without compressing the underlying tissues. To facilitate accurate re-measurement the skin was marked with a medium point semi-permanent black marker (Staedtler Lumocolor permanent, Germany) at the site of measurement. This was done prior to the first measurement, and remarked when the location marks became excessively light.
Blood sampling and analysis
Whole fresh blood was collected from a fingertip blood sample, using capillary puncture. The subject's hand was immersed in warm water for several minutes to encourage peripheral blood flow, before being removed and dried. The fingertips were wiped with an alcohol swab (Medi-Swab, UK) for pre-puncture site cleansing, and allowed to air dry. Capillary puncture was achieved using a medium-flow Lancet (GenieTM, BD Vacutainer Systems, USA), which was placed in contact with the skin then depressed, and the first drop of blood was wiped away with sterile gauze. The punctured finger was then gently and rhythmically massaged to encourage bleeding. Blood was collected using a plasma collection tube (MicrotainerTM, Becton Dickinson Vacutainer Systems, USA), which incorporates the anticoagulant additive lithium heparin at the optimal level for the specified volume of blood collected. The flo-top collector vent hole was navigated toward the blood drop until contact was made, allowing the blood to flow freely to the bottom of the collection vessel. Once the vessel was filled to the required level (~400IU), the tube was inverted ~ 8-10 times to facilitate thorough mixing of the whole blood with the lithium heparin additive and therefore assure anticoagulation, then refrigerated until centrifuging and separation of blood serum from red blood cells. Care was taken to avoid over or under filling the tube as this may result in clotting and/or erroneous test results, as the tubes contain additives in varying concentrations dependent upon their fill volumes and the required additive to blood ratio for the tube.
Capillary blood samples were obtained immediately pre and at 1, 24, 48 and 96h post-exercise. The samples were evenly loaded into a centrifuge (3K15, Sigma Laboratory Centrifuges, Germany), and centrifuged at 4,650rpm (relative g-value of 1,500) for 5 min to separate the red blood cells from the blood serum. The resultant blood serum was pipetted from the collection tubes into a plastic storage vessel, which was sealed and marked for identification purposes, then frozen at -20 °C until assayed.
Blood plasma was later measured for serum CK activity (U·l-1) using the rate of reaction assay developed by Swan & Wilkinson (1972). This method measures the rate of ATP formation, by reading the increase in extinction of NADPH at 340nm. The reagents were prepared prior to testing; the working solution comprised 1.2ml of reagent solution, 100ul NADP, 100ul of cysteine and 50ul of enzyme solution, which were pipetted into a 5ml tube and thoroughly mixed before being placed in a water bath at 37ºC for 10 minutes. The tubes were removed from the water bath and 50ul of serum was pipetted into each tube and then lightly shaken, before being decanted into a quart cuvette.
Six quart cuvette samples were loaded into a spectrophotometer (Beckman DV 7500, USA), which was set to an analytical wavelength of 340nm and a temperature of 37ºC. Samples were read for 0.5sec - at 30sec intervals over a period of 15min - using a 10mm light path. The assay results were printed out and serum CK activity (U·l-1) was calculated (Appendix E) using the formula below.
CK activity (U·l-1) = E340/min x total volume in cuvette (ml) = E340·min-1·4762
6.3 volume of serum taken (ml)
As the reaction rate may not exhibit linear qualities until ~ 6 to 8 mins after the addition of the serum the extinction rate was read over the 10-15min time period, which should correlate to the linear portion of the curve. All serum samples were analysed in duplicate, thereafter a mean CK value was calculated.
Exercise protocol
All subjects performed a continuous downhill running protocol, which simulated that used by Byrnes et al. (1985). Subjects were required to run for 30 min on a decline of -10 , once a pre-determined exercise intensity had been achieved on a level gradient - a treadmill speed corresponding to 85% of their AAHRM. As the test subjects exhibited a greater age range than those of Byrnes et al. (1985), the exercise intensity was correlated to an age adjusted maximum HR (Eston et al., 1996), rather than a fixed HR value. Subjects completed a familiarisation session to determine the treadmill speed that elicited a HR of ~85% of their AAHRM (220-age; ACSM, 1991). This speed was maintained for 5 min at a level gradient (0%), the treadmill slope was then declinated to -10 and the subjects continued to run for 30mins. The exercise protocol was designed to approximate the same relative workload for all the subjects.
The subjects were then reassigned to the opposite treatment group after a period of ~8-10 weeks, this allowed a suitable washout period and diminish any protective role elicited by the repeat bout effect. The performance of a prior bout of eccentric exercise has been shown to illicit a protective effect on subsequent bouts of eccentric activity (Nosaka & Clarkson, 1995; Schwane & Armstrong, 1983; Westerlind et al., 1992).. The longevity of the repeat bout effect has been shown to last several weeks (Ebbeling & Clarkson, 1989; Clarkson et al., 1992; McHugh et al., 1999), and possibly longer (Nosakaka et al., 2001; Nosaka & Clarkson, 1996). The subjects were required to perform a second bout of downhill running ~10-12 weeks after the firsts.
To minimise the risk of thermal stress several convection fan were positioned facing toward the subject. A large mat was placed behind the treadmill to reduce the chances of injury to the subject should they come of the back of the treadmill.
Muscle tenderness
Perceived tenderness was measured prior to testing to obtain a baseline value, immediately post-exercise, and at 24, 48 and 96h post-exercise. This was achieved using a digital algometer (Electronic Engineering Corporation, India). The site chosen corresponded to the midpoint of the anterior thigh - following the line of rectus femoris (Newham et al., 1983). This site was marked with a semi-permanent marker (Staedtler Lumocolor permanent, Germany) and two further sites were marked at ~2cm proximal and distal to the original one, also following the line of rectus femoris. The pressure that was required to initiate pain was observed and recorded for all three sites, this allowed a mean tenderness value to be calculated from the three measurements. Pressure measures were performed by the same person to ensure standardisation and reliability.
Isokinetic testing protocol
Subjects were tested for PT of the knee extensors in their non-dominant limb, using an isokinetic dynamometer (Cybex Norm ™, Finland). Pre-testing, subjects underwent a familiarisation session to facilitate the correct set-up of the dynamometer, this included ensuring correct joint alignment and allowing the subjects to become accustomed with the testing modality and the test protocol. The testing protocol consisted of performing 8 repetitions - 3 sub-maximal and 5 maximal - using the CON/ECC test protocol at an angular velocity of 90ºsec-1 (1.57 rad sec-1).
Pre-testing, the dynamometer was calibrated using the Cybex calibration software. The resistance arm was moved into a vertical position that corresponded with location marks on the dynamometer body. During assessment subjects were placed in a comfortable seated upright position. To minimise extraneous movement subjects were secured in position with the aid of a seat belt, thigh strap and lower limb strap. The chair settings were altered until the subjects lateral femoral epicondyle aligned with the axis of rotation of the dynamometer arm. The subject was instructed to flex and extend their knee to determine whether a comfortable and unrestricted range of motion could be obtained. Once this was achieved the following settings were taken: seat back angle, seat fore/aft position, seat back translation, resistance arm level and dynamometer height , rotation and angle were also recorded. These settings were recorded and saved in the Cybex software to facilitate retesting in a similar testing position throughout the course of the study. Gravity correction was obtained by having the subject extend their limb to the 45° position then relaxing it, allowing a gravity correction value to be obtained. Sub-sequent torque values were automatically adjusted by the Cybex software, taken into account the gravity correction value.
During testing, the cushion setting - which relates to the ends of ROM - was set to hard, this has the effect of reducing limb deceleration on the reciprocal motion (Taylor et al., 1991), and is suitable for subjects without injury. Subjects' were required to grip the handles at the side of the chair and were instructed to push maximally during the concentric phase and resist maximally during the eccentric phase (Figure 3).
Motivation and feedback were standardised for all subjects. Subjects' received verbal encouragement during testing and a computer monitor provided real-time performance feedback - allowing subjects to see the torque graphs from the previous repetitions. Baltzopoulos et al. (1991) showed that visual feedback can improve maximal torque generation during maximal isokinetic testing.
Statistical Analysis
Statistical analysis was performed on a personal computer, using the statistical
software package SPSS Version 11 (SPSS Inc., Chicago, IL, USA). Data were
analysed using a two-factor repeated measures of variance (ANOVA) to determine
the statistical significance of supplementation and time on the measured
parameters (Appendix F). When a significant F-ratio was detected, multiple
comparison were tested using a Scheffe post hoc test to detect the location
of any significant differences - this test protects against the experimentwise
error rate (type I error) unlike the standard t-test (Thomas & Nelson, 2001).
Statistical significance for all analyses was set at p < 0.05.
Results
As the digital algometer malfunctioned during the testing period this resulted in the incomplete collection of test data, and therefore no results regarding muscle tenderness are presented here.
The tests of within-subject's effects for CK activity revealed no significant
differences between the condition (p = 0.638), time (p = 0.543) or the interaction
between condition and time (p = 0.442). Even though the small sample size
(n = 4) makes establishing statistical significance less likely unless very
large differences exist, the resultant p values are still very high and
far removed from the significance level set (p < 0.05) . The between-subject
effects for serum CK activity were shown to be highly significant (p = 0.001),
this is in accordance with other studies that highlight the extensive intersubject
variability in CK response (Newham et al., 1983; Nosaka & Clarkson, 1996).
Fig 3. Comparison of CK activity between the supplement and placebo condition, immediately before (pre) and for up to 96h post-exercise. The values are mean ( SD).
The graph illustrating CK activity against time (Fig 3) appears to highlight a contrasting relationship between the supplement and placebo conditions and the resultant CK activity. The supplement rather than attenuate the CK response, would appear to have a negative influence in comparison to the placebo.
The magnitude of increases in the thigh girth between the supplement and placebo were not significantly different (p = 0.099). Analysis also revealed no significant differences for the within-subjects effects regarding time (p = 0.111), or the interaction between condition and time (p = 0.453). Although the results for within-subjects effects for condition and time were closer to the significance level set than the value for the interaction between condition and time. The tests for between-subject effects illustrated no significant differences (p = 0.153).
Fig 4. Comparison of increases in mid-thigh circumference between the supplement and placebo condition, immediately before (pre) and for up to 96h post-exercise. The values are mean ( SD).
There were also no significant differences for either concentric or eccentric peak torque for within subject effects pertaining to condition (p = 0.639, p = 0.417), time (p = 0.543, p = 0.306) or condition/time (p = 0.422, p = 0.229) respectively. Between subject effects proved highly significant for both concentric (p = 0.018) and eccentric (p = 0.006) peak torque.
As no significant differences were found for any of the within-subject effects for any of the variables tested, no follow up post hoc analysis were required. It seems likely that the results are compromised due to the small sample size (n = 4) making statistical significance less likely. Eysenck (2000) states that for a particular category having around 15 participants is usually adequate.
Fig 5. Comparison of eccentric peak torque between the supplement and placebo condition, immediately before (pre) and for up to 96h post-exercise. The values are mean ( SD).
Fig 6. Comparison of concentric peak torque between the supplement and placebo condition, immediately before (pre) and for up to 96h post-exercise. The values are mean ( SD).
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Discussion
The primary objective of this study was to assess the effects of a dietary supplement on markers of exercise-induced muscle damage. The findings of this study indicate that supplementation with QC (1000mg·d-1) - for 14 days pre-exercise and 5 days post exercise - does not significantly alter the post-exercise indirect markers of muscle damage. Similar results have been reported by other studies that used antioxidant supplementation (Helgheim et al., 1979; Kanter & Eddy, 1992), and measured indirect markers of muscle damage.
Exhaustive exercise that induces muscle damage has also been shown to increase free radical production (Alessio & Goldfarb, 1988). Free radical production has also been correlated to exercise intensity (Kanter et al., 1993; Lovlin et al., 1987). Lovlin et al. (1987) proposed that significantly heightened free radical production occurred during exhaustive maximal exercise and that sub-maximal exercise (less than 70% VO2max), may inhibit free radical production.
This study took subjects to ~ 85% of their AAHRM by manipulating the speed of the treadmill with them running on a level gradient - corresponding to ~ 75% VO2max (McArdle et al., 2001). The treadmill was then adjusted to a gradient of -10%, and the original treadmill speed was maintained for the duration of the test. Personal observation revealed a reduction in subject HR values following the treadmill being declinated to -10%. This would indicate that the relative intensity of the exercise protocol was also reduced, and may have fallen below a level that would induce sufficient oxidative stress. Altering the treadmill speed to elicit a HR ~ 85% of AAHRM during the downhill running would have maintained the oxidative insult at the originally prescribed level.
Several studies have reported a positive effect of dietary antioxidant vitamin supplementation on lipid peroxidation (Kanter et al., 1993; Sumida et al., 1989; Jakeman & Maxwell, 1993). However, Kanter et al. (1993) highlighted the difficulty with attributing positive results with vitamin mixture supplementation, to the effect of a single vitamin or the cumulative vitamin mixture. Ashton et al. (1999) demonstrated a reduction in oxidative stress following acute supplementation (1000mg 2h pre-exercise) with vitamin C. Surprisingly, as ascorbic acid (vitamin C) is a water-soluble antioxidant and perhaps would not be expected to inhibit lipid peroxidation or scavenge lipid derived radicals. A recent placebo controlled study demonstrated that supplementation with 1,000mg of ascorbic acid enhanced the radical scavenging capacity of plasma (Mullholland & Strain, 1992). One possible mechanism for this may be due to its properties as an effective reducing agent, donation of an electron from ascorbic acid to a peroxyl radical would result in a stable radical thus preventing propagation of lipid peroxidation (Ashton et al, 1999). Any positive results found in this study as a result of supplementation with QC would be equally difficult to attribute.
It has been demonstrated that exercise-induced muscle damage results in a decrease in the maximal force that the affected muscles can generate voluntary (Eston et al., 1996; Eston et al., 1994), or when electrically stimulated (Davies & White, 1981; Newham et al., 1983b). Downhill running with its functional eccentric muscular bias has been shown to results in an acute loss of strength, which continues for some days after the damage inducing exercise (Clarkson et al., 1992; Clarkson & Tremblay, 1988). Eccentric contractions are attributed with damaging the sarcoplasmic reticulum, which results in an impairment to generate normal levels of muscular force as a consequence of a breakdown in intercellular calcium regulation (Clarkson et al., 1992).
In this study muscle function was assessed by measuring the concentric and eccentric PT of the leg extensors, at an angular velocity of 90ºsec-1 (1.57rad sec-1). Eccentric and concentric muscle functions appear to be compromised immediately post-exercise for both the supplement and placebo conditions (Figures 5 & 6). These findings are supported by other studies that demonstrate an immediate decline in peak torque following maximal eccentric muscle contractions (Beaton et al., 2002; Hortobagyi et al., 1998) and downhill running (Eston et al., 1996). Surprisingly, for the placebo condition muscle function appeared to improve toward pre-exercise values by 24h post-exercise. In contrast, Eston et al. (1996) demonstrated reduced concentric and eccentric peak torque values even at 7 days post-exercise, following a 40 min intermittent downhill running program - 5 x 8 min. These anomalies in findings may be due to differences in the downhill running protocol durations (30min v's. 40min), as the gradient and exercise intensity were similar for both running protocols. They may also be due to differences in the training status between both sets of subjects, although this is hard to quantify given the limited amount of subject information.
For the supplement condition both concentric and eccentric PT continued to decline, reaching a minimum value at 24h post-exercise. Concentric PT recovered toward pre-exercise values by 48h post-exercise and was actually higher than pre-exercise values at 96h post-exercise. In contrast, Beaton et al. (2002) reported significant post-exercise eccentric and concentric strength loss, after performing 24 sets of 10 maximal isokinetic eccentric contractions of the leg extensors, but no significant differences between the vitamin E and placebo condition. A study that compared downhill and uphill running at a similar intensity (~ 80% HRM), found a large decrease in mean isokinetic strength of the knee extensors from 270 Nm to115 Nm immediately following downhill running, mean strength had not returned to pre-exercise values by 11 days post-exercise.
From personal observation many subjects recorded higher eccentric and concentric PT values post-exercise than pre-exercise. This may be due to a learning effect and have resulted in artificially low pre-exercise values, as the time between the initial familiarisation sessions and testing varied considerably. Familiarisation only took place prior to the first bout and therefore the learning effect may have been heightened during the performance of the second bout.
Unusually high levels of circulating enzymes such as CK are taken to reflect alteration in the integrity of muscle fibre membranes, such as damage or increased permeability of the membrane to the enzyme. There appears to be a difference in the magnitude of CK response between maximal, high force eccentric contractions and downhill running. Clarkson et al. (1992) reported maximal peak CK levels of ~2500 U·l-1 at 4 days post-exercise, following maximal eccentric exercise of the forearm flexors. However, in a previous study Clarkson and Tremblay (1988) demonstrated a significantly different peak CK response for the performance of 24 (~ 500 U·l-1) and 70 (~ 2300 U·l-1) maximal eccentric contractions of the forearm flexors. It appears that high force eccentric contractions have the potential to induce a heightened CK response over downhill running protocols, but that this potential is correlated to the quantity as well as the intensity of the eccentric contractions.
CK levels after downhill running would appear to peak earlier and be of a lesser magnitude (Byrnes et al., 1985; Schwane et al., 1983). In a study utilising 22 college students, Byrnes et al. (1985) reported peak CK values of 339± 62.7 U·l-1 at 18h post-exercise, following an initial bout of downhill running for 30 mins on a decline of 10 . These finding are different to those of Schwane et al. (1983) who reported peak CK values of ~ 470 U·l-1at 24h post-exercise, these differences could be due variations in exercise time (30 vs. 45min). Schwane et al (1983) also measured CK values at 0, 24, 48 & 72h post-exercise, whereas Byrnes et al. (1985) measured CK at 6, 18 & 42h post-exercise. If Byrnes et al. (1985) had measured CK at 24h post-exercise, it is possible that the magnitude of change for CK would have been similar to that of Schwane et al. (1983).
This study produced much lower peak CK values than those previously reported following downhill running protocols. The peak CK values measured for the placebo and supplement condition were 134.0 ± 73.3 U·l-1 at 96h and 160.3 ± 39.9 U·l-1 at 48h respectively. However, pre-exercise CK values (placebo = 111.3 ± 17.6 U·l-1; supplement = 140.5 ± 47.4 U·l-1) were comparable to those of other studies (Byrnes et al., 1985; Schwane et al., 1983). Surprisingly the supplement condition showed an earlier and heightened CK response compared to the placebo condition (Fig 3.). Although the results were not statistically significant this could easily be due to the very small sample size - 4 subjects for each condition. Another study has reported negative effects on CK activity following supplementation with a combination of bioactive nutrients including quercetin (Phillips et al., 2003). Although the results were not statistically significant, the CK and LDH post-exercise response was higher in the supplement condition than the placebo condition, peaking at 3d post-exercise and returning toward pre-exercise values at 7d post-exercise. However, the magnitude of the CK response was much greater, reaching peak values of ~ 600-800 U·l-1. These differences in the magnitude of the CK response are likely due to differences between the exercise protocols. Philips et al. (2003) utilised a high force eccentric exercise protocol - 3 sets of 10 eccentric repetitions at 80% of their eccentric one repetition maximum - which is consistent with other studies using a similar protocol and similar number of maximal contractions (Clarkson & Tremblay, 1988). In contrast, Beaton et al. (2002) demonstrated a general reduction in CK activity following vitamin E supplementation (1200 IU·d-1), at 3 days post-exercise CK activity was significantly reduced ( p less than 0.05) for the supplement condition in comparison to the placebo condition, this may have been due to CK peaking at day 3 for the placebo condition and at day 7 for the supplement condition. These findings are contrary to what this study found, there is also a lack of support for the time course of CK activity between this study and that of Beaton et al. (2002).
Muscle swelling is a classic sign of inflammation that accompanies muscle soreness after eccentric exercise (Nosaka & Clarkson, 1996). Following eccentric exercise the enlargement of the muscle has been documented by an increases in muscle circumference (Clarkson et al., 1992; Howell et al., 1985). It has been proposed that increases in muscle circumference are due to two different phenomena - inflammatory swelling and protein synthesis (Smith, 1991). The accumulation of tissue fluid is though to be the main cause of swelling up to 2 days post-exercise, which may be followed by the production of connective tissue (Ryan & Majno, 1977).
In this study muscle swelling was assessed by measuring changes in the mid-thigh circumference of the non-dominant leg. Mid-thigh circumference increased immediately post-exercise for the both the supplement and placebo conditions and continued to increase, peaking at 48h and 96h for the supplement and placebo conditions respectively (Figure 4). Support for the time course of muscle swelling for the placebo condition, comes from two studies that measured changes in upper arm circumference following voluntary (Nosaka et al., 2001) and electrically stimulated maximal eccentric contractions (Nosaka et al., 2002). Both these studies demonstrated an initial spike immediately post-exercise, a small decrease at 24h, and a progressive rise up to a peak value at 96h post-exercise. These studies differed only in the magnitude of the change in muscle circumference (~15mm), as compared to this study (~5mm). These variations are surprising given the differences in limb circumference between the upper arm and mid-thigh, although support for these findings comes from previous studies that have measured changes in upper arm (Chen, 2003; Nosaka et al., 2001) and mid-thigh circumference (Beaton et al., 2002) following maximal eccentric contractions.
For the supplement condition, increases in mid-thigh circumference continued until they peaked at 48h post-exercise, then showed a small decrease at 96h. It is difficult to draw comparisons with other studies that have investigated the effect of antioxidant supplementation on changes in muscle circumference. One study that supplemented with vitamin E simply reported no change in thigh diameter after the exercise protocol, but they did concede that supplementation may have altered the time course of inflammatory events and the muscle damage and repair cycle (Beaton et al., 2002)
Potential inadequacies of the present study included the small sample size, the intensity of the exercise protocol, and the CK analysis. The small sample size makes establishing statistical significance less likely unless a very large effect is present, to overcome this future studies should employ a larger sample size. The exercise protocol may not have been as aerobically demanding as envisaged. Subjects were initially taken to 85% of their age adjusted HR max on a level gradient by altering the treadmill speed. The treadmill gradient was then adjusted to a declination of -10% and the treadmill speed was maintained at the value that elicited 85% of the subjects AAHRM. A noticeable reduction in HR occurred after the treadmill gradient was set the -10% decline; therefore the exercise became less aerobic than initially intended. This would likely be partially offset by the phenomena of oxygen drift that is experienced during downhill running, several studies have reported a steady upward shift in VO2 during downhill running (Byrnes et al., 1985; Dick & Cavanagh, 1987; Westerlind et al., 1992)
In conclusion it is possible that QC supplementation may have altered the time course of the inflammatory response and muscle damage and repair cycles. Based on the findings of this study it is clear that further investigations are required to determine the viability of flavonoids or flavonoid complexes as antioxidant supplements, in reducing the markers of exercise-induced muscle damage. Although this study and prior studies provide an insight into the mechanism in vivo, further research would be necessary to clarify the exact mechanism and to establish the level of individual versus synergistic involvement of such natural compounds. Where a flavonoid/vitamin complex (FVC) is being investigated it may be prudent to include an additional group that supplements with the vitamin present in the (FVC), as a positive effect with the (FVC) could be down to an individual or synergistic effect.
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