| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Wastl Human Performance Laboratory, Department of Health and Kinesiology, Purdue University, West Lafayette, Indiana 47907; and 2Department of Nautical Hygiene, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433, People's Republic of China
Submitted 19 September 2002 ; accepted in final form 17 April 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
75% of energy intake for L+S and 50% for both S and
P. A 6% CHO-electrolyte solution (S and L+S) or placebo (P) was ingested
preexercise (6 ml/kg) and every 20 min during exercise (3 ml/kg). Blood
glucose was significantly higher at 40, 60, and 100 min during L+S, and at 60,
80, and 100 min during S compared with P (P < 0.05). Blood lactate
was significantly higher (P < 0.05) during L+S than S and P. Blood
glycerol was significantly lower (P < 0.05) at 20, 80, and 100 min
during L+S, and at 80 and 100 min during S than P. The proportion of CHO (%)
utilized during exercise was significantly higher (P < 0.05)
during L+S (71.3 ± 3.8%) and S (67.3 ± 4.3%) than P (59.2
± 4.6%). Performance times (P > 0.05) were 132.5 ±
6.3 min (S), 134.4 ± 6.3 min (L+S), and 136.6 ± 7.9 min (P). In
conclusion, it appears that when CHO availability in women is increased
through CHO loading and/or CHO supplementation, there is a concomitant
increase in CHO utilization. However, this may not necessarily result in
significantly improved performance. endurance performance; substrate utilization; glucose; glycerol; lactate
CHO supplementation during prolonged endurance exercise is thought to prevent a decline in blood glucose concentration, thus facilitating a high rate of CHO oxidation during the latter stages of exercise (4, 12, 16, 31, 35). Maintaining blood glucose concentration may also delay the onset of fatigue, allow exercise intensity to be sustained, and result in improved performance. The relationship among CHO ingestion, blood glucose, and performance enhancement has been extensively investigated (9, 12, 15, 22, 24, 31), particularly with respect to male athletes. Although there is an increased interest in understanding the interaction of CHO intake and exercise performance in women, investigators have primarily examined CHO loading (29, 32) with less attention being given to CHO supplementation (18). Thus the response of women to CHO feedings during endurance exercise, alone or in combination with CHO loading, requires more research.
It is important to understand how women respond to CHO augmentation during
endurance running for two reasons. First, female endurance runners believe
that CHO intake can be beneficial despite the fact that there is a paucity of
scientific evidence in support of this notion. Second, during prolonged
endurance exercise, men tend to utilize CHO to a greater extent than women,
whereas women tend to preferentially utilize more lipid
(13,
17,
18,
28). Possible reasons include
differences between men and women in the distribution and/or activation of
- and 
-adrenergic receptors
(5), aerobic capacity and/or
fitness level (13), exercise
intensity (13,
17), and the lack of a
sufficient amount of CHO ingested by women (compared with men) during and
before exercise (5,
13,
17,
30). However, the general
consensus among researchers is that it is likely mediated by endocrine
differences. Regardless of the mechanisms involved, it could be hypothesized
that CHO feedings would not enhance performance in female athletes to the
extent previously seen in similarly trained male athletes because of their
relatively greater reliance on lipid.
Cycling has been selected most often as the mode of exercise used to investigate the effect of CHO feedings on performance (18, 22, 24, 36). This may be due to the fact that athletes tend to have more difficulty ingesting fluid and experience a greater level of discomfort from fluid ingestion during running than cycling (9, 10). Additionally, it is logistically easier for researchers to obtain physiological and hematologic measures during cycling compared with running. To our knowledge, there is no information regarding the effect of CHO supplementation or the combination of CHO loading and supplementation on run performance in women. Thus the purpose of this study was to examine the combined effects of CHO loading and supplementation, as well as the effects of CHO supplementation alone, on metabolic, hematologic, and performance variables in endurance-trained female athletes during prolonged exercise. We hypothesize that augmenting CHO intake in women, either by supplementation alone or supplementation combined with loading, will result in greater CHO utilization during exercise but little increase in performance.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Experimental design. Subjects completed three different 24.2-km (15 miles) treadmill performance runs. With the use of a Latin square (counterbalanced) design, subjects completed each of the following three trials: no CHO loading + no CHO supplementation (placebo; P), CHO supplementation only (S), and CHO loading + CHO supplementation (L+S). Each trial was completed during the midfollicular phase of the menstrual cycle (between days 5 and 10 from the first day of menses) to minimize the effects of gonadotrophic hormones on fuel metabolism. Thus subjects performed one trial per month.
Preliminary measurements and procedures. Before experimental testing, subjects completed health, training, and menstrual cycle history questionnaires. They were also asked to complete a 3-day food diary and a 3-wk training log to ensure that they met the dietary and training criteria necessary for inclusion into the study. On meeting the study requirements, participants were given a list of food choices and asked to identify foods they consumed on a regular basis (familiar foods). From these choices, menus were generated for the women to follow for 4 days immediately before each experimental trial (Computer Planned Menus, Nutritional Computing Concepts, Indianapolis, IN). Additionally, subjects were given a dietary exchange book to help them approximate the amount of food they consumed on a daily basis and adhere to the prescribed dietary menus before each trial.
Maximal oxygen consumption
(
O2 max) was measured
2 wk before the first experimental trial by using a continuous,
incremental treadmill running test to exhaustion. On a separate day, subjects
completed a self-paced 60-min treadmill run to familiarize themselves with the
procedure for controlling treadmill speed and the drinking patterns and
measurements to be used during the experimental trials.
Exercise taper. Subjects ran for 60, 40, 40, 20, 20, and 0 min for the 6 days immediately before each trial, respectively. They were also required to log their daily activity during the week preceding each trial to ensure that they were following the prescribed tapering protocol.
Diet. Subjects followed a prescribed diet for 4 days before each experimental trial and recorded their dietary intake during this time to ensure they were ingesting a high-CHO diet (75% energy intake) before the L+S trial and a moderate-CHO diet (50% energy intake) before the S and P trials. Total energy intake was unchanged, but dietary composition was altered to either increase or decrease the percentage of dietary CHO. The high-CHO regimen required subjects to ingest a greater percentage of energy in the form of complex CHO and commercially available glucose polymers in solution (Gatorlode, Quaker Oats, Chicago, IL).
Experimental procedure. Experimental beverages were administered in a double-blind fashion during the performance trials. Subjects received a 6% CHO-electrolyte solution (Gatorade, Quaker Oats) during trials S and L+S and a similarly flavored artificially sweetened placebo during the P trial. The placebo solution was similar to the supplement solution in electrolyte and mineral content, coloring, and flavoring, but it did not contain the CHO.
Subjects arrived at the laboratory between 0700 and 0800 on the morning of the trial after a 10-h fast. Body weight was measured, a fingertip blood sample was obtained from a prewarmed hand, and 6 ml/kg of either the placebo or the CHO supplement was ingested. Subjects completed a 3-min self-paced warm-up run, rested for 2 min, and then began the 24.2-km time trial. Every 20 min throughout exercise, subjects ingested 3 ml/kg of the prescribed solution. The 24.2-km time trial was self-paced, and subjects were encouraged to complete the exercise in the shortest time possible. Pacing was accomplished through the use of photocells positioned at the front and rear of the treadmill, which allowed treadmill speed to be controlled by the subject simply by moving forward or "drifting" back on the treadmill to interrupt a light beam to increase or decrease speed, respectively. The treadmill was interfaced with a desktop computer and a software program that allowed subjects to see continuous updates of speed and distance on a nearby monitor.
Oxygen consumption
(O2), heart rate (HR),
and ratings of perceived exertion (RPE) were measured every 30 min during the
run starting at minute 25. Subjective level of gastrointestinal (GI)
discomfort was obtained throughout the run by using a nonvalidated scale (1 =
comfortable, 2 = partially full, 3 = full, 4 = uncomfortably full, 5 =
nauseous). A small blood sample (200 µl) was collected from a fingertip
every 20 min, with the beverage administered immediately after the blood
collection. No measurements were taken and no beverages were administered
after the 21-km (13 mile) mark; i.e., subjects ran uninterrupted for the
remainder of the run. On completion of the run, subjects were weighed and a
final blood sample was obtained 5 min postexercise.
Measurements. Expired air was collected into Douglas bags. Volume
was determined by forcing the contents of the bag through a dry-gas meter
(Parkinson-Cowan). Respiratory gases were measured by using O2
(paramagnetic) and CO2 (infrared) analyzers (ParvoMedics, Salt Lake
City, UT). The analyzers were calibrated before each series of analyses by
using gases with known concentrations.
O2 and carbon dioxide
production were determined from expired volume and the percentages of
O2 and CO2. Nonprotein respiratory exchange ratio (RER)
and O2 were used to
estimate the amount of CHO oxidized during the run. RPE was measured by using
the Borg 6–20 category-ratio scale of perceived exertion, and HR was
measured telemetrically (Polar Vantage XL, Polar, Stamford, CT).
Blood sample preparation, storage, and analysis. Blood collected before, during, and after the run was used for the subsequent determination of glucose, glycerol, and lactate concentration. Approximately 200 µl of whole blood (WB) were obtained at each sampling point and dispensed into a prechilled tube. Fifty microliters of WB were deproteinized in 200 µl of cold 8% perchloric acid. After centrifugation, an aliquot of the perchloric acid extract was stored at -80°C for subsequent analysis of lactate concentration. Of the remaining acid extract, 120 µl were mixed with 50 µl of cold potassium hydroxide and then stored at -80°C for subsequent analysis of glycerol. WB was centrifuged and stored at -80°C for future determination of serum glucose concentrations.
Glucose was determined spectrophotometrically by using a commercially
available glucose kit [Infinity Reagent, procedure no. 18-UV; standards and
controls (Accutrol); Sigma-Aldrich, St. Louis, MO]. The perchloric acid
extract was assayed for lactate by using an enzymatic technique
(23). Twenty microliters of
sample, standard, or controls were added to a cuvette with 1 ml of a reagent
cocktail containing excess NAD+ (12 mmol/l), lactate
dehydrogenase (bovine heart, >300 U/ml), and a 1 M glycine-0.8 M hydrazine
buffer (pH 9.2). Glycerol was analyzed fluorometrically by using an enzymatic
method (3).
Statistical analyses. Values are presented as means ± SE. A repeated-measures one-way ANOVA was used to determine the effects of the experimental treatments on performance time and total grams of CHO oxidized. Blood lactate, glucose, and glycerol values were analyzed by using separate two-way ANOVA with repeated measures. Tukey's post hoc tests were used where appropriate. Additionally, a Friedman's nonparametric matched-sample statistical test was conducted to test for a possible order or training effect. Statistical significance was accepted at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
53 km/wk for at
least 12 mo before the study.
|
Pretrial conditions. Training diaries collected on the morning of each trial showed that all subjects complied with pretrial training requirements; i.e., each subject followed the prescribed exercise taper. Diet records for the 4 days preceding each trial showed that subjects achieved the CHO intake goals for each experimental condition. Significantly greater CHO intake (P < 0.05) was consumed before the L+S trial (72.8 ± 2.0% CHO) than either the P trial (52.3 ± 1.2% CHO) or S trial (53.3 ± 1.4% CHO). This corresponded to 334.9 ± 29.0 g of CHO (5.45 ± 0.43 g/kg) for the L+S trial vs. 214.2 ± 15.1 g CHO (3.49 ± 0.21 g/kg) and 238.2 ± 15.3 g CHO (3.88 ± 0.21 g/kg) for the P and S trials, respectively. Total energy intake was not significantly different for the 4 days before each trial (P > 0.05).
Laboratory conditions. Laboratory temperature was 23.4 ± 0.6, 23.3 ± 0.5, and 23.6 ± 0.8°C, and relative humidity was 53.3 ± 3.3, 51.0 ± 4.3, and 50.4 ± 7.7% for the L+S, S, and P trials, respectively (P > 0.05). The women were cooled by an electric fan throughout the run.
Performance time. There was no significant difference in performance time among the trials (P > 0.05). Mean performance times were 132.5 ± 6.3 min for the S trial, 134.4 ± 6.3 min for the L+S trial, and 136.6 ± 7.9 min for the P trial. Six of the eight subjects completed the run faster with CHO augmentation (S and L+S trials) compared with ingestion of the placebo. Results of the Friedman's test indicated that there was no order effect for performance times (P > 0.05), suggesting that learning and/or training effects did not occur over the course of the investigation.
HR. No significant treatment effect or treatment x time
interaction was found. HR was maintained at 172.8 ± 3.5, 172.6
± 3.9, and 167.7 ± 3.1 beats/min during the L+S, P, and S
trials, respectively (P > 0.05).
RPE. The RPE response did not differ among experimental conditions, nor was there a treatment x time interaction (P > 0.05). There was a significant time effect such that RPE was higher (P < 0.05) at 120 min (16.0 ± 1.5) than at 30 min (11.6 ± 1.1) across all trials.
O2. There
were no treatment, time, or treatment x time effects for
O2 (P >
0.05). Exercise elicited an average
O2 of 36.2 ± 1.2,
37.2 ± 2.0, and 37.2 ± 1.6 ml · kg-1 ·
min-1, which corresponded to 68.5 ± 0.02, 69.8 ±
0.02, and 69.6 ± 0.02% of
O2 max for the S, P, and
L+S trials, respectively.
RER and substrate utilization. There was no significant time effect or treatment x time interaction for RER, but there was a significant treatment effect (P < 0.05). As shown in Fig. 1, RER values were significantly higher during the L+S (0.92 ± 0.01) and S (0.91 ± 0.01) trials compared with the P trial (0.89 ± 0.01).
|
A significant treatment effect was found (P < 0.05) for CHO oxidation, such that more CHO (expressed as a percentage of energy expenditure) was utilized during the L+S (71.3 ± 3.8%) and S (67.3 ± 4.3%) trials compared with the P trial (59.2 ± 4.6%). No significant time effect or treatment x time interaction was noted.
Blood glucose. Mean glucose values are presented in Fig. 2. A significant treatment by time interaction was noted (P < 0.05). Compared with the P trial, blood glucose levels were significantly higher during the L+S trial at 40 min, 60 min, 100 min, and postexercise, and during the S trial at 60 min, 80 min, 100 min, and postexercise. No significant differences were detected between the S and L+S trials. Plasma glucose was similar among treatments before each run, averaging 3.9 ± 0.2, 3.9 ± 0.2, and 4.0 ± 0.2 mmol/l for the P, L+S, and the S trials, respectively (P > 0.05).
|
Blood lactate. As shown in Fig. 3, blood lactate concentration was similar among treatments before each run (P > 0.05). A significant treatment effect was observed (P < 0.05) such that blood lactate during exercise was significantly higher in the L+S (3.0 ± 0.4 mmol/l) compared with both the P trial (2.6 ± 0.3 mmol/l) and the S trial (2.7 ± 0.3 mmol/l). There was no significant treatment x time interaction (P > 0.05).
|
Blood glycerol. Mean glycerol values are displayed in Fig. 4. Glycerol levels were similar among treatments before each run (P > 0.05). A significant treatment x time interaction was found (P < 0.05) such that glycerol levels were significantly higher at 20 min, 80 min, 100 min, and postexercise during the P trial compared with the L+S trial. Glycerol levels were also significantly higher at 80 min, 100 min, and postexercise during the P trial compared with the S trial (P < 0.05). No significant differences were detected between the S and L+S trials.
|
Fluid consumption and weight loss. Total fluid consumption was 1,434.8 ± 108.9 ml and did not differ among treatments. The initial bolus was 374.0 ± 13.3 ml, and the amount ingested every 20 min throughout the run was 191.8 ± 8.9 ml. Thus the hourly fluid consumption averaged 575.5 ± 26.7 ml. Total CHO intake during the S and L+S trials averaged 86.1 ± 6.5 g (1.40 ± 0.07 g/kg), which included 22.4 ± 0.8 g CHO (0.37 g/kg) in the initial bolus and an additional 28.8 ± 5.1 g/h (0.47 ± 0.04 g · kg-1 · h-1) during the run. Body weight was significantly lower after exercise (60.9 ± 2.1 kg) than before (62.2 ± 2.0 kg), with no significant difference in weight loss among the trials.
GI discomfort. GI discomfort was similar among the three trials, averaging 3.56 ± 0.37, 3.44 ± 0.49, and 3.44 ± 0.47 for the L+S, P, and S trials, respectively. On average, subjects felt somewhere between full and uncomfortably full throughout the run. They tended to experience increased GI discomfort later in the run during all three trials (P > 0.05), suggesting that the discomfort was a result of the volume rather than the type of beverage ingested.
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
4 and 2 min faster than the P trial, respectively, these differences
did not reach statistical significance. Additionally, the RPE, HR, and
O2 responses did not
differ among experimental conditions, indicating that subjects were running at
a similar level of physical exertion for each experimental condition. A post
hoc power analysis indicated that it would have taken 20 female subjects
to detect a performance difference in this study with 80% power at =
0.05. Therefore, it is possible that the inclusion of more subjects would have
resulted in a different outcome with respect to performance. However, despite
the lack of a significant difference in performance across the three exercise
trials, significant differences were noted in substrate utilization and in the
blood glucose, lactate, and glycerol responses. Blood glucose was maintained at or above resting values throughout exercise in all three trials and was significantly higher in the L+S and S trials compared with the P trial. Maintenance of blood glucose concentration during the P trial is not unusual and has been observed in previous run studies (6, 9, 27, 34). Lactate concentration was significantly higher during the L+S trial compared with both the P and S trials, indicating that CHO loading in addition to supplementation may significantly increase CHO oxidation, perhaps by utilizing muscle glycogen. It has been shown that the primary substrate for lactate production in muscle is glycosyl units derived from local glycogen stores (14). Previous researchers have reported higher blood lactate levels after CHO loading compared with normal or depressed glycogen levels (1, 21), indicating that glycogen levels influence its utilization during exercise. It has also been shown that when lactate and glucose are infused simultaneously during exercise, lactate turnover exceeds that of glucose, indicating that muscle glycogen provides most of the CHO oxidized during exercise (7). Muscle glycogen concentrations were not measured in the present study, so it is uncertain whether CHO ingestion throughout exercise had any effect on muscle glycogen utilization during exercise. However, the significantly higher RER values observed during the L+S and S vs. P trials are reflective of a higher rate of CHO metabolism, indicating that when CHO is made available through CHO augmentation, trained female runners will preferentially utilize CHO.
Glycerol concentrations were significantly higher in the P trial compared with the L+S and S trials, indicating a greater contribution of fat metabolism during the P condition. Although glycerol concentrations rose steadily during all trials, the increase was significantly smaller during both CHO trials, suggestive of a possible blunting effect as a result of CHO augmentation. Previous researchers have also reported decreased glycerol levels when CHO was consumed before or during an endurance run (6, 10, 31).
Few researchers have examined the combined effect of CHO loading and supplementation on endurance performance. Mean performance time of the L+S trial in the present study was not significantly different from the S or P trials, indicating that the combination of CHO loading and supplementation was of no added benefit to the women runners. These results are similar to those from a cycling study conducted by Burke et al. (8). However, other researchers found performance to be bettter in subjects when CHO loading and supplementation were combined before and during exercise (19), suggesting that loading and supplementation may have a synergistic effect on performance. In contrast, Widrick et al. (33) found that cycling performance was significantly improved when preexercise glycogen levels were elevated by CHO loading, regardless of whether CHO was ingested throughout exercise. It is unclear from these contradictory findings whether CHO loading provides any additional benefit to CHO ingestion.
Women in the present study consumed 335 g CHO/day, or 5.45 g CHO
· kg-1 · day-1, before the L+S trial.
Because only one subject performed optimally during that trial, it is possible
that the amount of CHO consumed was inadequate for improving performance.
These findings are similar to results of a previous study by Tarnopolsky et
al. (29), where CHO loading
failed to increase glycogen stores and also failed to significantly improve
cycling performance. The women in their study consumed 6.4 g CHO ·
kg-1 · day-1 (370 g CHO/day) during the CHO
loading regimen. However, in a study by Brewer et al.
(6), male and female subjects
significantly increased run time to exhaustion after consuming
7–7.5 g CHO · kg-1 · day-1 for
3 days before the run trial. In absolute terms, the subjects were consuming
462–507 g CHO/day. Because male and female data were pooled, the
relative and absolute amount of CHO the women were consuming is unclear. In
another study (32), female
cyclists had a significant increase in glycogen stores and a significant
improvement in performance after a CHO-loading regimen requiring them to
consume 8.14 g CHO · kg-1 · day-1 (464 g
CHO/day). Tarnopolsky et al.
(30) recently suggested that
perhaps women fail to increase their muscle glycogen stores to the same extent
as male athletes in response to CHO loading because they do not consume the
same absolute amount of CHO as men.
It has been suggested that a CHO dosage of 30–60 g/h is necessary for
subjects to benefit from CHO supplementation
(12,
15). However, Murray et al.
(26) found on two separate
occasions that the ingestion of 26 and 78 g CHO/h increased cycle performance
to a similar extent, suggesting that 26 g/CHO · h was adequate to
enhance performance. CHO dosage throughout the S and L+S trials of the present
study was 29 g/h (0.47 g · kg-1·h-1),
with an additional 22.4 g (0.37 g/kg) administered in the preexercise bolus.
Relative to body weight, this was comparable to the amount of CHO administered
to male runners in previous studies
(10,
31). Run time to exhaustion in
those men was significantly increased when ingesting CHO at a rate of 0.33 g
· kg-1 · h-1 (39 g/h) in addition to a
0.44 g/kg bolus (31), and 0.30
g · kg-1 · h-1 (35 g/h) plus an additional
0.55 g/kg in the bolus (10).
Women in the present study oxidized significantly more CHO with the ingestion
of 29 g CHO/h when compared with a placebo, suggesting that this dosage
was adequate to increase CHO utilization.
The optimal dose eliciting performance improvements when CHO feedings are administered throughout prolonged exercise has not been established. Hargreaves (15) has suggested there is little added benefit in ingesting CHO solutions more concentrated than 6–8% because consuming higher dosages does not increase the rate of exogenous glucose oxidation. Evidently, there is a plateau in exogenous CHO oxidation during prolonged exercise whereby additional CHO ingestion above a certain amount will have no effect. On the basis of findings from Mitchell et al. (25), ingesting a CHO beverage that is too concentrated (i.e., 18% CHO) can impair gastric emptying and cause discomfort. Similarly, ingesting a less concentrated beverage in a greater volume of fluid also causes discomfort to the subjects. The difficulty is to administer a beverage that will provide an adequate amount of CHO to the subject without causing GI discomfort. Subjects in the present study experienced GI distress late in exercise during all three experimental trials, indicating that fluid volume rather than CHO concentration caused the discomfort. Chryssanthopoulos et al. (11) suggested that one cause of GI distress is that the volume of fluid prescribed and consumed by runners in laboratory studies is greater than the volume consumed during competition. Therefore, it is possible that subjects in the present study were experiencing GI distress because they were consuming more beverage during the experimental trials than they would during training and/or competition.
In summary, the aim of the present study was to gain a better understanding and provide more information on the response of women runners to CHO augmentation. On the basis of the findings, it appears that when CHO availability was increased through CHO loading and/or CHO supplementation there was a concomitant increase in CHO utilization. However, this did not translate into significantly improved performance.
|
DISCLOSURES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
P significantly
different from L+S, P < 0.05.
Significantly higher than minute 20, P <
0.05.