Eur J Appl Physiol (2000) 81: 47±53
Ó Springer-Verlag 2000
ORIGINAL ARTICLE
Donna L. MacIntyre á W. Darlene Reid Donald M. Lyster á Donald C. McKenzie
Different effects of strenuous eccentric exercise on the accumulation of neutrophils in muscle in women and men
Accepted: 27 July 1999
Abstract The purpose of this study was to evaluate the sex dierences in delayed onset muscle soreness (DOMS), torque, and accumulation of technetium-99m (Tc-99m) neutrophils in eccentric-exercised muscle. A group of 10 female and 12 male subjects took part in this study. The subjects completed a pre-test using the descriptor dierential scale (DDS) to describe DOMS, and tests of concentric and eccentric torque of the right quadriceps. A volume of 100 ml of blood was taken by venipuncture for neutrophil labelling in the early morning of the exercise day. The Tc-99m neutrophils were re-infused intravenously before the eccentric exercise. The exercise stimulus consisted of 300 eccentric repetitions of the right quadriceps muscles. Radionuclide images of both quadriceps muscles (lateral views) were taken at 2 and 4 h. The DDS, and concentric and eccentric torques of the quadriceps were subsequently evaluated at 0 h, 2, 4, 20 and 24 h post-exercise. The presence of Tc-99m neutrophils was greater in the exercised leg than the non-exercised leg at 2 and 4 h postexercise (P £ 0.013) and greater in the exercised leg of the women compared to the men at 2 h (P = 0.03). The DOMS had increased post-exercise (P < 0.001) and torque had decreased post-exercise (P £ 0.002) but the patterns were dierent between the sexes. We concluded
D.L. MacIntyre (&) á W.D. Reid School of Rehabilitation Sciences, University of British Columbia, T325 2211 Wesbrook Mall, Vancouver, BC V6T 2B5, Canada Fax: 604 822-7624 e-mail:
[email protected] D.M. Lyster Division of Nuclear Medicine, University of British Columbia, Vancouver, BC, Canada D.C. McKenzie School of Human Kinetics, University of British Columbia, Vancouver, BC, Canada
that the sex in¯uences the presence of Tc-99m neutrophils in the exercised muscle following eccentric exercise. In addition, dierent patterns of DOMS and torque were observed between the sexes after eccentric exercise, and require further investigation. Key words Exercise-induced muscle injury á Torque á Delayed soreness
Introduction Delayed onset muscle soreness (DOMS) has been described as the sensation of discomfort associated with exertion-induced muscle injury, which is most evident in skeletal muscle 1 (Stauber 1989) to 3 days (Armstrong 1984) following eccentric exercise. While Lieber and Friden (1993) have reported that active strain during lengthening disrupts the muscle ®bres, Faulkner et al. (1993) have suggested that muscle injury from eccentric exercise is due to the increased tension per individual cross bridge causing mechanical disruption of the ultrastructural elements within the muscle ®bres. This exercise-induced muscle injury has been described as causing an immediate loss of muscle force for up to 1 h after eccentric exercise (FrideÂn et al. 1983; Newham et al. 1987; Clarkson and Tremblay 1988). Although there may be some involvement of a metabolic component, Faulkner et al. (1993) have suggested that the initial decline in force may be a function of both mechanical injury and fatigue. Recently a biphasic decline of maximal isometric force in the extensor digitorum longus muscle (EDL) and tibialis anterior (TA) muscle of mice (Faulkner et al. 1993) and eccentric torque in the quadriceps muscles of women (MacIntyre et al. 1996) has been reported. In the studies with mice it has been shown that, maximal isometric force declined at 1 h post-exercise, with a second decline between 3 h (TA) and 3 days (EDL) (Faulkner et al. 1993); in the women, the greatest decline in eccentric torque was shown to be immediately following
48
the eccentric exercise, the muscle recovered from 2 to 4 h, and then torque declined again at 20±24 h postexercise (MacIntyre et al. 1996). It has been reported that a secondary biochemical injury may occur in the muscle as a result of acute in¯ammation at the site of the original damage (Faulkner et al. 1993) which may, in part, contribute to the second loss of force. It has been found that acute in¯ammation, in response to injury, is characterized by an in¯ux of neutrophils, followed later by an accumulation of mononuclear cells (Kent and Hart 1993). The neutrophils accumulate at the site of the injury during the ®rst 6 to 12 h, their lifespan in the tissue usually being 1±2 days (Walker and Fantone 1993). After eccentric exercise, one study with 9 men has shown neutrophils in muscle at 45 min and 5 days post-exercise (Fielding et al. 1993); another study with 10 women has reported the presence of technetium-99m labelled white blood cells (Tc-99m WBC) in muscle over 24 h (MacIntyre et al. 1996); and a third study with 5 men and women has shown in®ltration of mononuclear cells from 7 to 14 days post-exercise (Jones et al. 1986). Although these studies have reported the presence of acute in¯ammatory cells in muscle following eccentric exercise, the time course, the types of in¯ammatory cells in the injured muscles, and the sex selection of the groups has varied. There has been con¯icting evidence of sex dierences in animals concerning the amount of muscle damage present in the exercised muscle following eccentric exercise (Amelink et al. 1991; Van Der Meulen et al. 1991). There have also been reports from animal studies that indicate dierences in susceptibility to oxidative stresses between males and females, and evidence of an anti-oxidant and membrane-stabilizing eect of oestrogens (Tiidus 1995). In another report of sex dierences in neutrophil function in endotoxic rats, the reproductive-aged female rats had a greater neutrophil phagocytic response to the endotoxin challenge than males or pre or post-reproductive female rats (Spitzer and Zhang 1996). In addition, there have been reports in the literature of sex dierences in pain perception (Feine et al. 1991). Therefore, the primary purpose of this study was to evaluate and contrast the sex responses of DOMS, torque, and the accumulation of Tc-99m neutrophils in the exercised muscle compared to the contralateral non-exercised muscle following eccentric exercise. A second purpose was to examine the relationships among the presence of neutrophils and work, DOMS and torque.
Methods Subjects A group of 10 healthy women (ages 19±33 years) and 12 healthy men (21±29 years) participated in the neutrophil labelling procedure during the ®rst 4 h following eccentric exercise. In addition, 4 women (21±24 years) participated in the red blood cells (RBC) labelling procedure within the ®rst 4 h post-eccentric exercise to
determine if the increased presence of labelled cells was in the circulation or the muscle. The subjects were included if they participated in less than 6 h of recreational exercise each week. The women were excluded from the study if they were pregnant or breast-feeding, or if they had missed a menstrual cycle because these are precautions necessary for the labelling procedure; individuals were also excluded if they were taking anti-in¯ammatory medication, if they were engaged in recreational exercise of more than 6 h a week, if they were jogging, running, or weight training, or if they were involved in competitive sport. Experiment protocol Approval for this study was granted by the Clinical Screening Committee for Research Involving Human Subjects at the University of British Columbia. All the subjects provided written informed consent. The experiments complied with the laws of Canada, the country in which the experiments were performed. Protocol for the neutrophil labelling procedure The subjects completed a pre-test, 1±3 weeks before the exercise stimulus, using the descriptor dierential scale (DDS) to describe DOMS, and a test of concentric and eccentric torque of the right quadriceps muscles. A volume of 100 ml of blood was taken by venipuncture for neutrophil labelling in the early morning of the exercise day. The Tc-99m neutrophils were re-infused intravenously 3.5 h later. This was followed by 300 repetitive fatiguing eccentric repetitions of the right quadriceps muscles on the KinCom isokinetic dynamometer (Chattanooga Group, Hixson, Tenn.). The DDS and subsequent tests of concentric and eccentric torque were repeated at 0 h (immediately following the exercise stimulus), 2, 4, 20, and 24 h after the exercise stimulus. Radionuclide images of both quadriceps muscles (lateral views) were taken at 2 and 4 h post-exercise. Protocol for the RBC labelling procedure A volume of 3 ml of blood was taken for RBC labelling in the morning of the exercise day. The Tc-99m RBC were re-introduced 1.5 h later. The eccentric exercise was the same for this group as for the neutrophil labelling protocol. To determine that DOMS was present, it was evaluated using DDS at the pre-test, 0 h, 2, 4, 20 and 24 h post-exercise but concentric and eccentric torques were not evaluated. The radionuclide images of both quadriceps muscles (lateral views) were taken at 2 and 4 h post-exercise. Descriptor dierential scale The intensity of quadriceps muscle soreness (DOMS) was assessed using the sensory intensity scale of DDS (Gracely and Kwilosz 1988). This scale contained 12 descriptor items with 21 points along the line for each descriptor. For all assessments the subjects completed the scale immediately after the test of concentric and eccentric torque (two sessions of four contractions). The DDS was scored by assigning scores of 0±20 for each point along the line of each descriptor. The scores for each descriptor were added together and averaged over the 12 descriptors to arrive at a single score for each assessment of DOMS. Reliability of the sensory intensity scale has been established at r = 0.82 (Gracely and Kwilosz 1988). Concentric and eccentric torques of the quadriceps The subjects were seated on the isokinetic dynamometer, with their hips at 80°, their back supported and the pelvis stabilized on the
49 bench with strapping as has been reported previously (MacIntyre et al. 1996). In this study the subjects exercised through a range of motion of 35±95° of knee ¯exion and the assessment of concentric torque was added to the data collection. Both torque and torque normalized to body mass were calculated. The exercise stimulus The subjects were seated on the KinCom as has been described previously (MacIntyre et al. 1996) and performed 300 eccentric repetitions of the right quadriceps muscles (35±95° of ¯exion) at a slow velocity (30° á s)1) using the continuous eccentric/eccentric mode of the machine. The subjects were instructed to push back on the shin pad to initiate the movement and to let the machine straighten their knees during the upward movement of the lever arm. When the machine changed direction they were verbally encouraged maximally to resist the eccentric movement of the lever arm from 35 to 95° of range of motion. Work and work normalized to body mass were calculated over the 300 repetitions. Radionuclide investigation of acute in¯ammation Neutrophil labelling A volume of 100 ml of whole blood was taken by venipuncture from each of the subjects. Radionuclide labelling of the neutrophils was performed using a modi®ed technique that has been outlined previously (Thakur et al. 1984; MacIntyre et al. 1996). The neutrophils were further separated using the percoll gradient method (Thakur et al. 1984). Percoll gradients of 65%, 60% and 50% were prepared. The neutrophils were harvested from the 50%±60% interface and the 60% layer, and washed using platelet poor plasma. Radiolabelling with Tc-99m-hexamethylpropylene amine oxime (Tc-99m HMPAO) proceeded as has been described previously (MacIntyre et al. 1996). As the subject dose was drawn a quality control procedure was performed. In the dose calibrator, the activities of the supernatant and the cells were measured to obtain a labelling eciency. Labelling eciency should be between 50% and 60%, so the activity of neutrophils injected into each subject varied. Once the criteria for the quality control procedures had been met, the doses were drawn for the subjects and ranged from 92 to 384 MBq (2.49± 10.38 mCi).
RBC labelling A volume of 3 ml of whole blood were drawn by venipuncture from the subjects. The RBC were labelled with Tc-99m in vitro using the Brookhaven kit and procedures that have been described elsewhere (Srivastava and Chervu 1984). Nuclear imaging Following the eccentric exercise, a series of images of the quadriceps muscles (lateral views) were taken at 2 and 4 h post-exercise. Images were collected over 5 min per view for all tests. After the series of images had been completed, computer analysis of regions of interest (ROI) was used to determine the average count per pixel of gamma radiation. A marker on each patella served as a reference point. The area of the quadriceps muscle chosen as ROI was the antero-distal area from the lateral view (Fig. 1). The antero-distal ROI represented the muscle to tendon junction of the quadriceps. Tidball (1991) has reported that the muscle to tendon junction is the primary site of injury in many muscle lesions. The analyses of ROI were performed on three separate occasions by two scorers to reduce the intra-observer variability that has been previously reported by MacIntyre et al. (1996). The scorers used a pre-selected map of the ROI to be drawn (Fig. 1). Finally, as the doses of Tc-99m HMPAO neutrophils varied among subjects, the counts per pixel were normalized to 100 MBq of Tc99m HMPAO neutrophils and Tc-99m RBC for each subject for each ROI and presented as the average counts per pixel per 100 MBq. Statistical analysis A two-way repeated measures analysis of variance (ANOVA, SPSS-X 3.0; SPSS Incorporated, Chicago, Ill.) was used on each of the dependent variables (DDS intensity of soreness score, concentric torque, eccentric torque) to test for signi®cant dierences Fig. 1 Regions of interest for the radionuclide scanning. On the left is the anterior view of both thighs. In the middle is the lateral view of the right thigh (exercised leg), illustrating the antero-distal region of interest (ROI), and on the right is the lateral view of the left thigh (non-exercised leg), illustrating the antero-distal ROI
50 between groups (women and men ) and with time. A paired Student's t-test was used to examine the dierences in the presence of Tc-99m neutrophils in the quadriceps muscles between legs. An independent t-test was used to examine dierences in Tc-99m neutrophils and muscle work between groups (women and men). Posthoc analyses examined contrasts at each test time compared to the pre-test for DOMS, concentric torque and eccentric torque. Pearson product moment correlation coecients were used to assess relationships between the presence of Tc-99m neutrophils and work, DDS intensity of soreness score, concentric torque, and eccentric torque in both groups. The signi®cance levels were set at P < 0.05.
Results The presence of Tc-99m neutrophils in the antero-distal ROI was signi®cantly greater in the exercised leg than in the non-exercised leg for both women (P £ 0.013) and men (P £ 0.002; Table 1). The women had a greater presence of Tc-99m neutrophils in the exercise leg than the men at 2 h post-exercise (P = 0.03). There were no dierences between the exercised and non-exercised legs for Tc-99m RBC at 2 and 4 h post-exercise (Table 1). The men performed signi®cantly more muscle work during the eccentric exercise protocol than the women (P = 0.002) but when normalized to body mass there Table 1 Technetium-99 m (Tc-99m) neutrophils and Tc-99m red blood cells (RBC) in muscle
were no dierences between the sexes (Table 2). The men also had signi®cantly higher eccentric torque than the women in all strength tests (P = 0.006). With time, eccentric torque decreased signi®cantly for both the women (P = 0.002) and men (P < 0.001; Table 2). Eccentric torque was signi®cantly lower at 2 (P = 0.006) and 20 h post-exercise (P = 0.001) compared to the pre-test for the women and at all test times from 2 to 24 h post-exercise compared to the pre-test for the men (P £ 0.007). The post-hoc tests and the patterns of the torque responses are illustrated in Fig. 2a. The bimodal decrease in eccentric torque is shown by the signi®cant decrease at 2 h and 20 h post-exercise in the women. However, the men did not show this same pattern, their responses remaining uniformly decreased from 2 h to 24 h post-exercise. In addition to the sex dierences in eccentric torque, there were also dierences in concentric torque (P = 0.001) and signi®cant changes with time for the women and men (P < 0.001; Table 2). All post-exercise tests were signi®cantly lower than the pre-test for the women (P £ 0.05) and the men (P £ 0.005; Fig. 2b). There was also a signi®cant sex and time interaction (P = 0.005) indicating that the pattern of decline in concentric tor-
2h
4h
Exercise Mean Tc-99m Neutrophils Women (n = 10) Men (n = 12)
10.83 5.98
Tc-99m RBC Women (n = 4)
69.78
SD 8.42* 2.53* 30.34
Non-exercise
Exercise
Mean
Mean
4.0 3.69 67.9
SD 0.95** 0.57** 34.24
Non-exercise SD
Mean
9.83 6.7
7.05 2.62
3.86 4.07
69.57
21.49
71.72
SD 0.98** 0.53** 23.59
* Signi®cant dierences between the exercise legs of women and men P = 0.03 ** Signi®cant dierences between the exercise and non-exercise legs P £ 0.013 Table 2 Strength and muscle soreness Work Women (n = 10) Men (n = 12)
Mean
SD (J)
Mean
SD (J á kg-1)
24.895 33.499
5.719 b 6.886
1.89 1.96
0.44 0.29
Pre Mean
0h SD
2h Mean
4h SD
Mean
20 h
Mean
SD
SD
Concentric torque (N á m) Women 78.4 20.1 Men 133.67 33.65
61 110.75
18.27 31.9
64.9 98.92
19.5 32.24
68 94.92
15.66 30.87
Eccentric torque (N á m)a,b Women 116.4 26.93 Men 167.17 43.75
100.7 150.42
21.17 43.75
93.3 127.08
18 41.34
97.4 123.83
18.17 44.06
4.5 4.67
8.48 9.75
4.1 3.59
9.98 10.04
4.02 3.43
Mean
24 h SD
Mean
SD
17.79 28.82
67.2 106.25
14.47 33.56
16.74 44.51
95.6 123.67
11.58 37.48
3.65 3.35
12.92 9.1
3.82 3.63
a,b,c
Intensity of soreness (maximal score of 20)a,c Women 0.18 0.42 6.19 Men 2.29 2.72 9.46 a
62.2 99.25 90.1 124.5 11.87 9.98
Signi®cant changes over time P £ 0.002. b Signi®cant dierences between the sexes P £ 0.006. c Signi®cant sex ´ time interaction P £ 0.005
51
Fig. 3 Delayed muscle soreness ± the responses of the women are represented by a solid line and the responses of the men by a broken line. Values are means and SD. The lowest score on the scale, 0, represents no soreness. **Signi®cantly dierent from the pre-test P < 0.01; ***signi®cantly dierent from the pre-test P £ 0.001
Fig. 2 Torque. The responses of the women are represented by a solid line and the responses of the men by a broken line. a Eccentric torque ± values are means and SD. **Signi®cantly dierent from the pre-test P < 0.01; ***signi®cantly dierent from the pre-test P £ 0.001. b Concentric torque ± values are means and SD. *Signi®cantly dierent from the pre-test P < 0.05; **signi®cantly dierent from the pre-test P < 0.01; ***signi®cantly dierent from the pre-test P £ 0.001
que was not the same for the women and men, and that the responses depended on the test time. Concentric torque for the women was lowest at 0 h post-exercise, increased up to 4 h, declined to 20 h and increased again at 24 h post-exercise. For the men, concentric torque declined to 4 h post-exercise and then increased again at 24 h post-exercise (Fig. 2b). When concentric and eccentric torque responses for both the sexes were normalized to body mass there were no dierences between groups. However, the decline in
strength and the sex dierences in the patterns of the responses were the same as for the non-normalized torque responses illustrated in Fig. 2a, b. There were signi®cant changes with time for DDS (P < 0.001; Table 2), but there were no dierences between the sexes. After the exercise stimulus, the DDS was signi®cantly higher at all subsequent test times compared to the pre-test for the women (P £ 0.003) and the men (P £ 0.001; Fig. 3). However, there was a signi®cant sex versus time interaction (P < 0.001) indicating that DDS response was not the same for the two groups. The responses of the women continued to increase from 0 to 24 h post-exercise while those of the men increased at 0 h and then remained the same for 24 h (Fig. 3). Table 3 illustrates the DDS of the women evaluated for the presence of Tc-99m RBC at 2 and 4 h post-exercise. Even though the protocol of the eccentric exercise sessions was the same as for the previous groups of women and men, DDS was evaluated to con®rm a response that increased up to 24 h post-exercise. Similar to the response of the women as illustrated in Fig. 3, the DDS steadily increased up to 24 h post-exercise. The only signi®cant correlations were between the Tc-99m neutrophils and eccentric torque for the women at 2 h (r = )0.57) and at 4 h (r = )0.52) post-eccentric exercise. All other relationships between Tc-99m neutrophils and work, concentric torque and DDS were less than r = 0.38 for both the women and men.
Table 3 Muscle soreness in women with technetium-99m red blood cells Pre
Intensity of soreness Women (n = 4)
0h
2h
4h
20 h
24 h
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
4.41
3.6
5.22
7.9
7.2
6.9
10.43
5.1
12.1
5.7
12.01
7.2
52
Discussion This study showed an increased presence of neutrophils in the eccentric exercised quadriceps muscles of both the men and women at 2 and 4 h post-exercise. The women showed a greater presence of neutrophils in their exercised quadriceps muscles despite the fact that, as expected, the men did signi®cantly more work than the women during the eccentric exercise stimulus. The increase in neutrophils in the exercised quadriceps muscle was unlikely to have been related to increased circulation to the muscle following exercise because there was no dierence in the presence of Tc-99m RBC at either 2 or 4 h between the exercised and non-exercised muscles. Thus, the increased presence of neutrophils in the exercised muscles was probably due to increased adherence and migration of neutrophils into the muscle as has been described by Walker and Fantone (1993) and Pyne (1994). The results of this study indicated a greater neutrophil response in women which has not been reported previously in humans following eccentric exercise. To determine whether the women did more work for their body mass than the men, the work was normalized to body mass but no dierences were found. In another study with humans, the investigators have concluded that there were no sex eects on muscle protein turn-over following eccentric exercise (Phillips et al. 1997) but no other reports on sex dierences in exercise-induced muscle injury in humans have been found. The paucity of literature would suggest that investigation of the sex dierences in each stage of the events leading to exercise-induced muscle injury, and the role of sex hormones in these events, are important directions for future research. In this study, DDS was greater for both sexes up to 24 h; however, the responses of the women progressively increased between 0 and 24 h whereas those of the men reached a high plateau from 0 to 24 h. There have been reports in the literature of sex dierences in pain perception, with women having lower pain detection thresholds and pain tolerance than men (Feine et al. 1991). In addition, there have been studies that have reported sex-based dierences in animals in endogenous pain inhibition, or analgesia (Mogil et al. 1993). In the evaluation of a health-related ®tness assessment, Suni et al. (1998) have reported that inactive women were more prone to severe DOMS than men when they participated in the one-leg squat test. In contrast to their report, the results of this study would indicate that when making sex comparisons of DOMS the timing of that comparison is important. As expected, the men had higher eccentric and concentric torques than the women at all times, and there were signi®cant decreases in torque with time after the exercise stimulus for all groups. An unexpected result was the dierent pattern of responses between the women and men. The women showed a bimodal decrease in concentric and eccentric torque within 24 h,
which has previously been reported for eccentric torque (MacIntyre et al. 1996), while the men did not. In evaluating the relationships between Tc-99m neutrophils and the other outcomes, only the relationship between the neutrophils and eccentric torque in the women was above )0.50. As the Tc-99m neutrophils were greater in numbers at 2 and 4 h post-exercise, eccentric torque was less. The moderate relationship between the two measurements in the women lends support to the hypothesis that the eccentric torque response is in part related to the in¯ammatory response taking place in the muscle. For the men, all of the correlational analyses were less than 0.28, indicating no relationship between the Tc-99m neutrophils and any other measurement. A greater in¯ux of neutrophils in the eccentric-exercised muscle of the women may have led to a greater phagocytic response, and a closer relationship between the in¯ammatory process and muscle strength in the women. Lowe et al. (1995) have reported that maximal isometric tetanic force declined and remained the same from 6 to 72 h post-injury in mice even though protein degradation and phagocytic in®ltration continued to increase up to 48 h post-exercise. They have concluded that the in¯ammatory process was focal, tightly controlled and restricted to sites of damage, and that muscle force remained level despite protein degradation and phagocytosis. The torque response of the men over 24 h in this study more closely resembled the ®ndings of Lowe et al. (1995) where the muscle strength response was found to be dissociated from the intracellular responses. The dierences between the sexes found in this study requires further investigation. Although the men did more work, when normalized to body mass, there were no dierences between the sexes. The men took a longer time to develop the greatest loss of torque and had little evidence of recovery over 24 h, while the women showed a greater presence of neutrophils in the muscle and a moderate relationship between Tc-99m neutrophils and eccentric torque at 2 and 4 h post-exercise. The limitations of this study were that the single reinfusion of Tc-99m neutrophils may have underestimated the circulating pool at a later time because neutrophils have been shown to have a relatively short half-life of 6 h (Walker and Fantone 1993). As experimental time progresses, more neutrophils will be entering the circulation from the marginating pool and the bone marrow. Although work and torque were normalized to body mass, a better normalization procedure would have been to body mass index. Other limitations of this study were in not knowing the percentage of muscle ®bres injured during the eccentric exercise or the timing of the oestrogen and progesterone cycles for the women. In summary, there was a greater presence of neutrophils in the eccentric-exercised muscle compared to the non-exercised muscle for up to 4 h post-exercise, and a greater presence of neutrophils in the exercised muscle of the women compared to the men at 2 h post-exercise.
53
The presence of Tc-99m neutrophils, and the patterns of torque responses and delayed soreness were dierent between the sexes. In addition, there was a modest relationship between Tc-99m neutrophils and eccentric torque for the women. The results of this study would suggest that the sex of the subject in¯uenced the responses to eccentric exercise. Acknowledgements This study was supported by a grant from the British Columbia Medical Services Research Foundation. We gratefully acknowledge the technical support of the Nuclear Medicine Departments in the Vancouver Hospital and Health Science Centres, and the assistance of Ms. Kim Keays and Ms. Martha Moscovich with the data collection. We also express our appreciation to Dr. Jonathan Berkowitz for the statistical analysis.
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