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Medicine & Science in Sports & Exercise:
Special Communications: Methods

Comparison of methods for evaluating exercise-induced changes in thromboxane B2 and β-thromboglobulin

TODD, M. KENT; GOLDFARB, ALLEN H.; BURLESON, CATHY

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Department of HPERD, Longwood College, Farmville, VA 23909; and Exercise and Sport Science Department, University of North Carolina at Greensboro Greensboro, NC 27412

Submitted for publication February 1996.

Accepted for publication November 1996.

Supported by an equipment donation from BodyGuard Inc. The authors wish to thank Wesley Long Community Hospital in Greensboro for their assistance with this project.

Address for correspondence: M. Kent Todd, Department of HPERD, Longwood College, 201 High Street, Farmville, VA 23909.

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Abstract

This study compared the effects of exercise on TXB2 and β-TG when evaluated by four methods: 1) not adjusted; 2) adjusted for plasma volume changes (PV); 3) standardized per 105 platelets (PC); 4) or both PC and PV (PC-PV). Blood was collected from 16 men (41.3 ± 8.1 yr) at rest after 30 min of exercise (IPE) and after 30 min recovery. Resting TXB2 and β-TG concentrations were 62.0 ± 6.2 pg·mL-1 and 129.8 ± 12.5 ng·mL-1, respectively. When expressed on a per 105 platelet basis, resting PCTXB2 was 23.8 ± 2.8 pg·mL-1·105-1 platelets and PCβ-TG was 50.77± 6.0 ng·mL-1·105-1 platelets. At IPE, TXB2 decreased 20.5% and β-TG increased 13.6%. Thirty minutes after exercise TXB2 was 4.2% lower than resting values, whereasβ-TG was 26% higher. TXB2, β-TG, PVTXB2, and PVβ-TG were not significantly altered by exercise. The only significant changes in TXB2 occurred at IPE when values were adjusted for changes in platelet count. At IPE, PCTXB2, and PC-PVTXB2 decreased 32.8% and 33.6%, respectively (P < 0.05). Similarly, β-TG were not altered significantly by exercise except when the samples taken after 30 min of recovery were adjusted for changes in platelet count. At 30 min post-exercise PCβ-TG and PC-PVβ-TG were 21.2% and 28.4% greater(P < 0.05) than the resting β-TG values. These data suggest that methods used to adjust concentrations of platelet derived substances for changes owing to exercise may influence conclusions about the effect of exercise on platelet function. Thus, it is imperative that researchers consider the purpose for which they are collecting TXB2 and β-TG, as well as other constituents derived from blood cells, before they determine which methods of analysis to use.

Exercise-induced changes in substances known to influence platelet function and markers of platelet activity are frequently reported without adjustment for changes in plasma volume or platelet count. This practice may lead to reporting data that suggest one result when, in fact, another may be true. For example, reporting a significant increase in plasmaβ-thromboglobulin (β-TG) attributable to an exercise-induced reduction in plasma volume and a concomitant increase in platelet count may lead to the erroneous conclusion that platelet activation increased when in fact no change occurred. Similarly, increases in plasma concentrations of thromboxane B2 (TXB2) are frequently reported as a pro-aggregatory event without consideration to changes in platelet count.

A review of procedures in numerous studies(1,3,4,6-15) shows that adjustments for plasma volume changes were made in three studies in which the effects of exercise on TXB2, β-TG, or other substances released by platelets were under investigation(10,13,14). Of these three, two made adjustments for alterations in plasma volume and platelet count(13,14), and the third discussed the relevance of changes in platelet count (10). Details of procedures used in the other studies were not specific enough to ascertain whether adjustments were made for changes in plasma volume and/or platelet count.

The present study demonstrates the importance of making the correct decision concerning what adjustments, if any, should be made when reporting the effects of exercise on circulating concentrations of substances that influence platelet function or represent platelet activation. Four methods of evaluating changes in TXB2 and β-TG in response to exercise were considered. These include: changes in absolute concentrations, adjustments for changes in plasma volume, adjustments of changes in platelet count, and adjustments for changes in both plasma volume and platelet count.

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METHODS

Subjects. Sixteen healthy male volunteers (x = 41.3 ± 8.1 yr) participated in the present study. Individuals who smoked or were diagnosed with coronary artery disease, diabetes, or other chronic disorders or symptoms of disorders that may have predisposed them to risk during this study were not allowed to participate. All procedures conformed to the University of North Carolina at Greensboro's Internal Review Board policies for human subjects.

Each subject exercised on a motorized treadmill for 30 min between 70 and 75% of previously measured ˙VO2max. Expired air was periodically collected and analyzed for oxygen (Beckmann OM-11, Beckman Instruments, Palo Alto, CA) and carbon-dioxide (Beckmann LB-2) to ensure that subjects were exercising at the desired intensity. Each submaximal test was conducted between 0800 and 1100 hours. Subjects reported in a 10-h post-absorptive state.

Collection of blood samples. Two whole blood samples (i.e., 10 mL Vacutainer) were collected 15 min after the subject had rested, 1 min following 30 min of exercise, and 30 min after the cessation of exercise. A single venipuncture with a 21g needle was used during each collection period. A tourniquet was not used in the blood collection procedures. Each Vacutainer was gently rotated to ensure proper mixing.

The first Vacutainer of whole blood collected during each observation period was used only for the determination of platelet counts, hemoglobin, and hematocrit. The second Vacutainer collected during each observation was used for the determination of TXB2 and β-TG. Blood used for determination of platelet counts, hemoglobin, and hematocrit was collected in Vacutainers containing 0.5 mL of 4.5 mM EDTA as an anticoagulant. Blood used for the determination of TXB2 and β-TG was drawn into Vacutainers containing 0.5 mL of 4.5 mM EDTA as an anticoagulant, 30 mM acetylsalicylic acid to inhibit in vitro catabolism of arachidonic acid, and 1 μM prostaglandin E1 to inhibit in vitro platelet activation.

Blood collected for determination of TXB2 and β-TG was centrifuged at 3000 rpm for 15 min at 4°C. Plasma was removed and frozen at - 70°C until assayed.

Analysis of blood samples. Platelets were counted with a Unopette Microcollection System (Fisher Scientific, Pittsburgh, PA) and hemocytometry. Hemoglobin was evaluated by the colormetric methods outlined by Sigma Diagnostics Co. (Mississauga, Ontario, Canada). Plasma volume changes were determined by the following calculations, which were previously outlined by Dill and Costill (2).

In this formula, hemoglobin1 and hematocrit1 represent resting values whereas hemoglobin2 and hematocrit2 represent values from a sample taken after the resting value (i.e., immediate post-exercise or 30 min post-exercise).

Extraction of TXB2 was begun by adding a 7.0% solution of hydrochloric acid to plasma (0.5 mL) to adjust pH to 3.5. Plasma was then separated into organic and aqueous phases by adding 1.5 mL of ethyl acetate and centrifuging each sample at 3000 rpm for 10 min. The organic phase, which contained TXB2, was removed and the ethyl acetate evaporated. The residue was reconstituted in a phosphate buffer.

Plasma TXB2 and β-TG were measured by radioimmunoassay. Phosphate buffer, lyophilized standards of TXB2, 125I tracer, and TXB2 antibodies were acquired from Advanced Magnetics Inc.(Cambridge, MA) for determination of TXB2. Phosphate buffer, lyophilized standards of β-TG, 125I tracer, and β-TG antibodies were acquired from Abbott Laboratories, Inc. (Abbott Park, IL) for the determination of β-TG. Standard curves for both TXB2 andβ-TG were established for both of these constituents. Recovery was determined by measuring plasma sample to which known quantities of TXB2 and β-TG were added. Recovery for TXB2 was 84% and for β-TG was 91%. All measurements were made in duplicate.

Adjustments to TXB2 and β-TG. Concentrations of TXB2 and β-TG were adjusted according to three different procedures. An example of how constituent values were adjusted follows the explanation of each procedure.

For the first procedure, plasma volume changes were determined according to the procedures previously specified by Dill and Costill(2). The TXB2 and β-TG samples collected immediately following exercise and 30 min after exercise were then adjusted to reflect changes that were independent of plasma volume changes. For example, TXB2 was increased from 50 pg·mL at rest to 60 pg·mL with exercise and decreased 3% in plasma volume after exercise. A portion of the increase in TXB2 is attributable to the decrease in plasma volume or hemoconcentration, whereas the remaining portion is more directly attributable to the exercise. Thus, to determine the effect of the exercise on circulating concentrations of TXB2, the exercise value of 60 pg·mL must be multiplied by 0.97 (i.e., 1 {minus] 0.03). The product, 58.2 pg·mL-1, indicates that independent of plasma volume changes the exercise stimulus led to an 8.2 pg·mL-1 increase in TXB2, not a 10 pg·mL increase as suggested by the absolute rise in TXB2. Hereafter, TXB2 and β-TG concentrations adjusted using these procedures are denoted as PVTXB2 and PVβ-TG, respectively.

For the second procedure, resting samples and those collected immediately following exercise and 30 min after exercise were standardized on a per 100,000 platelet basis. This adjustment is made by dividing the constituent value for any given sample by the corresponding platelet count. For example, for a TXB2 value of 60 pg·mL and a platelet count of 300,000 platelets·mL of whole blood, the TXB2 concentration on a per platelet basis would be 20 pg·mL-1·105-1 platelets. This adjustment permits ascertaining the proportion of exercise-induced change in TXB2 and other constituents that are attributable to factors other than alterations in platelet count. TXB2 and β-TG concentrations adjusted using this procedures are subsequently denoted as PCTXB2 and PCβ-TG, respectively.

For the third procedure, resting TXB2 and β-TG, immediate post-exercise PVTXB2 and PVβ-TG, and 30 min post-exercise PVTXB2 and PVβ-TG concentrations were standardized on a per 100,000 (i.e., 105) platelet basis. Thus, TXB2 and β-TG were first adjusted for corresponding changes in plasma volume and then adjusted for corresponding changes in platelet count. These values are denoted as PC-PVTXB2 and PC-PVβ-TG.

Statistical analysis. Four separate 1 × 3 repeated measures ANOVA were used to test for main effects of exercise on the on TXB2 andβ-TG. Analyses were conducted to determine effects of exercise on unadjusted TXB2 and β-TG values, PVTXB2 and PVβ-TG, PCTXB2 and PCβ-TG, and PC-PVTXB2 and PC-PVβ-TG. Multiple comparisons were made using Tukey's procedure and the Student's range statistic to further elucidate the effects of exercise on TXB2 andβ-TG (7). Data are presented as means ± SE.

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RESULTS

Plasma, cell, and total blood volume. Plasma and cell volumes are presented as a percentage of initial blood volume. The value for initial blood volume is set at 100%. Plasma volumes were 53.73 ± 0.68% at rest, 51.54± 1.17% immediately following 30 min of exercise, and 57.67 ± 1.71% 30 min after exercise. Thirty minutes after exercise plasma volume was significantly higher than at rest and immediately after exercise (P< 0.01). Cell volumes were 46.28 ± 0.67 at rest, 46.25 ± 0.98 immediately following exercise, and 46.27 ± 0.71 after 30 min of recovery. There were no significant differences in cell volume. Blood volume was 2.22% less immediately after exercise and expanded 3.92% after 30 min recovery (Fig. 1).

Figure 1-Effect of e...
Figure 1-Effect of e...
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Platelet count. Resting platelet count was 268,875 ± 9,601·mL-1 whole blood. Platelet count increased significantly immediately after exercise to 325,844 ± 13,727·mL-1 whole blood (P < 0.001). After 30 min of recovery, platelet count was 275,968 ± 16,892·mL-1 whole blood, which was back close to resting levels (Fig. 2).

Figure 2-Effect of e...
Figure 2-Effect of e...
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Effect of exercise on TXB2 concentrations. Absolute concentrations of TXB2 at rest were 62.00 ± 6.24 pg·mL-1. After 30 min of exercise, TXB2 declined 20.5%. Thirty minutes following the cessation of exercise TXB2 was similar to resting concentrations. These values were not significantly different(P = 0.146; Table 1).

Table 1
Table 1
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When adjustments were made to account for changes in plasma volume, PVTXB2 decreased 22.2% immediately after 30 min of exercise. Thirty minutes after cessation of exercise, PVTXB2 had returned to resting concentrations. These values were not significantly different (P = 0.067, Table 1).

When standardized for platelet count, resting PCTXB2 was 23.80± 2.81 pg·mL-1·105-1 platelets. The concentration of PCTXB2 decreased significantly by 32.6%, immediately following exercise. Thirty minutes after exercise, PCTXB2 was only 5.4% below resting values. The immediate post-exercise values were significantly greater than both the resting and recovery values (P < 0.05,Table 1).

When adjusted for changes in plasma volume then standardized for platelet count, PC-PVTXB2 declined by 33.7% immediately after 30 min of exercise. Thirty minutes following exercise TXB2 concentrations had returned to resting values. The immediate post-exercise values were significantly greater than the resting and the recovery values (P< 0.05; Table 1).

Effect of exercise on β-TG concentrations. Concentrations ofβ-TG at rest were 129.83 ± 12.53 ng·mL-1. After exercise β-TG was 13.5% above resting values. Thirty minutes following exercise β-TG was 26.8% above resting values; however, these values were not significantly different (P > 0.05, Table 1).

When corrections were made for changes in plasma volume only, PVβ-TG had increased by 11.1% immediately following 30 min of exercise. PVβ-TG concentration 30 min after exercise was 33.3% above resting concentrations. However, owing to large variations in resting values, statistical significance was not obtained for β-TG concentrations using this method (P> 0.05, Table 1).

When standardized for platelet counts, resting PCβ-TG concentration was 50.77 ± 5.95 ng·mL-1·105-1 platelets. Immediately following 30 min of exercise, PCβ-TG did not change. After 30 min of recovery, PCβ-TG was 21.2% greater than resting values. The 30-min recovery values were significantly greater than the resting and immediate post-exercise values (P < 0.05, Table 1).

When β-TG was adjusted for changes in plasma volume and subsequently standardized for platelet count, PC-PVβ-TG declined by 6.2% immediately after 30 min of exercise. Thirty minutes following exercise the PC-PVβ-TG concentrations were 28.4% greater than resting values. The recovery values were significantly greater than both resting and immediate post-exercise values (P < 0.05, Table 1).

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DISCUSSION

These data clearly indicate that the methods used to adjust for the effects of exercise on circulating concentrations of TXB2 and β-TG may influence any conclusions drawn from the study's results. As demonstrated in the present study, when no adjustments were made or when adjustment for changes in plasma volume were made, no statistically significant differences were found. However, when TXB2 and β-TG concentrations were adjusted for exercise-induced changes in platelet count only and both plasma volume and platelet count, concentrations of each substance were found to be significantly different.

These findings are important for several reasons. First, TXB2 (i.e., the stable metabolite of TXA2) is frequently measured to ascertain predisposition to platelet aggregation. In isolation, such predisposition is more accurately reflected by the concentration of the platelet aggregating agents relative to the platelet count as opposed to the absolute circulating concentration.

Secondly, reporting plasma concentrations of β-TG and perhaps otherα-granule constituents (e.g., platelet factor IV) on a per platelet basis is logical since these substances are released almost exclusively by platelets (5) and have been used as markers of platelet activation (6,11,14). More specifically, a significant increase in circulating β-TG resulting from an exercise-induced increase in platelet count, accompanied by no change in the level of platelet activation, may lead to the erroneous conclusion that platelet activation increased. The significant alterations in plasma volume and platelet count found in this study concur with findings reported by others(6,10), as well as work previously conducted in the authors' laboratory (13,14). These and other data further suggest that acute exercise may lead to significant changes in circulating concentrations of platelet-derived substances in the absence of any change in the relative production or release of such substances.

Although hematological adaptations to exercise training was not the topic of this investigation, one should note that plasma volume expansion typically accompanies training. Thus, when examining the effects of exercise training on resting concentrations of platelet-derived substances and mediators of platelet activation, plasma volume adaptations should be considered.

One should also note that in this study a dilution of whole blood occurred during the sampling process for which no adjustment was made. Specifically, whole blood samples were drawn into Vacutainers containing 0.5 mL of anticoagulant solution. Although the containers are designed to hold 10 mL of whole blood, there is a possibility that the Vacutainers were not equally filled; thus, no adjustment was made. This methodology is unlikely to have impacted the outcome of the present study since plasma volumes, platelet count, TXB2, and β-TG would have all been diluted equally within any sample. Also, comparisons were made among the constituents measured in identical samples. Sample dilution, however, should be adjusted for in any study in which comparisons are made among different samples.

This study demonstrates that the purpose of investigating the effects of exercise on substances influencing or representing platelet function should be clear before the decision is made regarding how the data will be reported. Measurement of absolute concentrations may be useful for determining whether a change occurred; however, inferences about platelet function should be limited when using this technique. Reporting changes in TXB2, β-TG, and other substances related to platelet function on a per platelet basis is appropriate if one is investigating the effects of exercise on platelet function. Finally, if one wishes to partial out the direct effect of exercise on platelet function, adjustments for exercise-induced changes in plasma volume should be made before concentrations are further adjusted for changes in platelet count.

In conclusion, failure to appropriately match the method of determining exercise-induced changes in TXB2, β-TG, and other substances related to platelet function with the purpose of the investigation may lead to erroneous interpretations. In addition, the results of this study may be generalized to the measurement of other substances derived or released from blood cells, providing that the substances in question are released primarily from blood cells and that the substance has limited permeability to the vascular compartment.

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REFERENCES

1. Davis, R. B., D. G. Boyd, M. E. McKinney, and C. C. Jones. Effects of exercise and exercise conditioning on blood platelet function. Med. Sci. Sport Exerc. 22:49-53, 1990.

2. Dill, D. B. and D. L. Costill. Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration. J. Appl. Physiol. 37:247-248, 1974.

3. Green, L., E. Seroppian, and R. Handin, Platelet activation during exercise-induced myocardial ischemia. N. Engl. J. Med. 302:193-197, 1980.

4. Laustiola, K., E. Seppala, T. Nikkari, and H. Vapaatalo. Exercise-induced increase in plasma arachidonic acid and thromboxane B2 in healthy men: effect of adrenergic blockade. J. Cardiovasc. Pharmacol. 6:449-454, 1984.

5. Sauder, H., J. Slot, B. Bouma, P. Bolhaus, D. Pepper, and J. Sixma. Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglobulin in thin frozen sections of human blood platelets.J. Clin. Invest. 72:1277-1284, 1983.

6. Mant, M., C. Kappagoda, and J. Quinlan. Lack of effect of exercise on platelet activation and platelet reactivity. J. Appl. Physiol. 57:1333-1337, 1984.

7. McGill, D., J. McGuiness, J. Lloyd, and N. Ardlie. Platelet function and exercise-induced myocardial ischaemia in coronary heart disease patients. Thromb. Res. 56:147-158, 1989.

8. Metha, J., P. Metha, and C. Horalek. The significance of platelet-vessel wall prostaglandin equilibrium during exercise-induced stress.Am. Heart J. 105:895-900, 1983.

9. Ohnishi, A., T. Ishizaki, H. Echizen, K. Yasuda, H. Fujiwara, and T. Tanaka. Effects of a sustained thromboxane synthase inhibition on exercise-induced changes in eicosanoid formation, catecholamine concentration, and platelet aggregation in humans. Clin. Pharmacol. Ther. 51:454-464, 1992.

10. Piret, A., G. Niset, E. Depiesse, et al. Increased platelet aggregability and prostacyclin biosynthesis by intense physical exercise. Thromb. Res. 57:685-695, 1990.

11. Rudman, S. V. and R. C. Bates. The effect of acute exercise on vivo platelet release of beta-thromboglobulin and platelet factor 4 in women. Lab. Med. Jan: 28-31, 1989.

12. Taniguchi, N., H. Furui, K. Yamauchi, and I. Sotobata. Effects of treadmill exercise on platelet functions and blood coagulating activities in healthy men. Jpn. Heart J. 25:167-180, 1984.

13. Todd, M. K., A. H. Goldfarb, and B. T. Boyer. Effect of exercise intensity on 6-keto-PGF, TXB2, and 6-keto-PGF/TXB2 ratios. Thromb. Res. 65:487-493, 1992.

14. Todd, M. K., A. H. Goldfarb, R. Kauffman, and C. Burleson. Combined effects of age and exercise on thromboxane B2 and platelet activation. J. Appl. Physiol. 74:1548-1552, 1994.

15. Viinikka, L., J. Vuori, and O. Ylikorkala. Lipid peroxides, prostacyclin, and thromboxane A2 in runners during acute exercise.Med. Sci. Sports Exerc. 16:275-277, 1984.

β-THROMBOGLOBULIN; PLASMA VOLUME; PLATELET COUNT; THROMBOXANE B2

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©1997The American College of Sports Medicine

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