Serum Enzyme Changes During Marathon Training

Prévia do material em texto

716 STAMPFER ET AL. A.J.C.P. • June 1983 
vitamin E, but rather by their rise in those taking pla-
cebo. There is other evidence in the literature that vi-
tamin E in moderate doses does not lower total serum 
cholesterol. In a small study, Harman8 found no signif-
icant effect of vitamin E on total cholesterol or LDL. 
Farrell and Bieri6 found slightly increased cholesterol 
levels (9% higher) among habitual vitamin E users, and 
this, was confirmed by Tsai,14 in a large placebo-con-
trolled trial, who found a slight, though nonsignificant 
increase.in cholesterol among those assigned to vita-
min E. 
Both Tsai and co-workers and Farrell and Bieri ob-
served elevations in serum triglycerides among those 
taking vitamin E. Our findings of a modest elevation 
relative to placebo are consistent with those observa-
tions. 
Acknowledgments. Roche.Pharmaceuticals kindly provided the vi-
tamin E and placebo capsules. We are grateful to the study participants 
for their cooperation, to Noemia Bravo and Albina Mariano for expert 
technical assistance, and to Kelly Hallett for preparation of the manu-
script. 
References 
1. Abell LL, Levy BB, Brodie BB, Kendall FE: A simplified method 
for the estimation of total cholesterol in serum and demon-
stration of its specificity. J Biol Chem 1952; 195:357-366 
2. Burstein M, Saimaille T: Sur un dosage rapide du cholesterol lie 
aux a-aux et /J-lipoproteines du serum. Clin Chim Acta 1960; 
5:609 
3. Carew TE, Koschinsky T, Hayes SB, Steinberg D: A mechanism 
by which high density lipoproteins may slow the atherogenic 
process. Lancet 1976; 1:1315 
4. Castelli WP, Doyle JT, Gordon T, et al: HDL cholesterol levels 
(HDL-C) in coronary heart disease (CHD)—cooperative li-
poprotein phenotyping study. Circulation 1975; 52(Suppl II): 
97 
5. Editorial. High-density lipoprotein. Lancet 1981; 1:478-480 
6. Farrell PM, Bieri JG: Megavitamin E supplementation in man. 
Am J Clin Nutr 1975; 28:1381 
7. Friedewald WT, Levy RI, Fredrickso'n DS: Estimation of the con-
centration of low-density lipoprotein cholesterol in plasma, 
without use of the preparative ultracentrifuge. Clin Chem 1972; 
18:499-502 
8. Harman D: Vitamin E effect on serum cholesterol and lipopro-
teins. Circulation 1960;22:151-152 
9. Hermann WJ, Ward K, Faucett J: The effect of tocopherol on 
high-density lipoprotein cholesterol. Am J Clin Pathol 1979; 
72:848-852 
10. Kessler G, Lederer H: Fluorometric measurements of triglycer-
ides. Technicon symposia: automation.in analytic chemistry. 
Edited by Skeggs LT Jr. New York, Mediad 1965; p 341 
11. Manual of laboratory operations: Lipid Research Clinics Program. 
Vol 1, Washington, DC: Government Printing Office, 1974. 
(DHEW publication no. [N1H] 75-628) 
12. Miller GJ, Miller NE: Plasma-high-density-lipoprotein concentra-
tion and development of ischaemic heart disease. Lancet 1975; 
1:16 
13. Sundaram GS, London R, Manimekalai S; Nair PP, Goldstein P: 
Tocopherol and serum lipoproteins. Lipids 1981; 4:223-227 
14. Tsai AC, Kelley JJ, Peng B, Cook N: Study on the effect of mega-
vitamin E supplementation in man. Am J Clin Nutr 1978; 
31:831-837 
Serum Enzyme Changes During Marathon Training 
FRED S. APPLE, PH.D., AND MATT K. McGUE, PH.D. 
Blood enzyme activities that might be suggestive of organ-spe-
cific damage were measured over a six-week period in two, 
male, long-distance runners training for a marathon. Striking 
changes were observed in the CK and LD isoenzymes. Runner 
B exhibited a flipped LD,/LD2 isoenzyme ratio that paralleled 
his persistent MB CK elevation. The hepatic enzyme ALT was 
transiently elevated in Runner A. These data suggest that in-
creases of cardiac isoenzymes (MB CK and LD|) and the spe-
cific hepatic enzyme ALT could lead to an inappropriate lab-
oratory diagnosis of a myocardial infarction or liver disease, 
respectively, in a healthy runner during intense training for a 
marathon. (Key words: MB CK; LD,/LD2 flip; Isoenzymes; 
Myocardial infarction; ALT; Marathon) Am J Clin Pathol 
1983; 79: 716-719 
Received September 7, 1982; received revised manuscript and ac-
cepted for publication November 22. 1982. 
Presented in part at the 1982 annual meeting of the American Col-
lege of Sports Medicine, May 1982, Minneapolis, Minnesota. 
Address reprint requests to Dr. Apple: Clinical Laboratories, Hen-
nepin County Medical Center, 701 Park Avenue South, Minneapolis, 
Minnesota 55415. 
Department of Laboratory Medicine and Pathology, 
Hennepin County Medical Center, University of Minnesota 
School of Medicine, Minneapolis, Minnesota, and Division of 
Biostatistics, Washington University School of Medicine, 
St. Louis, Missouri 
REGULARLY PERFORMED VIGOROUS EXER-
CISE induces a variety of adaptations in the training 
athlete. Skeletal muscle adapts to endurance exercise, 
such as long-distance running, by tending to develop 
enzyme patterns more characteristic of heart muscle.5 
In an attempt to provide further information in the field 
of diagnostic enzyrhology, this study correlates the ef-
fects of strenuous training for a marathon race (42 km) 
with blood enzyme activities suggestive of damage to the 
heart, liver, and/or skeletal muscle. We discuss the ele-
vations of creatine kinase MB (MB CK) and lactate de-
hydrogenase 1 (LD|) isoenzymes as false biochemical 
0002-9173/83/0600/0716 $01.00 © American Society of Clinical Pathologists 
 by guest on June 6, 2016
http://ajcp.oxfordjournals.org/
D
ow
nloaded from
 
http://ajcp.oxfordjournals.org/
Vol. 79 • No. 6 
Number of Weeks 
in Training 
1 
2 
3 
4 
5 
6 
7 
8 
Table 1. 
A* 
50 
47 
62 
64 
63 
65 
42 
Miles 
Marathon 
Serum 
B* 
73 
52 
84 
93 
103 
103 
70 
BRIEF SCIENTIFIC 
Enzyme 
REPORTS 
Activity Profiles during Premarathon Training 
Total CK 
A B 
(IO-70)t 
306 
488 
600 
750 
748 
394 
145 
595 
560 
1150 
738 
558 
412 
175 
% 
A 
NA§ 
4.1 
3.3 
5.3 
5.9 
4.6 
NA 
MB* 
B 
NA 
7.5 
7.0 
9.2 
4.7 
7.8 
NA 
Total LD 
A B 
(100-220) 
224 
208 
355 
270 
272 
230 
171 
328 
275 
350 
350 
315 
295 
230 
LD, 
LD2 
A 
0.8 
0.8 
0.9 
0.9 
0.8 
0.9 
0.9 
B 
0.8 
0.9 
0.9 
1.1 
1.2 
1.1 
1.3 
717 
ALT 
A B 
(5-35) 
31 22 
32 17 
38 26 
36 24 
42 22 
32 17 
30 16 
• A = Runner A: B = Runner B. 
t (. ) = normal range. U/L. 
X The per cent MB in both Runners A and B during the two weeks prior to the start of 
training were less than 2% of their total CK activity. Their respective weekly mileages were 
41 and 38 miles for A. and 51 and 46 miles for B. 
§ NA = not assayed. 
evidence for acute myocardial infarction. Abnormalities 
in serum activities of the liver-specific enzyme alanine 
aminotransferase (ALT) also are discussed. Finally, ev-
idence is presented that confirms enzyme release from 
skeletal muscle during premarathon training. 
Subjects and Methods 
Two subjects (both male) for this investigation were 
two, trained, long-distance runners. Runner A (28 year 
old, sub-three-hour marathoner) and Runner B (29 years 
old, sub-two-hour 40-minute marathoner) have been 
participating in long-distance running events for the past 
four years. At the time of these tests, both runners were 
training for a marathon. Runner A averaged 56 miles 
per week while Runner B averaged 82.6 miles per week 
for seven weeks prior to the marathon race. Blood sam-
ples were obtained between 12 and 24 hours after train-
ing runs. The samples were centrifuged, the serum re-
moved, and analyzed within 24 hours. There was no 
evidence of hemolysis in any of the samples. Neither 
Runner A or B experienced clinical symptoms charac-
teristic of acute myocardial infarction21 during the entire 
training period. However, EKGs were not obtained. 
Total enzymatic activities were measured on a kinetic 
enzyme analyzer for CK,13 LD,9 alkaline phosphatase 
(ASP),10 gamma glutamyltransferase (GGT),16 aldo-
lase," ALT,20 arid aspartate amino transferase (AST).20 
MB CK was measured with an ion-exchange chromato-
graphic procedure.19Separation of CK isoenzymes also 
was performed by electrophoresis on cellulose acetate. 
CK isoenzyme samples for electrophoretic analysis were 
preserved by adding /3-mercaptoethanol to the serum. 
The isoenzymes were located by NADPH fluorescence 
with a lower limit of sensitivity of 15 U/L.15 The elec-
trophoretic strips were interpreted for MB CK as positive 
or negative. There were no positive BB CK isoenzymes 
in any of the samples. LD isoenzyme separation was 
performed by electrophoresis on cellulose acetate.14 
Prior to analysis, samples were stored at room temper-
ature to prevent decreases in fractions LD4 and LD5. 
The isoenzymes were located by visualization using col-
orimetric procedures. Areas of enzymatic activity were 
determined by scanning densitometry. 
Results 
The cardiac enzyme activities measured in serum dur-
ing premarathon training are shown in Table 1. Runner 
B (82.6 miles per week average) had an average of MB 
CK of 7.2% (range, 4.7-9.2%) of the total CK activity. 
The maximum MB CK activity occurred during his 
major increase in mileage to 93 miles. In addition, dur-
ing the last four weeks of intensive training before the 
marathon, he had both a consistently elevated MB CK 
and LD,/LD2 flip. Runner A (56 miles per week average) 
had an average MB CK of 4.6% (range, 3.3-5.9%) during 
his entire training period. In both runners, the rise in 
MB CK activity paralleled the increase in total CK ac-
tivity and the increase in weekly mileage. 
Parallel rise of CK (Runner A, 2.5-fold; runner B, 2.0-
fold) and aldolase (Runner A, 1.6-fold; Runner B, 2.0-
fold) during the initial mileage buildup (Runner A, 40-
65 miles; Runner B, 60-100 miles) confirms that en-
zymes are released from skeletal muscle during this pe-
riod. Similar increases were observed in AST and LD 
activities (1.6-fold, Runners A and B). There were no 
substantial changes in GGT or ALP activities. However, 
Table 1 shows that Runner A had a transient elevation 
of the hepatic enzyme ALT to 42 U/L (1.2-fold) during 
the initial mileage buildup, while Runner B showed no 
substantial changes. 
Discussion 
Elevated activity of the MB CK isoenzyme in serum 
is a sensitive and accurate indicator for the diagnosis of 
 by guest on June 6, 2016
http://ajcp.oxfordjournals.org/
D
ow
nloaded from
 
http://ajcp.oxfordjournals.org/
718 APPLE AND MCGUE A.J.C.P.. June 1983 
acute myocardial infarction (AMI) due to the release 
from myocardial tissue.12 In addition, a coincident de-
tection of a flipped LDi/LD2 ratio increases the predic-
tive value for the diagnosis of an AMI.8 During the pres-
ent study, the magnitude of the MB CK activities, as 
well as a flipped LDi/LD2 ratio, were similar to those 
found during acute myocardial infarction. The sustained 
serum level of MB CK for Runner B (7.2% average) is 
the highest caused by strenuous exercise reported in the 
literature. However, MB CK has been shown to be pres-
ent in skeletal muscle samples717 as well as in the serum 
of patients with and without skeletal muscle disorders 
without myocardial involvement.3-416 Siegel and co-
workers16 have reported an 8-9% MB CK activity from 
homogenized lateral' gastrocnemius muscle from 25 
marathon runners. Although they suggest a correlation 
between per cent tissue MB CK activity and per cent 
serum MB CK activity, presently there is no firm evi-
dence whether the skeletal muscle or myocardial tissue 
or both are releasing the MB CK isoenzyme caused by 
the stress of long-distance running. Furthermore, the per 
cent of LD, of the total LD activity has been found to 
increase concomitantly with an increased percentage of 
slow-twitch muscle fibers.18 Coincidently, long-distance 
runners have been shown to have a higher per cent of 
slow-twitch muscle fibers and thus an increase in the 
relative activity of the LD, isoenzyme. While the ap-
pearance of both a flipped LTVLD2 pattern and a pos-
itive MB CK makes the diagnosis of an AMI biochem-
ically appropriate for Runner B, it seems clinically in-
appropriate in this asymptomatic individual. 
Recent evidence suggests that ALT testing is a poten-
tially useful method of screening blood donors to reduce 
the incidence ofhon-A, non-B hepatitis.1'2 The findings 
of Alter and associates show that the risk of non-A, non-
B hepatitis is proportional to the concentrations of ALT 
in donor blood.2 In their series of patients, donors whose 
ALT activities were at least 20% elevated above the up-
per limits of normal accounted for 29% of the post-trans-
fusion hepatitis cases. In our study, Runner A showed 
transient elevations in his ALT activity (Table 1). Dur-
ing training week 5, his ALT activity was elevated 20% 
(absolute value 42 U/L) above the upper limit of normal 
(35 U/L) and, thus, he could be considered a high-risk 
donor. 
The increases we observed in CK, LD, aldolase, and 
AST are consistent with those reported earlier. The si-
multaneous rises of CK and aldolase confirms enzymatic 
release from skeletal muscle. The source of the increase 
of AST is less certain since this enzyme can be found 
in many tissues. The hepatic enzymes, GGT and ALP, 
showed no changes in both runners during the premar-
athon training period. This is in agreement with pre-
viously published data. 
On the basis of what is known about changes in blood 
enzyme chemistries, enzyme activities from runners are 
often suggestive of a pathologic condition. This study 
yields two findings of practical implications. First, de-
spite the utility of cardiac isoenzyme profiles in the di-
agnosis of an AMI (serial CK and LD isoenzymes), the 
effects of long-term strenuous exercise on serum CK and 
LD isoenzymes create uncertainty in the interpretation 
of enzyme patterns obtained from patients presenting 
to a physician during an extensive period of intensive 
training. Whether the source of the MB CK and LD, 
increase is myocardial or skeletal needs to be investi-
gated. Second, evidence is presented that suggests tran-
sient elevations in the hepatic enzyme ALT can occur 
in a runner who is training for a marathon. This increase 
in ALT may pose a diagnostic problem for the clinician 
who is screening blood for use in transfusions in regard 
to non-A, non-B hepatitis infectivity. Thus, it is impor-
tant that changes in blood enzymatic activities incurred 
during premarathon training are not misinterpreted as 
being indicative of a disease state. 
References 
1. Aach RD, Szmuness W, Mosely JW, et al: Serum alanine ami-
notransferase of donors in relation to the risk of non-A, non-
13 hepatitis in recipients. N Engl J Med 1981; 304:989-994 
2. Alter HJ, Purcell RH, Holland PV, Ailing DW, Koziol DE: Donor 
transaminase and recipient hepatitis. JAMA 1981; 246:2340-
2344 ' 
3. Apple FS: Presence of creatine kinase MB isoenzyme during mar-
. athori training. J Engl J Med 1981; 305:764-765 
4. Brownlow K, Elevitch F: Serum creatine phosphokinase isoen-
zyme (CPK 2) in myositis: JAMA 1974; 230:1141-1144 
5. Holloszy JO: Adaptation of skeletal muscle to endurance exercise. 
Med Sci Sports 1975; 7:155-164 
6. Huseby NE; Stromme JH: Practical points regarding routine de-
termination of 7-glutamyl transferase in serum with a kinetic 
method at 37°C. Scand J Clin Lab .Invest 1974; 34:357-363 
7. Jockers-Wretou E, Pfleiderer G: Quanitation of creatine kinase 
isoenzymes in human tissues and sera by an immunological 
method. Clin Chem Acta 1975; 58:223-232 
8. Lott JA, Stang JM: Serum enzymes and isoenzymes in the di-
agnosis and differential diagnosis'of myocardial ischemia and 
necrosis. Clin Chem 1980; 26:1241-1250 
9. Morgenstern S, Flor R, Kessler G.̂ Klein B: The automated de-
termination of NAD-coupled enzymes. Clin Chem 1966; 
12:274-281 
10. Morgenstern S, Kessler G, Auerbach J, Flor R, Klein B: An au-
tomated p-nitrophenylphosphate-serujn alkaline phosphatase 
procedure. Clin Chem 1965; 11:876-882 
11. Pinto. PV, Kaplan A, VanDreal PA: Aldolase: spectrophotometry 
determinationusing an ultraviolet procedure. Clin Chem 1969; 
15:349-360 
12. Roberts R, Sobel BE: Creatine kinase isoenzymes in the assess-
ment of heart disease. Am Heart J 1978; 95:521-528 
13. Rosalki SB: An improved procedure for serum creatine phospho-
kinase determination. J Lab Clin Med 1967; 69:696-701 
14. Rosalki SB: Standardization of isoenzyme assays with special ref-
erence to lactate dehydrogenase isoenzyme electrophoresis. 
Clin Biochem 1974; 7:29-40 , ' 
15. Shell WE, Kligerman M, Rorke MP; Burhan M: Sensitivity and 
specificity of MB creatine kinase activity determined with col-
umn chromatography. Am J Cardiol 1979; 44:67-75 
 by guest on June 6, 2016
http://ajcp.oxfordjournals.org/
D
ow
nloaded from
 
http://ajcp.oxfordjournals.org/
Vol. 79 • No. 6 BRIEF SCIENTIFIC REPORTS 719 
16. Siegel A, Silverman L, Evans W, Madar D: Elevated skeletal mus-
cle creatine kinase MB isoenzyme levels in marathon runners. 
Med Sci Sports Exer 1982; 14:171 (Abstract) 
17. Siegel AJ, Silverman LW, Holman L: Elevated creatine kinase 
MB isoenzyme levels in marathon runners. JAMA 1981; 
246:2049-2051 
18. Sjodin B, Thorstensson A, Frith K, Karlsson J: Effect of physical 
training of LDH activity and LDH isoenzyme pattern in hu-
man skeletal muscle. Acta Physiol Scand 1976; 97:150-157 
The measurement of /J-thromboglobulin (BTG) and platelet 
lactic dehydrogenase (LDH) as markers for a-granule release 
and platelet lysis, respectively, has been reported to correlate 
with the amount of damage incurred during the preparation 
and storage of platelet concentrates. We compared the con-
centrations of these markers in the supernatant plasma of 
platelets stored on platform, elliptical, and circular rotators. 
Significant BTG release occurred after 24 hours on all three 
rotators (P < 0.01), but LDH discharge after 24 hours was 
significant (P < 0.05) only with elliptical rotation. BTG and 
LDH levels were not significantly different at any time when 
platform and circular rotations were compared, but both mark-
ers were significantly (P < .05) higher during elliptical rotation. 
The lower levels of BTG release and LDH discharge during 
platform and circular agitation implies that these rotators pro-
duce less in vitro platelet activation and damage than occurs 
with elliptical agitation. (Key words: /$-thromboglobulin re-
lease; Lactic dehydrogenase discharge; Platform, circular, and 
elliptical rotation) Am J Clin Pathol 1983; 79:719-721 
PLATELET VIABILITY during liquid storage has been 
evaluated by in vitro and in vivo methods to assess 
changes that occur in platelet concentrates under dif-
ferent storage conditions. Early studies established the 
importance of maintaining the pH greater than 6, with 
considerations in this respect being the type of plasti-
cizer, storage temperature, and the number of platelets 
in the concentrate.6,9 Platelet agitation also was found 
to be necessary for successful storage and various meth-
ods have been described.3'79 However, platelet altera-
tions were noted during agitation, with the damage pre-
Received August 23, 1982; received revised manuscript and ac-
cepted for publication October 11, 1982. 
Supported in part by a grant from National Red Cross Blood Ser-
vices. Washington, D.C. 
Address reprint requests to Dr. Kalmin: American Red Cross Blood 
Services, 1925 Monroe Drive, Atlanta, Georgia 30324. 
19. Storey JD, Sass ML, Grady HJ, Bailie EE, Jax SM, Moore JJ: An 
evaluation of a creatine kinase isoenzyme method for the 
duPont aca. duPont Company, August 1979 
20. Wilkenson JH, Baron DN, Moss DW, Walker PG: Standardiza-
tion of clinical enzyme assays: a reference method for aspartate 
and alanine transaminases. J Clin Pathol 1972; 25:940-943 
21. World Health Organization: Report on working group on isch-
emic heart disease registers. Annex I. WHO Regional Office 
for Europe. Copenhagen, 1971, pp 26-32 
American Red Cross Blood Services, Atlanta Region, 
Atlanta, Georgia 
dominately related to shear stress effects rather than to 
platelet-surface interactions.12 Studies showed a pro-
gressive release of/3-thromboglobulin (BTG) from plate-
lets at lower stress levels with a simultaneous, progressive 
increase of the platelet cytoplasmic constituent, lactic 
dehydrogenase (LDH), as measured in the supernatant 
plasma. Release of these platelet constituents has been 
correlated with a change in platelet morphology from 
the normal discoid to spherical shape, with more or less 
complete loss of normal intracellular structures. This 
finding has been used as an in vitro test to demonstrate 
platelet damage during preparation and storage of plate-
let concentrates.10" 
In order to determine the optimum type of agitation 
for platelet storage in this center, three types of rotators 
were compared, using in vitro measurement of BTG and 
LDH release from platelets stored on each rotator for 
72 hours. Variables in platelet concentrates were min-
imized by aliquoting a common donor unit into three 
equal portions for storage on each of the three rotator 
types. 
Materials and Methods 
Platelet Preparation 
Twelve normal blood donors, with no history of as-
pirin ingestion during the previous 72 hours, were bled 
into Fenwal PL-146 quad bags containing CPD adenine-
1 (Fenwal Laboratories, Deerfield, IL). Platelets were 
In Vitro Assessment of Platelet Damage 
During Rotator Storage 
NORMAN D. KALMIN, M.D., M. JANE WILSON, MT(ASCP)SBB, AND BOBBIE A. LILES, MT(ASCP)SBB 
0002-9173/83/0600/0719 $00.95 © American Society of Clinical Pathologists 
 by guest on June 6, 2016
http://ajcp.oxfordjournals.org/
D
ow
nloaded from
 
http://ajcp.oxfordjournals.org/