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(1988)
“Flipped”Patterns of Lactate Dehydrogenaselsoenzymesin Serum of Elite College Basketball
Players
hi Rotenberg,’ Richard Seip,2 Larry A. Wolfe,
and DavidE. Bruns4
We kinetically measured total lactate dehydrogenase (LD,
EC 1.1.1.27), total creatine kinase (CK, EC 2.7.3.2), and
aspartate aminotransferase (AST, EC 2.6.1.1.) in 16 elite
college basketball players, before the competition season
and not in close temporal relation to near-maximal exercise,
and in 17 healthy non-athlete controls. LD isoenzymes were
determined by both electrophoretic and immunoprecipitation
methods. CK-MB isoenzyme was measured electrophoretically. We found significantlly higher mean LD-1 values and
LD-1/LD-2 ratios in the players than the controls: 31.6 (SD
3.7)% vs 25.8 (SD 3.2)% (P <0.005) and 1.1 (SD 0.13) vs
0.87 (SD 0.16) (P <0.001), respectively. A “flipped” LD
pattern (LD-1 > LD-2) was found in half the players and in six
of the eight black athletes, but in only two of the control group
and in none of the black controls. Mean CK activity in serum
exceeded normal values in the serum of the athletes and was
higher in comparison with the control group [274 (SD 156) vs
103 (SD 82) U/L]. Mean CK was significantly higher in the
eight athletes with the flipped LD pattern than in those with
LD-1 <LD-2 [322 (SD 163) vs 180 (SD 98) U/L; P
0.05],
and also in comparison with CK in the two controls with
flipped LD pattern. We saw no significant difference in mean
CK between the nine players with normal immunochemical
LD-1/LD ratios and the seven players with above-normal
ratios. CK-MB was not detected in either athletes or controls.
Departments
of Pathology and2 Health and Physical Education,
Schools of Medicine and Education, University of Virginia, Charlottesville, VA 22908.
Present address: ‘Massada Center for Heart Diseases, Beiinson
Medical Center, Petah Tikva 49100, Israel; and3 School of Physical
and Health Education, Queens University,
Kingston, Ontario,
Canada, K7L 3N6.
‘Address correspondence to this authoi- Box 168, Department of
Pathology, University of Virginia Medical School, Charlottesville,
VA 22908.
Received June 29, 1988; accepted August 8, 1988.
None of the players had any clinical or electrocardiographic
evidence for myocardial ischemia or infarction. Evidently the
flipped LD pattern usually found in patients with acute
myocardial infarction and reported in some athletes after
extreme exercise such as ultra-marathon running may also
be found in athletes who are in their “basal fitness shape” but
who are not involved in competitive physical activity.
Numerous reports have been published on the increase in
serum enzyme activities following various types of exercise
(1-10). The most commonly described
increase
is that in
serum creatine kinase (CK, EC 2.7.3.2) (1-4, 6-9, 11).
Increases
in aspartate
aminotransferase
(AST, EC 2.6.1.1)
(1, 7) and lactate dehydrogenase
(LD, EC 1.1.1.27) (2, 6, 7,
11) have also been seen, but the changes were generally
smaller than for CK. Most studies have reported increased
LD only after vigorous exercise, such as marathon running.
Some (4, 9, 12) have even described a “flipped” LD pattern
(LD-1 > LD-2) in certain athletes after long-distance
running, similar
to that described
in patients
with acute
myocardial
infarction (13-16).
The present study was prompted by the unexpected
finding of abnormalities of both CK and LD isoenzymes in
college basketball players. In particular, five of 16 players
demonstrated
a ffipped LD ratio when the athletes underwent routine examinations
on the second day of team
practice. The present prospective study was undertaken
in
the same group a year later, to determine a “basal” proffle of
LD isoenzymes
before the competition season.
Patients and Methods
We studied 16 members of a highly ranked U.S. basket(mean age 20.3, SD 1.3 years), and 17 healthy, tall
ball team
5Nonstandard
abbreviations:
dehydrogenase; AST, aspartate
CK, creatine kinase; LD, lactate
aminotransferase.
CLINICALCHEMISTRY, Vol. 34, No. 11, 1988 2351
non-athletes
who agreed to act as control subjects (mean age
21.6, SD 2.2 years). Eight of the athletes and seven of the
controls were black. The players were evaluated in the late
fall, immediately
before the competition season but after
completion of pre-season physical conditioning. None of the
control subjects was a varsity athlete and none engaged in
intensive sports training,
but they were similar in height to
the varsity athletes other than the team’s starting
center.
Each subject (athletes and controls) underwent
stress
electrocardiography
and echocardiography
studies and no
signs of cardiovascular
pathology were found.
The foflowing blood tests were obtained in each subject,
12 h after the last bout of exercise: LI) and LD isoenzymes,
AST, CK, and CK isoenzymes.
Total LD activity in serum was assayed at 30#{176}C
by a
kinetic assay (17), pyruvate-to-lactate,
adapted to a centrifugal analyzer.
The reference interval
in our laboratory
is
100-350 UIL. The proportions of the LD isoenzymes were
determined as described previously (18) by use of electrophoreals with the “ACI System” (Corning Medical,
Medfield,
MA 02052). Reference
intervals
for the LI) isoenzymes,
expressed in percent of total LD, are: LD-1, 18-33%; LD-2,
28-40%; LD-3, 18-30%; LD-4, 6-16%; and LD-5, 2-13%. We
also quantified LD-1 by unimunoprecipitation
as described
earlier (18), using commercially
available reagents (Roche
Diagnostics,
Nutley, NJ 07110). The reference range for LD1 was 36-73 U/L and for LD-1/LD ratio 21-35% (18).
The activity of total CK was measured with use of an
optimized assay reagent based on that described by Szasz et
al. (19) and obtained
from Boehringer-Mannheim
(BioDynamics/bmc,
Indianapolis,
IN 46250). The reference interval in our laboratory is 0-110 UIL. CK-MB activity was
measured
by electrophoresis,
with the ACI system (20). AST
activity was measured
spectrophotometrically
in a centrifugal analyzer (21). The reference interval is 7-40 U/L.
We compared the above enzymatic variables in the players and control groups. Differences between means, assessed
by Student’s t-test, were considered significant at P <0.05.
Enumerative
data were evaluated
by chi-square
analysis of
2 x 2 contingency tables, with the Yates correction.
Results
Table 1 compares the enzymes and isoenzymes in serum
from the two groups studied. No differences between the two
groups were found for AST, total U), or LI) isoenzymes
2
and 3. However, mean electrophoretic LD-1 values were
significantly
higher in the athletes as compared with the
controls: 31.6 (SD 3.7)% vs 25.8 (SD 5.2)% (P <0.005). The
mean LD-1/LD-2 ratio also was significantly
higher in the
athletes: 1.1 (SD 0.13) vs 0.87 (SD 0.16) (P <0.01). Half of
the athletes showed a ifipped LI) pattern, as compared with
only two of the controls (P <0.05). Six of the eight players
with ffipped LI) pattern were black; the two controls with
this pattern were white.
The immunochemically
measured LD-1ILD ratio was also
significantly
increased
in the players as compared with the
controls. In seven of the athletes and in none of the controls
it exceeded the reference interval.
The mean CK activity
was above normal in sera of the athletes and higher than in
the control group. CK-MB was not detected in either group.
Table 2 gives the individual data for LD and CK in the
eight players and two controls with a ifipped LD pattern. All
players had CK values >110 UIL, although no CK-MB was
detected. The value for mean CK was significantly higher in
these eight players than in those with LD-1 <LD-2322
2352 CLINICALCHEMISTRY, Vol. 34, No. 11, 1988
Table 1. Serum Enzymes In ElIte College Basketball
Players vs a Control Group (Mean ± SD)
Basketball
players
(n = 16)
Total LD, U/L
LD-1/LD, % (E)
LD-2/LD, % (E)
LD-3/LD,% (E)
LD-4/LD, % (E)
LD-5/LD, % (E)
LD-1/LD-2 (E)
No. withflipped
LD
LD-1, U/L (I)
LD-1/LD,% (I)
No. with LD-1/LD
(I) >35%
AST, U/L
CK, U/L
CK-MB (E)
251
31.6
28.8
23.9
6.7
6.3
1.1
Controls
(n = 17)
91
P value
265
25.8
29.4
26
±
45
3.7
2.2
2.7
1.4
2.5
±
0.13
0.87 ± 0.16
<0.001
2
<0.05
NS
<0.005
±
±
±
±
±
±
±
±
±
5.2
1.9
3.6
10.3 ± 3
8.3
±
2.8
8
89±16
73±23
36±5
28±4
7
0
21±6
274 ±
16±3
103 ±
156
NS
<0.005
NS
NS
<0.001
<0.05
<0.01
NS
82
<0.005
Negative
Negative
“E, electrophoretcmethod;I, immunochemical
method;NS, nots;gnificant.
(SD 163) vs 180 (SD 98) U/L (P = 0.05)-and
also in
with the two controls with the ffipped LI)
pattern. No difference in mean CK values was found between the two controls with a flipped LD pattern [65 (SD
4.2) U/L] and the 15 controls with LD-1 <LD-2 [111 (SD 88)
U/LI.
Table 3 shows the serum enzymes in relation to the
immunochemical
LD-1 ratio. No significant difference was
found in mean LD or CK or in the LD-1/LD-2 ratio between
the nine players with normal LD-1/LD ratio and the seven
with an increased ratio. A significant difference was found,
however, both in the mean LD-1/LD-2 ratio and the mean
CK between the seven players with an increased LD-1/LD
ratio and the control group, all with normal LD-1/LD ratio.
comparison
DIscussIon
Estimation of the isoenzymes of LD in serum in combination with CK has become a well-established
laboratory
procedure
for helping in the diagnosis of acute myocardial
infarction
(12). The most common methods for measuring
U) isoenzymes are of two major types: the electrophoretic
(22) and the immunochemical
(23), which measures LD-1
only. The myocardium
has a preponderance of LD-1 with
lesser amounts of LD-2. Necrosis of the myocardium
results
in the release of relatively more U)-1 than LD-2 into the
blood, reversing (ifippung) the normal ratio of U)-1 to LD-2
in serum. A similar situation may occur with hemolysis and
renal injury. After acute myocardial infarction, the LD-1/LD
and LD-1/LD-2 ratios in serum increase, usually within 24 h
(24,25), and U) assumes the LD-1 >LD-2 proffle, usually
between 24 and 48 h after the infarct (12). A similar ffipped
U) pattern has been described only after extreme physical
activity, and in only a few subjects. Kaman et al. (4)
reported a ffipped U) pattern in five trained healthy middleaged men 8 h after near-maximum
runs of eight to 10 miles.
Basal values for LD isoenzyme
activities were within the
normal reference interval. They reported also a large increase in CK values attributable to the skeletal muscle type
(MM) alone. Kielbiock et al. (9) sampled blood from 20
runners
on completion or partial completion of a 160-km
marathon
(ultra long distance running) and could detect
CK-MB in the sera of 65% of these subjects. Five subjects
(25%) had a flipped LD pattern. Three subjects exhibited
CK-MB in the presence of a ffipped U) pattern. No electro-
Table 2. IndIvidual Lactate Dehydrogenase and Creatlne Kinase Data In 10 SubJects with a Flipped LD-1/LD-2
Pattern
Race
Total LO, OIL
LD-1/LD-2 (E)’
LD-1, O/L (I)
LD-1/LD, % (I)
CK, O/L
CK-MB (E)
1
2
3
4
5
7
B
B
B
B
B
B
288
211
190
310
244
264
1.01
1.19
1.23
1.19
1.21
1.35
100
81
90
105
88
102
36
38
47
34
36
39
Negative
9
C
239
1.13
10
C
290
1.06
76
97
32
34
540
402
264
546
338
173
133
Subject
A. Athletes
Mean
±
SD
253
±
40
1.17 ± 0.10
92 ± 10
1.28
38
180
322 ± 163
4.6
37 ±
B. Controlgroup
1
9
C
C
141
27
28
214
60
1.08
Mean±SD
177±52
1.18±0.14
49±16
a, electrophoretic method; I, immunochemicalmethod; B, black; C, caucasian (white).
Negative
68
62
28±1
65±4.2
Table 3. Serum Enzymes In Relatlon to immunochemical Lactate Dehydrogenase-1 Ratio
Play..,
LD-1/LD >35%
LD-1/LD <35%
No.
7
9
LD-1/LD (I)
39 ± 4
32 ± 2
LD, U/L
238 ± 49
270 ± 37
LD-1/LD-2 (E)
1.12 ± 0.15
1.06 ± 0.10
CK, U/L
287 ± 149
258 ± 182
1E, electrophoretic; I, Immunochemical;
NS, notsignificant.
cardiographic evidence of irreversible cardiac damage could
be established after the race. Apple and McGue (12) measured blood enzyme activities that might be suggestive of
organ-specific damage over a six-week period in two male
long-distance
runners training for a marathon.
Total CK
activity was supranormal
in both. One of them exhibited a
flipped LD-1/LD-2 ratio, which paralleled his persistent
CKMB increase. By contrast, Rumley et al. (26) reported no
ifipped pattern in 38 sedentary middle-aged men after a 30week marathon
training
program.
The most striking feature in our study is that, although
the baskethall players were evaluated in their “basal fitness
shape”-i.e.,
before the competition season-half
of them
showed a typical ffipped U) pattern. Although CK-MB
activity was normal in all players, mean total CK was
higher in the “flipped” LI) group of players than in the “nonflipped” group of players and it was higher in comparison
with mean CK in the two controls with a flipped U) pattern.
Although most of the players with the flipped pattern were
black, the abnormal patterns in these black athletes did not
reflect simply a race-related
difference in reference intervals, because none of the black control subjects showed a
flipped ratio.
The methodology
used in detecting LI) isoenzymes is
highly important.
There may be method variation in the
electrophoretic
method used (27) and, of course, between the
immunochemical
method compared with the electrophoretic. We showed previously (18) that the U)-1/LD ratio
provides a good separation between the LD-1 > LD-2 and
LD-1 <LD-2 categories as determined by the electrophoretic method. However, there is some overlap, which is to be
expected because the two assays give somewhat different
information. Thus, when we separated the players into two
subgroups according to LD-1ILD-2 ratio, we found the mean
value for CK to be significantly higher in the eight players
P
value
Control LD-1I
LD <35%
P value: controls vs
players with LD-11
LD >35%
17
0.002
28
NS
265
NS
± 4
± 91
± 0.16
0.87
103 ± 82
NS
0.0002
NS
0.002
0.001
with a ffipped U) pattern than in the eight with LD-1 <U)2-whereas
when we separated the data on the players into
two groups according to U)-1/LD ratio, no significant difference was found in mean CK values between the two
subgroups of athletes.
What pathophysiological
mechanisms
could explain the
ffipped LI) pattern in our group of players?
Intravascular
hemolysis due to sustained physical exertion has been described (9) and could explain the increased
ID-i. Although we saw no clinical or laboratory signs of
hemolysis in our group of subjects, we cannot exclude
subclinical mild hemolysis as a possible cause for the flipped
LI) pattern. Another mechanism
has been suggested by
Rumley et al. (26, 28). They have shown that endurance
athletes have more slow-twitch
muscle fibres than nonathletes and that these fibers have a higher proportion of
the LD-H subunits than do fast-twitch
fibers. Thus one
might expect to find, in the serum of athletes, a higher
proportion of those isoenzymes that contain predominantly
the H subunits (U)-1 and U)-2). Alternatively,
extreme
physical stress may cause release of enzymes from the heart
in the absence of acute myocardial
infarction
(13) and
produce a ffipped U) pattern.
This explanation
seems
unlikely in our players, because no CK-MB was detected.
Finally, severe rhabdomyolysis
reportedly produced ffipped
LD-1JU)-2 ratios in two patients (29,30), in contrast to the
more common finding of increased LD-5 in such patients.
Possibly, the athletes
in our group experienced
muscle
trauma from unofficial off-season games or other activities,
but none had renal failure or other evidence of severe
rhabdomyolysis
as was seen in the two rhabdomyolysis
patients reported to have ifipped ratios (29, 30).
In conclusion, even if we are unable to give at the moment
a single satisfactory explanation for the ffipped LI) pattern,
CLINICAL
CHEMISTRY,
Vol. 34, No. 11, 1988
2353
it must be recognized that such a pattern may be found, not
only in the serum of athletes after performing
extreme
exercise but also in athletes
who are in their “basal fitness
shape” but not involved in extreme or unaccustomed
physical activity. When such a pattern is found, other clinical and
enzymatic measurements
such as CK-MB and immunochemical ID-i determination
should be undertaken,
to
exclude acute myocardial infarction. When results of other
tests are negative, the athlete can be assured that he has a
“benign” athletic
pattern of U) isoenzymes.
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