Eccentric exercise and muscle damage markers • 39

Evaluation of serum leaking enzymes andinvestigation into new biomarkers for exerciseinduced muscle damage
Kazue Kanda1, Kaoru Sugama2,3, Jun Sakuma4, Yasuo Kawakami5,
Katsuhiko Suzuki1,5
1 Cooperative Major in Advanced Health Science, Tokyo University ofAgriculture and Technology/ Waseda University, Japan.

2 Consolidated Research Institute for Advanced Science and Medical Care

(ASMeW), Waseda University, Japan.

3 Division of Cell Regeneration and Transplantation, Department of Functional

Morphology, Nihon University School of Medicine, Japan.

4 Institute for Sport Sciences, Waseda University, Japan.
5 Faculty of Sport Sciences, Waseda University, Japan.

ABSTRACTThis investigation determined whether existing muscle damage markers andorgan damage markers respond to an acute eccentric exercise protocol and areassociated with affected muscle symptoms. Nine healthy-young men completed
one-leg calf-raise exercise with their right leg on a force plate. They performed
10 sets of 40 repetitions of exercise at 0.5 Hz with a load corresponding to half of
their body weight, with 3 min rest between sets. The tenderness of medial gastrocnemius, lateral gastrocnemius and soleus, and the ankle active range of motion(ROM) were assessed before, immediately after, 24 h and 48 h, 72 h, 96 h and
168 h after exercise. Blood and urine were collected pre-exercise and 2 h, 4 h, 24
h, 48 h, 72 h and 96 h post-exercise. Serum was analyzed for creatine kinase(CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate
dehydrogenase (LDH) and aldolase (ALD) activities. We also determined hearttype fatty acid-binding protein (H-FABP), intestinal-type fatty acid-binding pro-

Address for correspondence:
Katsuhiko Suzuki, MD, PhD, Faculty of Sport Sciences, Waseda University,
2-579-15, Mikajima, Tokorozawa, Saitama 359-1192, Japan.
Katsuhiko Suzuki, Adjunct Professor, Email katsu.suzu@waseda.jp
EIR 20 2014

40 • Eccentric exercise and muscle damage markers
tein (I-FABP) and liver-type fatty acid-binding protein (L-FABP), neutrophil
gelatinase-associated lipocalin (NGAL), interleukin (IL)-17A, IL-23, nerve growth factor (NGF), soluble-Endothelial (sE)-selectin, s-Leukocyte (L)-selectin, sPlatelets (P)-selectin, and 8-isoprostane in plasma and urine. The tenderness ofproximal and middle gastrocnemius increased significantly 72 h (p<0.05,
p<0.01) after exercise. Ankle active ROM in dorsal flexion decreased significantly 48 h (p<0.05) and 72 h (p<0.01) after exercise. CK and ALD activities significantly increased at 72 h (p<0.05) and remained elevated at 96 h (p<0.01) postexercise compared to pre-exercise values. Also, ALD which showed relatively
lower interindividual variability was significantly correlated with tenderness of
middle gastrocnemius at 72 h. LDH activity significantly increased 96 h postexercise (p<0.01), whereas the increase in AST and ALT activities 96 h post-exercise was not significantly different from pre-exercise values. There were no significant changes in FABPs, NGAL, IL-17A, IL-23, NGF, selectins and 8-isoprostanes in plasma and urine. In conclusion, calf-raise exercise induced severe local
muscle damage symptoms which were accompanied by increases in both serum
CK and ALD activities, but we could not detect any changes in examined markers
of organ damage, inflammation and oxidative stress. Further research is needed
to determine other more sensitive biomarkers and the underlying mechanisms of
exercise-induced muscle damage.

Key words: exercise-induced muscle damage, organ damage markers, acuteeccentric exercise, creatine kinase, aldolase

INTRODUCTION

Exercise-induced muscle damage has been one of the most important targets insport science research. Direct assessment of muscle damage involves histologicalexamination of muscle tissue via biopsy. However, there is difficulty gatheringtissue samples with biopsy technique and demonstrating disruption of muscle
cells (1, 3, 48), in combination with the process being an invasive experience forresearch participants. Therefore, the less invasive measures for the accurate
assessment of exercise-induced muscle damage are needed. As indirect indicatorof muscle damage, delayed-onset muscle soreness (DOMS) is a poor reflector of
eccentric exercise-induced muscle damage, and changes in other indicators ofmuscle damage are not necessarily accompanied by DOMS (27). On the other
hand, the changes of range of motion (ROM) have been used as an indirect variable of DOMS (8, 9). Although a large number of studies have reported theeffects of exercise on muscle damage, DOMS and inflammatory responses inhumans (26, 30, 31, 34, 41, 44, 46), the mechanisms of exercise-induced muscle
damage are not fully understood at present. DOMS is characterized by tenderness
and movement-related pain, that is, mechanical hyperalgesia in the exercisedmuscle (22), and ROM might be affected by muscle soreness (33). Therefore, it isnecessary to assess the muscle symptoms in a multifaceted manner, and investigate the associations with other muscle damage markers carefully.
Serum enzyme activities such as creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase
EIR 20 2014

Eccentric exercise and muscle damage markers • 41
(LDH), aldolase (ALD) and myoglobin which leak into circulation from damaged muscle have been used as indirect markers of exercised-induced muscle
damage (5, 28). Serum levels of these damage indicators depend on gender, muscle mass, exercise intensity and duration in addition to the individual training
state, and there is a remarkable interindividual variability in the degree to which
serum enzyme activities increase with exercise (30). Instead of the existing leaking enzymes, fatty acid-binding protein (FABP) and neutrophil gelatinase-associated lipocalin (NGAL) have been introduced as organ damage markers. FABPs
are present in almost all tissues, and were named after the tissue in which they
were discovered or are prominently expressed: L-FABP, liver-type fatty acidbinding protein; I-FABP, intestinal-type fatty acid-binding protein; H-FABP,
heart-type fatty acid-binding protein (50). H-FABP is mainly expressed in theheart, but to a lesser extent also in the skeletal muscle (47). NGAL was identified
as a 25 kDa protein secreted by neutrophils (45), and is expressed in human tissues, including kidneys, lungs, stomach, and colon (10). NGAL is focused inrecent years as a biomarker in several benign and malignant diseases, especiallyas biomarker in acute kidney injuries (11), and may have the potential to protect
against cellular injury mediated by reactive oxygen species (ROS) (36, 37).
Although we have observed that neutrophils produce ROS following exercise(14, 42-44), NGAL might become a good alternative variable which is easier tomeasure simply in plasma and urine by conventional enzyme-linked immunosorbent assays (ELISA) than the existing neutrophil functional analyses that containrather complex procedures and need to be determined ex vivo soon after blood
sampling.
Exercise-induced muscle damage causes local inflammation which degenerates and regenerates muscle and surrounding connective tissue (30). Briefly,
neutrophils are mobilized into the circulation after exercise, and soon infiltrate
into the damaged tissue (2). Neutrophils are primed by the chemoattractants suchas complement 5a (C5a) and interleukin (IL)-8 from the exercise-induced damaged muscle, causing rolling, arrest and transmigration which is mediated byintercellular adhesion molecules, selectins (16), initially. Neutrophils are present
in muscle within a day after exercise (3, 17, 18, 19, 34, 40), and after neutrophils’
infiltration, macrophages are replaced and present in muscle from 1 to 14 days
after exercise (2, 3, 12, 13, 19, 32, 38). Neutrophils and macrophages produce
ROS to degrade the damaged muscle tissue (23, 24), and may produce proinflammatory cytokines (6). The pro-inflammatory cytokines such as IL-1β,tumor necrosis factor (TNF)-α, and IL-6 are expressed in skeletal muscle following eccentric exercise (30), but we could not detect any changes of thesecytokines in plasma and urine in our previous study (14). On the other hand,
Sugama et al. reported that IL-17 induced by IL-6 and activated by IL-23 might
promote neutrophil activation and muscle damage in a different way from theclassical pro-inflammatory cytokines IL-1β and TNF-α following prolonged
endurance exercise (41). Furthermore, nerve growth factor (NGF) which is
involved in pathological pain conditions (15, 49) increased in the muscle after
lengthening contraction in rats (22). 8-isoprostane is a prototypical biomarker ofoxidative stress by exercise-induced free radical production (21). Fluctuations ofthese substances may be detected following exercise-induced muscle damage andinflammation, and can be one of the underlying mechanisms of it.
EIR 20 201442 • Eccentric exercise and muscle damage markers
We have shown in a previous study that increased myoglobin concentration
at 72 h after exercise was correlated with the appearance of DOMS at 72 h(r=0.73, p<0.05), and there was a positive correlation between exercise-induced
increases in neutrophil migratory activity at 4 h and increases in myoglobin at 48
h (r=0.67, p<0.05) following one-leg calf-raise exercise (14), suggesting that neutrophils may be involved in muscle damage and that myoglobin is a very sensitive
muscle damage marker. Based on these findings, the aims of the present study
were to examine 1) changes in serum leaking enzyme activities and their correlation with DOMS, 2) concentrations of novel organ damage markers, novel proinflammatory cytokines and an oxidative stress marker and their relationship tomuscle damage, and 3) urinary excretion rates of these markers as potential surrogate non-invasive indicators of muscle damage, using a local muscle damage
model of one-leg calf-raise exercise.

METHODS

Subjects
Nine untrained healthy males (age 24.8±1.3 (mean±SD) yrs, body mass 62.3±6.3
kg and height 1.72±0.05 m) volunteered for this study. At the time of the study,
the subjects had not been involved in any hard exercise or resistance training forat least two weeks before the exercise bout, and were not taking any supplements,
or participating in recovery strategies such as massage, stretching or cryotherapy.
The subjects were instructed to maintain their usual daily schedule during theexperiment. All subjects completed a medical questionnaire and gave written
informed consent. The experimental procedure was approved by the ethics committee of Waseda University.

Experimental design
Subjects performed a calf-raise exercise, including repetitive eccentric muscle
contractions with their right leg on a force plate. The range of motion of the ankle
joint during the exercise was regulated from -20° (dorsiflexion position) to 15°(plantar flexion position) using a goniometer (SG 110/A, Biometrics, Newport,UK) with its ends attached onto the skin over the tibia and calcaneus. They performed 10 sets of 40 repetitions of exercise at 0.5 Hz with a load corresponding to
the half of their body weight, with 3 min rest between sets. The tenderness of theexercised muscle correlative to DOMS was assessed using the FP meter (SN-402,
Navis, Japan) at 1 kg and rated subjectively using a visual analogue scale (VAS)
that has a 100-mm line with “no pain” on one end and “extremely sore” on theother. The points of measurements were the proximal, the middle and the distal
points of medial gastrocnemius (MG) and lateral gastrocnemius (LG), the middle
points between MG and LG, and the middle and the distal points of soleus (SOL).
The ankle active ROM was assessed using the goniometer. The tenderness of theexercised muscle and the ankle active ROM were assessed before, immediately
after 24 h and 48 h, 72 h, 96 h and 168 h after and exercise. The blood and urine
samples were collected before and 2 h, 4 h, 24 h and 48 h, 72 h and 96 h after the
exercise. Participants were supposed to urinate 2 h before each sampling, and theurine samples were collected in measuring cylinders.
EIR 20 2014

Eccentric exercise and muscle damage markers • 43
Blood and urine sampling and analyses
Approximately 12 ml of blood was drawn by standard venipuncture techniquefrom the antecubital vein. Blood samples were collected into serum separation
tubes and vacutainers containing heparin and EDTA. A portion of whole blood
was used to measure haemoglobin, haematocrit and complete blood cell counts
using an automatic blood cell counter (PocH100i, Sysmex, Kobe, Japan). The
serum separation tubes were left to clot at room temperature for 30 min, and thevacutainers containing EDTA for plasma separation were immediately centrifuged at 1000×G for 10 min. Serum and plasma samples were then removed
and stored at –80°C for later analyses. Serum CK, AST, ALT, LDH and ALD
activities were measured using an automated analyzer (Model 747-400, Hitachi,
Tokyo, Japan). Urine samples were centrifuged immediately at 1000×G for 10
min to remove sediments, and the supernatants were stored at –80°C for later
analyses. Urinary concentration of creatinine was measured using an automatedanalyzer (Model 747-400, Hitachi, Tokyo, Japan). Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the plasma and urine concentrations of H-FABP, I-FABP, L-FABP and NGAL (Human H-FABP, I-FABP, LFABP and NGAL ELISA kit, Hycult biotechnology, Uden, The Netherlands),
IL-17A (Human IL-17A ELISA kit, Gen-Probe Diaclone SAS, Besancon,
France), IL-23 (Human IL-23, R&D Systems, Inc. MN, USA), nerve growth factor (NGF) (Human Nerve Growth Factor ELISA kit, Cusabio Biotech Co. Ltd.,
Wuhan, China), E-selectin (Soluble), sL-selectin and sP-selectin (Life technologies Co. CA, USA) and 8-isoprostane (Detroit R&D, Inc., MI, USA). The measurements were performed according to the instructions for each ELISA kit using a
microplate reader (VERSAmax; Molecular Devices, Sunnyvale, CA, USA). The
urinary data were corrected for the gross amount (raw concentration × urine volume) per minute (excretion rate).

Statistical analysis
Data are presented as mean ± SD. Statistical validation was made using Friedman’s test. When significant time effects were evident, multiple comparisonswere analyzed with Scheffe test. Associations between data were analyzed with
Pearson’s correlation coefficient (r). Statistical significance was accepted at
p<0.05.

RESULTS

Changes in the tenderness of the exercised muscle, and the ankle joint active
range of motion (ROM)As shown in Table 1, tenderness of the exercised muscle developed on subsequent days after calf-raise exercise and was principally sensed on the MG. It
increased significantly 72 h (proximal MG; p<0.05, middle MG; p<0.01) after
exercise compared with the pre-exercise values. The ankle joint active ROM in
the dorsal flexion decreased significantly at 48 h (p<0.05) and 72 h (p<0.01). The
lowest ROM was shown at 72 h after exercise in dorsal flexion, whereas there
was no significant difference in plantar flexion compared with the pre-exercise
values.
EIR 20 2014

44 • Eccentric exercise and muscle damage markers
Changes in the muscle and the organ damage markers
As shown in Table 2, CK and ALD activities significantly increased at 72 h after
exercise (p<0.05), thereafter, they remained elevated for 96 h after exercise(p<0.01). LDH activity significantly increased at 96 h after exercise (p<0.01).
However, AST and ALT activities showed no significant difference when compared with pre-exercise values. As listed in Table 3, plasma concentrations of HFABP, I-FABP and L-FABP were below the detectable levels of the assays andurinary concentrations exhibited no significant change. Plasma and urinary
NGAL were not significantly different compared with pre-exercise values.

Changes in the pro-inflammatory cytokines and other indicators
As shown in Table 4, plasma concentration of IL-17A was not significantly
changed and urinary excretion rate of IL-17A was below the detectable level.
Plasma concentration of IL-23 was below the detectable level, whereas urinary
excretion rate of IL-23 showed no significant change. Plasma sE-selectin concentration was below the detectable level, and sL-selectin and sP-selectin concentrations were not significantly different. Plasma concentration of 8-isoprostane was
below the detectable level, and urinary excretion rate of 8-isoprostane and plasma
concentration of NGF did not change significantly.

Relationships between enzyme activities and the tenderness of exercisedmuscle and the ankle active range of motions (ROM)As shown in Table 5, there were no significant correlations between the tenderness of proximal MG 72 h and CK, LDH and ALD for the percent changes of thepeak values. The tenderness of middle MG 72 h was significantly correlated with
ALD 72 h (r=0.78, p<0.05). The dorsal flexion of ankle active ROM was not significantly correlated to the tenderness of MG or to CK, LDH and ALD.

DISCUSSION

Muscle-derived leaking enzymes such as CK, AST, ALT, LDH and ALD havebeen used in many studies as indirect markers of exercise-induced muscle damage. However, there are generally poor correlations between DOMS and muscle
damage indicators (27). In this study, CK and ALD were significantly increased
earlier than LDH. Also, ALD showed relatively lower interindividual variations
and was significantly correlated with tenderness of middle MG at 72 h, whereas
there were only moderate, non-significant correlations between tenderness andthe other leaking enzyme activities. These results suggest that ALD rather than
CK and LDH might be a more accurate and objective muscle damage indicator.
There are a few studies that have investigated the organ damage markers,
FABPs and NGAL following exercise-induced muscle damage. FABPs are present in almost all tissues (50), but H-FABP is present in skeletal muscle (47).
Sorichter et al. reported that plasma H-FABP and myoglobin increased earlier (30min) than CK (2 h) following 20 min of downhill running (39). These results suggest that plasma H-FABP concentrations reflect exercise-induced muscle damage
earlier, but plasma H-FABP seems to have no advantage in view of its similarityof changes in the appearance of myoglobin. Also, the change might be attributed
EIR 20 2014








   

 



 
 




                 



 
 

                
  



 
 

 
                                     
          





  


 



  
                              



 
 

              
  

          !"    # $ %&&'( $$%&&(  )      *     *(  )  +  & ( ,-   +  ,- ( -.   +  -. ( /,  +  /, ( "0   +  "0 ( 0.   +  0. (                
 
   

 

 




   

    





 



 



   

Eccentric exercise and muscle damage markers • 45

EIR 20 2014

EIR 20 2014



#$

#$

#$

#$

&'(

&)(

)*+

&)*

%  %%  %

%  %

%  %

%   %


%

%

%  %% 

%  %

 %  %

% %  %

 %  %

%  %

 %  %%  %        2 3

%  %

%  %

%  %

%  %

 %  %%

%  %

 % %  %

%  %%    %

  ,   
 
%



%  %

 %  %

%  %

% 





% 

%

%  % 

%  % %  %          !  "# !  $ %  & ''() %%  & '')   *     +     +) -   ,  - ) .   ,  . ) -.   ,  -. ) ./  ,  ./ ) 0-   ,  0- ) #1   ,  #1 )   2    4)      5!)        56)   *    67)   56

#$



!"

 -46 • Eccentric exercise and muscle damage markers

$
$

$
$

) -&. ) -&. #/1&) 
/1&) #/* /*
%  %/* 

%  %
%  %







 %  %
%  %/*   /*%  %/*  

%  %
%  % /*  

/*%  %/*  

 %  %
%  %

/*   /* %  %/*
  

%  %
%  %/*   /*%  %/*   

%   %%  %/*   /*
%  %/*   %  %
%  % /*   /*%  %/*         !  "#    +) -   ,  - ) .   ,  . ) -.
  ,  -. ) ./   ,  ./ ) 0-   ,  0- ) #1   ,  #1 )  3     9)   +)   :)  7) ;    ;)  6) * ,  <5=+)     ,   9>56: *  3        3    ,        

$
$

0 -&. 
0 -&. #       *  2     $
$

+ -&. + -&. # 8Eccentric exercise and muscle damage markers • 47

EIR 20 2014

EIR 20 2014

$
$
$
$

$
$

/* %   %
 %  %
%  %

/*%  %

/*%  %%  %
/*



/*
 %   %%  %
%  %

/*
%  %/*%  %

%  %/*/*
 %  %
%  %%  %

/*%  %/*
%  %%  %/*/* %  %
%  %
%  %/*
 %  %/*
%  %%   %
/*/*%  %%  %%  %/*
 %  %/*%  %

%  %/*/* %   %%  %%  %/*
%  %

/*
%  %

%  %
/*



/*
 %  %
%  %%  %/*
%   %

/*
%  %%  %
/*   

      !  "#    +) -   ,  - ) .   ,  . ) -.
  ,  -. ) ./   ,  ./ ) 0-   ,  0- ) #1   ,  #1 )  3     9)   +)   :)  ?    ?)  62*,?   6)  +  + : *  3        3    ,    

2 3 ) 3  3 /4 5 3   #$
$

0)  
0)  #       *  * 2 $$0) & 0) & # ..

48 • Eccentric exercise and muscle damage markers



  







 

















 

















 

































 

































 

















                                  
!     "# #         $%&&'      $()'  #    $*+,'    $*+'        $-.' 
     $--.'            $/0-' 12       $12 ' 34      $34 ' 56       $56 ' 









 

 

 









 



 

 

 

                              

Eccentric exercise and muscle damage markers • 49

EIR 20 2014

50 • Eccentric exercise and muscle damage markers
to not only eccentric exercise-induced local muscle damage, but also systemic
factors such as dynamic exercise-induced haemoconcentration. In this study, oneleg calf-raise exercise caused severe muscle damage as shown by the precise local
assessment of the muscle symptoms, but we could not detect any changes in
FABPs and NGAL nor any correlations with the symptoms. In any case, theseorgan damage markers are not considered to be indicative of muscle damage
resulting from eccentric exercise loading on the calf, thus they cannot be used as
local muscle damage markers at least.
Endurance exercise induces peripheral blood neutrophilia (41-44), andenhances the capacity of neutrophils to produce ROS (29, 42-44). In a study using
myeloperoxidase (MPO) knockout mice, exercise-induced muscle damage wasfacilitated by MPO-containing neutrophils and their activating factors such as
proinflammatory cytokines (25). On the other hand, Maruhashi et al. reported thatthe antioxidant capacity was affected by the type and intensity of exercise, specifically, low-load eccentric exercise did not reduce antioxidant capacity, but conversely low-load concentric exercise temporarily reduced antioxidant capacity(20). We investigated neutrophil activation-related markers such as selectins, 8isoprostane, IL-17A and IL-23, but they were not significantly changed, suggesting that inflammatory responses and oxidative stress were not changed, at least, inthe examined markers following one-leg calf-raise exercise. The enhanced eccentric exercise-induced neutrophil migratory activity independent of ROS production and MPO degranulation observed in our previous study (14) might be due tothe mobilization of functionally different heterogeneous neutrophils possibly
from the bone marrow reserve (42, 44). Since we could not detect any significant
changes of a wide range of proinflammatory and oxidative stress markers in thisexercise mode other than neutrophil mobilization and migration (14), it is necessary to focus more on the involvement of this earlier step of inflammation as theunderlying mechanisms and the point of target for potential preventive countermeasures against exercise-induced muscle damage in the future studies.
In conclusion, it is confirmed in the present study that not only are serum
CK but also ALD activities more reliable indicators for exercise-induced muscle
damage than the other examined variables, but there are lower correlations withmuscle symptoms, and novel organ damage markers of FABPs and NGAL could
not be alternative indicators for muscle disruption, neutrophil mobilization andmigration. Also, we could not detect any perturbations of novel proinflammatory
cytokines and soluble adhesion molecules, and the inflammatory mechanisms arestill not clear. Therefore, further research is needed to determine whether there
are more sensitive indicators including urinary biomarkers as non-invasive
assessment of exercise-induced muscle damage and the underlying mechanisms
as well.

ACKNOWLEDGEMENTS

The authors thank volunteers who participated in the study. Also, we are very
grateful for the technical support of Dr. Noriaki Kawanishi and Dr. Masaki Takahashi, and for the English editing of Dr. Cecilia Shing. This study was supported
by grants from the Ministry by Education, Culture, Sports Science and TechnoloEIR 20 2014

Eccentric exercise and muscle damage markers • 51
gy of Japan, the Grant-in-Aid for the Global COE Program “Sport Science for the
Promotion of Active Life”, the Scientific Research (A) 23240097, (B) 21300217,
and Challenging Exploratory Research 24650410.

REFFERENCES
1.
2.

3.

4.

5.
6.
7.

8.

9.
10.

11.
12.

13.

14.

15.

Armstrong, R. B. Mechanisms of exercise-induced delayed onset muscular soreness:a brief review. Med. Sci. Sports Exerc. 16: 529-538. 1984.
Beaton, L. J., D. A. Allan, M. A. Tarnopolsky, P. M. Tiidus, and S. M. Phillips. Contraction-induced muscle damage is unaffected by vitamin E supplementation. Med.
Sci. Sports Exerc. 34: 798-805. 2002.
Beaton, L. J., M. A. Tarnopolsky, and S. M. Phillips. Variability in estimating eccentric contraction-induced muscle damage and inflammation in humans. Can. J. Appl.
Physiol. 27: 516-526. 2002.
Beaton, L. J., M. A. Tarnopolsky, and S. M. Phillips. Contraction-induced muscle
damage in humans following calcium channel blocker administration. J. Physiol.
544: 849-859. 2002.
Brancaccio, P., N. Maffulli, R. Buonauro, and F. M. Limongelli. Serum enzyme
monitoring in sports medicine. Clin. Sports Med. 27(1): 1-18. 2008.
Cannon, J. G., and B. A. St. Pierre. Cytokines in exertion-induced skeletal muscle
injury. Mol. Cell Biochem. 179: 159-167. 1998.
Chakraborty, S., S. Kaur, S. Guha, and S. K. Batra. The multifaced roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer.
Biochim. Biophys. Acta. 1826(1): 129-69. 2012.
Chapman, D., M. Newton, P. Sacco, and K. Nosaka. Greater muscle damage inducedby fast versus slow velocity eccentric exercise. Int. J. Sports Med. 27: 591-598.
2006.
Clarkson, P. M., K. Nosaka, and B. Braun. Muscle function after exercise-induced
muscle damage and rapid adaptation. Med. Sci. Sports Exerc. 24: 512-520. 1992.
Cowland, J. B., and N. Borregaard. Molecular characterization and pattern of tissue
expression of the gene for neutrophil gelatinase-associated lipocalin from humans.
Genomics. 45(1): 17-23. 1997.
Halawa, A. The early diagnosis of acute renal graft dysfunction: A challenge weface. The role of novel biomarkers. Ann. Transplant. 16(1): 90-98. 2011.
Hamada, K., E. Vannier, J. M. Sacheck, A. L. Witsell, and R. Roubenoff. Senescenceof human skeletal muscle impairs the local inflammatory cytokine response acuteeccentric exercise. FASEB. J. 19: 264-266. 2005.
Jones, D., D. Newham, J. Round, and S. Tolfree. Experimental human muscle damage: morphological changes in relation to other indices of damage. J. Physiol. 375:
435-448. 1986.
Kanda, K., K. Sugama, H. Hayashida, J. Sakuma, Y. Kawakami, S. Miura, H. Yoshida, Y. Mori, and K. Suzuki. Eccentric exercise-induced delayed-onset muscle soreness and changes in markers of muscle damage and inflammation. Exerc. Immunol.
Rev. 19: 74-87. 2013.
Lewin, G. R., and L. M. Mendell. Nerve growth factor and nociception. Trends Neurosci. 16: 353-359. 1993.
EIR 20 2014

52 • Eccentric exercise and muscle damage markers
16.

17.

18.

19.

20.

21.

22.

23.

24.
25.

26.

27.

28.

29.

30.

Ley, K., C. Landanna, M. I. Cybulsky, and S. Nourshargh. Getting to the site of
inflammation: the leukocyte adhesion cascade update. Nat. Rev. Immunol. 7: 678689. 2007.
MacIntyre, D. L., W. D. Reid, D. M. Lyster, I. J. Szasz, and D. C. Mckenzie. Presence of WBC, decreased strength, and delayed soreness in muscle after eccentric
exercise. J. Appl. Physiol. 80: 1006-1013. 1996.
MacIntyre, D. L., W. D. Reid, D. M. Lyster, and D. C. Mckenzie. Different effects ofstrenuous eccentric exercise on the accumulation of neutrophils in muscle in womenand men. Eur. J. Appl. Physiol. 81: 47-53. 2000.
Malm, C., P. Nyberg, M. Engstrom, B. Sjodin, R. Lenkei, B. Ekblom, and I. Lundberg. Immunological changes in human skeletal muscle and blood after eccentric
exercise and multiple biopsies. J. Physiol. 529: 243-262. 2000.
Maruhashi, Y., K. Kitaoka, Y. Yoshiki, R. Nakamura, A. Okano, K. Nakamura, T.
Tsuyama, Y. Shima, and K. Tomita. ROS scavenging activity and muscle damage
prevention in eccentric exercise in rats. J. Physiol. Sci. 57: 211-216. 2007.
Morrow, J. D., K. E. Hill, R. F. Burk, T. M. Nammour, K. F. Badr, and L. J. Roberts
2nd. A series of prostaglandin F2-like compounds are produced in vivo in humans by
a non-cyclooxygenase, free radical-catalyzed mechanism. Proc. Natl. Acad. Sci.
USA. Biochemistry. 87: 9383-9387. 1990.
Murase, S., E. Terazawa, F. Queme, H. Ota, T. Matsuda, K. Hirate, Y. Kozaki, K.
Katanosaka, T. Taguchi, H. Urai, and K. Mizumura. Bradykinin and nerve growth
factor play pivotal roles in muscular mechanical hyperalgesia after exercise(delayed-onset muscle soreness). J. Neurosci. 30(10): 3752-3761. 2010.
Nguyen, H. X., and J. G. Tidball. Interactions between neutrophils and macrophages
promote macrophage killing of rat muscle cells in vitro. J. Physiol. 547: 125-132.
2003.
Nguyen H. X., and J. G. Tidball. Null mutation of gp91phox reduces muscle membrane lysis during muscle inflammation in mice. J. Physiol. 553: 833-841. 2003.
Nguyen, H. X., A. J. Lusis, and J. G. Tidball. Null mutation of myeloperoxidase inmice prevents mechanical activation of neutrophil lysis of muscle cell membranes invitro and in vivo. J. Physiol. 565: 403-413. 2005.
Nieman, D. C., J. M. Davis, D. A. Henson, J. Walberg-Rankin, M. Shute, C. L.
Dumke, A. C. Utter, D. M. Vinci, J. A. Carson, A. Brown, W. J. Lee, S. R. McAnulty,
and L. S. McAnulty. Carbohydrate ingestion influences skeletal muscle cytokine
mRNA and plasma cytokine levels after a 3-h run. J. Appl. Physiol. 94: 1917-1925.
2003.
Nosaka, K., M. Newton, and P. Sacco. Delayed-onset muscle soreness does notreflect the magnitude of eccentric exercise-induced muscle damage. Scand. J. Med.
Sci. Sports. 12(6): 337-46. 2002.
Paulsen, G., U. R. Mikkelsen, T. Raastad, and J. M. Peake. Leucocytes, cytokines
and satellite cells: what role do they play in muscle damage and regeneration following eccentric exercise? Exerc. Immunol. Rev. 18: 42-97. 2012.
Peake, J. M. Exercise-induced alterations in neutrophil degranulation and respiratory
burst activity: possible mechanisms of action. Exerc. Immunol. Rev. 8: 49-100.
2002.
Peake, J., K. Nosaka, and K. Suzuki. Characterization of inflammatory responses to
eccentric exercise in humans. Exerc. Immunol. Rev. 11: 64-85. 2005.

EIR 20 2014

Eccentric exercise and muscle damage markers • 53
31.

32.

33.
34.

35.
36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

Pedersen, B. K., A. Steensberg, P. Kelller, C. Keller, C. Fischer, N. Hiscock, G. vanHall, P. Plomgaard, and M. Febbraio. Muscle-derived interleukin-6: lipolytic, antiinflammatory and immune regulatory effects. Eur. J. Physiol. 446(1): 9-16. 2003.
Peterson, J. M., T. A. Trappe, E. Mylona, F. White, C. P. Lambert, W. J. Evans, and F.
X. Pizza. Ibuprofen and acetaminophen: effect on muscle inflammation after eccentric exercise. Med. Sci. Sports Exerc. 35: 892-896. 2003.
Proske, U., and T. J. Allen. Damage to skeletal muscle from eccentric exercise.
Exerc. Sport Sci. Rev. 33: 98-104. 2005.
Raastad, T., B. A. Risoy, H. B. Benestad, J. G. Fjeld, and J. Hallen. Temporal relation between leukocyte accumulation in muscle and halted recovery 10-20 h afterstrength exercise. J. Appl. Physiol. 95: 2503-2509. 2003.
Rosen, S. D. Ligands for L-selectin: homing, inflammation, and beyond. Annu. Rev.
Immunol. 22:129-156. 2004.
Roudkenar, M. H., R. Halabian, Z. Ghasemipour, A. M. Roushandeh, M. Rouhbakhsh, M. Nekogoftar, Y. Kuwahara, M. Fukumoto, and M. A. Shokrgozar. Neutrophil gelatinase-associated lipocalin acts as a protective factor against
H(2)O(2)toxicity. Arch. Med. Res. 39: 560-566. 2008.
Roudkenar, M. H., Y. Kuwahara, T. Baba, A. M. Roushandeh, S. Ebishima, S. Abe,Y. Ohkubo, and M. Fukumoto. Oxidative stress induced lipocalin 2 gene expression:
addressing its expression under the harmful conditions. J. Radiat. Res (Tokyo). 48:
39-44. 2007.
Round, J., D. Jones, and G. Cambridge. Cellular infiltration in human skeletal muscle: exercise induced muscle damage as a model of inflammatory muscle disease. J.
Neurol. Sci. 82: 1-11. 1987.
Sorichter, S., J. Mair, A. Koller, M. M. Pelsers, B. Puschendorf, and J. F. Glatz. Early
assessment of exercise induced skeletal muscle injury using plasma fatty acid binding protein. Br. J. Sports Med. 32: 121-124. 1998.
Stupka, N., M. A. Tarnopolsky, N. J. Yardley, and S. M. Phillips. Cellular adaptation
to repeated eccentric exercise-induced muscle damage. J. Appl. Physiol. 91: 16691678. 2001.
Sugama, K., K. Suzuki, K. Yoshitani, K. Shiraishi, and T. Kometani. IL-17, neutrophil activation and muscle damage following endurance exercise. Exerc.
Immunol. Rev. 18: 116-127. 2012.
Suzuki, K., S. Naganuma, M. Totsuka, K. J. Suzuki, M. Mochizuki, M. Shiraishi, S.
Nakaji, and K. Sugawara. Effects of exhaustive endurance exercise and its one-weekdaily repetition on neutrophil count and functional status in untrained men. Int. J.
Sports Med. 17: 205-212. 1996.
Suzuki, K., H. Sato, T. Kikuchi, T. Abe, S. Nakaji, K. Sugawara, M. Totsuka, K.
Sato, and K. Yamada. Capacity of circulating neutrophils to produce reactive oxygen
species after exhaustive exercise. J. Appl. Physiol. 81: 1213-1222. 1996.
Suzuki, K., K. Totsuka, S. Nakaji, M. Yamada, S. Kudoh, Q. Liu, K. Sugawara, K.
Yamaya, and K. Sato. Endurance exercise causes interaction among stress hormones, cytokines, neutrophil dynamics, and muscle damage. J. Appl. Physiol. 87:
1360-1367. 1999.
Triebel, S., J. Blaser, H. Reinke, and H. Tschesche. A 25 kDa 2-microglobulin-related protein is a component of the 125 kDa form of human gelatinase. FEBS. Lett.
314: 386-388.1992.

EIR 20 201454 • Eccentric exercise and muscle damage markers
46.

47.

48.
49.

50.

Uchida, M. C., K. Nosaka, C. Ugrinowitsch, A. Yamashita, E. Martines, A. S.
Moriscot, and M. S. Aoki. Effect of bench press exercise intensity on muscle soreness and inflammatory mediators. J. Sports Sci. 27(5): 499-507. 2009.
Van Nieuwenhoven, F. A., A. H. Kleine, W. H. Wodzig, W. T. Hermens, H. A.
Kragten, J. G. Maessen, C. D. Punt, M. P. van Dieijen, G. I. van der Vusse, and J. F.
Glatz. Discrimination between myocardial and skeletal muscle injury by assessmentof the plasma ratio of myoglobin over fatty acid-binding protein. Circulation 10:304-309. 1995.
Warren, G. L., D. A. Lowe, and R. B. Armstrong. Measurement tools used in the
study of eccentric contraction-induced injury. Sports Med. 27: 43-59. 1999.
Woolf, C. J., B. Safieh-Garabedian, Q.-P. Ma, P. Crilly, and J. Winter. Nerve growth
factor contributes to the generation of inflammatory sensory hypersensitivity. Neuroscience 62: 327-331. 1994.
Zimmerman, A. W., and J. H. Veerkamp. New insights into the structure and functionof fatty acid binding proteins. Cell Mol. Life Sci. 59:1096-1116. 2002.

EIR 20 2014