OsteoArthritis and Cartilage (2006) 14, 71e76
ª 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.joca.2005.08.003
International
Cartilage
Repair
Society
Sport-related differences in biomarkers of bone resorption and cartilage
degradation in endurance athletes1
J. W. O’Kane M.D.y, E. Hutchinson M.D.z, L. M. Atley Ph.D.y2 and D. R. Eyre Ph.D.y*
y Department of Orthopaedics and Sports Medicine, University of Washington, USA
z University of Washington College of Medicine, USA
Summary
Objective: By measuring urinary cross-linked N-telopeptide (NTx) as a bone resorption marker and urinary C-telopeptide of type II collagen
(CTx-II) as a cartilage degradation marker, we asked whether differences in skeletal stresses in college athletes undergoing high-intensity
training for diverse types of aerobic sports affect their skeletal metabolism and, if so, differentially or in unison.
Methods: The study was cross-sectional at a Division 1 college campus with 60 student athletes representing crew, cross-country running and
swimming. Controls were 16 non-athlete undergraduates. Urine samples were collected for NTx and CTx-II analysis by enzyme-linked immunosorbent assay, normalizing results to creatinine. Two-way analysis of variance models and pair-wise comparisons were used to test whether
biomarker levels differed by sport and the significance when adjusted for body mass index (BMI).
Results: NTx and CTx-II showed significant differences between groups before and after adjusting for BMI. NTx was highest in the rowers, and
higher in rowers and runners than in swimmers or controls. CTx-II was significantly higher in runners than in crew, swimmers or controls, when
unadjusted for BMI. After adjusting for BMI, these group differences remained significant except for runners over crew.
Conclusion: Athletes in-training in the three sports show significant differences in these markers of bone resorption and cartilage collagen
degradation. The results suggest that crew undergo the highest bone remodeling and runners the highest cartilage degradation. The results
also show how these markers can vary physiologically between individuals, at extremes of skeletal exercise.
ª 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Key words: Collagen, Cartilage, Cross-links, Biomarkers, Athletes.
and joint disorders. The cross-linked N-telopeptide (NTx)
of type I collagen in blood or urine has seen extensive
use as a marker of systemic bone resorption1e4. The NTx
peptide is a neoepitope generated from bone collagen during resorption by osteoclasts, and cathepsin K, a protease
abundantly expressed by osteoclasts, has been shown to
make the proteolytic cleavage that generates the NTx epitope5. NTx levels are elevated in post-menopausal women,
in patients with metabolic bone disease and in growing children2. Type I collagen in non-mineralized tissues such as
skin is degraded by a different proteolytic pathway hence
explaining the specificity of urinary NTx as a bone resorption marker. The specificity to bone resorption is seen in
the response of NTx to antiresorptive therapies for osteoporosis, for example to the bisphosphonate, alendronate3,4.
In a related approach, a fragment of type II collagen found
in urine has been targeted as the basis of a biomarker assay for cartilage collagen breakdown6,7. In this case, the
neoepitope was a peptide from the C-telopeptide of type II
collagen (CTx-II) of sequence EKGPDP, where the lysine
residue (K) is part of a cross-link. Using a monoclonal antibody 2B4 that recognizes the C-terminal sequence (GPDP),
the proteolytic epitope (CTx-II) can be detected in synovial
fluid, serum, and urine8e11. Various collagen cleavage
products, including the pyridinoline cross-links themselves
have been pursued as markers of joint pathology in osteoarthritis12. Atley et al.7 found very high levels of the 2B4 epitope in the urine of growing children, presumably from
growth plate activity, and levels were higher in patients
with rheumatoid arthritis and osteoarthritis than in controls.
Introduction
Rowing, cross-country running, or swimming competitively
at the college level requires outstanding aerobic fitness
and extensive aerobic training. In all three sports, most of
the training is sport-specific. While the cardiovascular challenges for the three sports are similar, the musculoskeletal
challenges vary significantly. Runners expose their lower
extremity bones and joints to large repetitive axial loads.
While crew athletes expose their entire skeleton to both axial and non-axial loads, their lower extremity bones and
joints experience lower peak axial loads than those of runners. Swimmers’ bones and joints experience the lowest axial loads. Such extremes of chronic loading might be
expected to affect the turnover rate of bone and cartilage
in affected parts of the skeleton and be detectable using
systemic biomarker assays.
Various molecular markers have been reported as indicators of bone turnover and cartilage metabolism in human
subjects and in patients enrolled in clinical studies of bone
1
Support provided by NIH grants AR37318 and AR36794.
Current address: Murdoch Children’s Research Institute, Royal
Children’s Hospital, Parkville VIC, Australia.
*Address correspondence and reprint requests to: David R. Eyre,
Department of Orthopaedics and Sports Medicine, University
of Washington, Box 356500, Seattle, WA 98195-6500, USA. Tel:
1-206-543-4700; Fax: 1-206-685-4700; E-mail: [email protected].
edu
Received 31 May 2005; revision accepted 8 August 2005.
2
71
72
J. W. O’Kane et al.: Collagen cross-link markers in athletes
binding. Various matrix metalloproteinases are able to
make this cleavage in vitro from intact type II collagen7
but the tissue source(s) and pathway of origin of the collagen type II peptides measured in urine are not yet clearly
defined. The assay is a competition ELISA and the results
are expressed as ng of peptide equivalents per mg of creatinine in urine. Intra- and inter-assay coefficients of variation
for the CTx-II/Cr assay were !6% and !13%, respectively.
In contrast to the many reports on NTx and other collagen
degradation markers in metabolic bone disease, there have
been few studies on the effects of extremes of normal skeletal stress on NTx, for example in high activity athletes, and
none on a cartilage collagen marker. Here, we designed
a study to compare whether these two markers were differentially affected in three groups of intercollegiate athletes
undertaking training for different aerobic sports and compared with non-athlete controls.
STATISTICAL ANALYSIS
Methods
The study was cross-sectional in design. Spearman rank
correlation coefficients were used to determine any association between the athlete characteristics (height, weight,
BMI, and age) and biomarker measures. Using the KolmogoroveSmirnov test, both NTx/Cr and CTx-II/Cr data sets
passed the test of normality. Two-way analysis of variance
(ANOVA) models were used to test whether biomarker levels differed by sport. When significant differences were
found, further pair-wise comparisons were made. Differences in athlete characteristics by sport were assessed by
the KruskaleWallis test. ANOVA was used to assess the
significance of group biomarker values adjusted by BMI.
SUBJECTS
The study was approved by the Human Subjects Review
Board at the University of Washington with participant informed consent. Sixty collegiate athletes (NCAA Division I)
representing crew, cross-country runners, and swimmers
were recruited at their routine fall physical to participate in
the study. Three groups of athletes each consisting of 10
males and 10 females were enrolled. All athletes had
been actively training for their sports for several weeks before specimen collection. In addition, one swimmer, one
cross-country runner, and six rowers participated in some
cross training. Individual heights and weights were measured to determine body mass index (BMI). In the control
group were 16 University of Washington college students,
8 males and 8 females, recruited as volunteers and randomly
selected from the university student population. None of the
controls participated in collegiate athletics, and they participated in sports-related activity %3 times per week.
Results
Table I shows the mean (GSD) height, weight, BMI and
age profiles of the athlete and control groups. There are
no significant differences in age among the male or female
groups. There are small differences in height between the
groups and larger differences in weight and subsequently
BMI.
The biomarker data were examined for confounders that
included known injuries at the time of sample collection,
concurrent illness, recent surgeries, history of amenorrhea,
use of medications including oral contraceptive pills, and
history of recent fractures. None of these potential confounders appeared to have any influence on the data.
Mean NTx/creatinine values were significantly different
between sports groups for both male and female athletes
[Fig. 1, Table II(a)]. The male rowers had a higher NTx excretion than all other groups (cross-country P Z 0.04, swimmers P ! 0.01, control P ! 0.01). After adjustment for BMI,
NTx excretion of the male rowers remained significantly
higher than the male swimmers (P ! 0.01), and male controls (P ! 0.01) but were not significantly different from
the cross-country group. For female rowers, NTx was
higher than for the swimmers (P ! 0.01) and the controls
(P ! 0.01). NTx for the swim group was significantly lower
PROCEDURES
Spot urine samples were collected in the afternoon and
stored at 20(C before assay as a batch. NTx was measured in urine using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Osteomark, Ostex
International) and the values were normalized to creatinine
measured by a commercial assay based on the Jaffe reaction (Sigma Diagnostics). Intra- and inter-assay coefficients
of variation for the NTx/Cr assay are !5% and !8%,
respectively.
The CTx-II assay is based on a monoclonal antibody,
2B4, which recognizes a peptide sequence, EKGPDP, derived from the C-telopeptide domain of type II collagen
and in which the lysine residue (K) is part of a pyridinoline
cross-link7,9. The epitope is a proteolytic neoepitope, in
that the C-terminal sequence PDP is required for antibody
Table I
Mean (GSD) height, weight, BMI and age profiles of the athlete and control groups
Cross-country
Male
Female
Swimming
Crew
Controls
P*
No. of participants
Height
Weight
BMI
Age
10
71.5 (2.5)
153.8 (14.1)
21.2 (1.2)
18.7 (0.9)
10
73.3 (2.2)
188.4 (17.8)
24.7 (2.1)
19.4 (2.1)
10
74.4 (4.2)
201.2 (36.7)
25.4 (2.6)
20.1 (0.9)
8
69.8 (2.7)
175.6 (10.2)
25.5 (0.8)
20.5 (1.5)
0.02
!0.001
!0.001
0.10 (NS)
No. of participants
Height
Weight
BMI
Age
10
66.4 (2.4)
121.2 (11.2)
19.2 (1.2)
19.8 (1.4)
10
67.7 (2.5)
150.5 (14.0)
23.0 (2.3)
19.4 (1.0)
10
68.1 (1.3)
162.2 (16.2)
24.9 (2.9)
19.4 (1.8)
8
65.3 (2.4)
132.4 (19.4)
21.9 (2.9)
19.9 (1.4)
0.05
!0.001
!0.001
0.81 (NS)
KruskaleWallis non-parametric test used to test for group differences.
*Significance of variability across all groups.
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Osteoarthritis and Cartilage Vol. 14, No. 1
For males, the cross-country group had higher levels than
both the swimmers (P ! 0.01) and controls (P Z 0.02). After adjusting the CTx-II of males for BMI, however, there
were no statistically significant sport group differences.
But for females, there were significant group differences in
CTx-II, even after adjusting for BMI, and in the same pattern
as the trend for males. From pair-wise analysis, the female
cross-country group had significantly higher CTx-II values
than all three other groups (swimmers P ! 0.01, crew
P Z 0.02, and control P ! 0.01) in unadjusted analyses. After adjusting for BMI, the cross-country CTx-II was still significantly higher than for swimmers (P ! 0.01) and controls
(P ! 0.01), but not for crew. The combined male and female results [Table III(b)] showed significant group differences in CTx-II both before and after adjusting for BMI. The
cross-country runners had significantly higher CTx-II than
each of the three other groups (swimmers P ! 0.01, crew
P Z 0.01, control P ! 0.01) in unadjusted analyses. These
pair-wise differences remained statistically significant (at
P Z 0.02) when adjusted for BMI, except for the crosscountry to crew comparison. The combined male and female analyses presumably reflect the larger sample size
and hence increased statistical power. The sport-related
effects on both biomarker levels also remained highly significant when the data were log transformed or a nonparametric test was applied (not shown).
Fig. 1. Differences between sport groups in the bone collagen resorption marker, urinary NTx. Results are plotted by sex for each
sport and control group as medians (bar), second and third quartiles (extent of box), bottom and top quartiles (whiskers) and outlier
points.
Discussion
than for the cross-country runners (P Z 0.2). Pair-wise analysis showed that the differences remained statistically significant after adjusting for BMI (P ! 0.01). When
combined male and female NTx results were adjusted for
BMI, the cross-country group was significantly higher than
the swimmer and control groups and rowers were higher
than swimmers and controls [Table II(b)].
Table III(a) and Fig. 2 show the differences in collagen
type II peptide excretion (CTx-II) between sport activities.
Both biomarkers, for bone and cartilage degradation,
showed significant differences among the three sports.
The high NTx values in crew athletes are probably a result
of the high axial and non-axial forces applied to the entire
skeleton. Similarly, the high NTx values in runners compared with controls, swimmers and the published normal
range (39 G 3)13, probably result from the high amplitude
loading of the lower skeleton. Swimming clearly stresses
the entire body, but apparently not the skeleton in a way
that stimulates bone remodeling. Although we have
Table II
Urinary NTx/creatinine levels for sport activity
(a) Mean (S.E.M.) for each sport activity (nmol/mmol creatinine)
Cross-country
Swimming
Crew
Controls
P*
Male
Unadjusted
Adjusted for BMI
61.7 (10.1)
66.9 (13.4)
45.9 (10.1)
44.9 (10.3)
92.1 (10.1)
89.8 (10.8)
46.9 (11.3)
44.4 (12.1)
0.010
0.012
Female
Unadjusted
Adjusted for BMI
62.5 (10.3)
61.1 (10.5)
31.7 (8.5)
32.1 (8.8)
79.6 (11.0)
80.8 (10.1)
37.0 (7.8)
36.8 (9.7)
0.001
0.001
Combined
Unadjusted
Adjusted for BMI
62.1 (6.5)
65.1 (7.9)
38.8 (6.5)
38.1 (6.6)
85.8 (6.5)
83.8 (7.1)
41.9 (7.3)
41.4 (7.4)
!0.001
!0.001
(b) Statistical significance of pair-wise comparison for different sports and controls (combined males and females)
P
Unadjusted
Adjusted for BMI
0.01
0.01
0.04
NS
!0.001
!0.001
0.01
NS
0.04
NS
!0.001
!0.001
XC, Swimming
XC, Crew
XC, Controls
Swimming, Controls
Swimming, Crew
Crew, Controls
NS Z Not statistically significant.
*Significance of variability across all groups.
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J. W. O’Kane et al.: Collagen cross-link markers in athletes
Table III
Urinary CTx-II/creatinine levels for sport activity
(a) Mean (S.E.M.) for each sport activity (ng/mg creatinine)
Cross-country
Swimming
Crew
Controls
P*
Male
Unadjusted
Adjusted for BMI
33.5 (3.3)
31.7 (4.4)
20.2 (3.3)
20.6 (3.4)
27.3 (3.3)
28.1 (3.5)
20.2 (3.7)
21.0 (3.9)
0.023
0.14 (NS)
Female
Unadjusted
Adjusted for BMI
36.7 (3.1)
36.4 (3.9)
17.8 (3.1)
17.9 (3.2)
25.7 (3.1)
26.0 (3.7)
17.3 (3.5)
17.3 (3.5)
!0.001
0.001
Combined
Unadjusted
Adjusted for BMI
35.1 (2.2)
34.7 (2.7)
19.0 (2.2)
19.1 (2.3)
26.5 (2.2)
26.8 (2.4)
18.7 (2.5)
18.8 (2.5)
!0.001
!0.001
(b) Statistical significance of pair-wise comparison of different sports (combined males and females)
P
Unadjusted
XC, Swimming
XC, Crew
XC, Control
0.01
0.01
0.01
Adjusted for BMI
0.02
NS
0.02
NS Z Not statistically significant.
*Significance of variability across all groups.
measured only NTx, a specific marker of bone resorption,
not a formation marker, the two processes tend to be tightly
coupled in the long term whether the net result is a gain (adolescence) or loss (menopause) in bone mass. Therefore,
NTx can be interpreted as a systemic index of bone remodeling activity. Many studies have reported a positive association between load-bearing exercise and bone mineral
density14, implying that high NTx values in this young adult
population likely reflect net increases in bone mass.
The collagen CTx-II results suggest that swimming, compared with rowing and running, minimally stresses joints,
Fig. 2. Differences between sport groups in the cartilage collagen
degradation marker, urinary CTx-II. Results are plotted by sex for
each sport and control group as medians (bar), extent of second
and third quartiles (box), bottom and top quartiles (whiskers) and
outlier points.
similar to the conclusion from the NTx results for bone.
This is consistent with other studies suggesting that sports
involving high bone and joint stresses such as distance running and rowing cause cartilage remodeling or degradation
beyond that seen in swimming and control subjects15,16.
The CTx-II marker is less well characterized than NTx in
terms of tissue and processing origins, but elevations in
clinical studies are consistent with higher cartilage degradation, for example from active growth plates in children or
joints in adults with rheumatoid or osteoarthritis6e8. In the
present study, the higher CTx-II levels are presumably
from accelerated remodeling in stressed joints. Whether
from collagen type II forming the framework of the articular
cartilage bearing surfaces, or primarily from the deep calcified interface with bone as we suspect10, is unknown. It has
been shown that in patients with osteoarthritis, those with
higher CTx-II measured by a different antibody have more
rapidly progressive joint destruction17e19. Remodeling of
joint tissues probably occurs in response to the effects of
normal mechanical loading and injuries from high loads,
the latter potentially leading to subsequent degenerative
changes20,21. In interpreting the CTx-II results, however, it
is important to recognize that joint cartilages account for
only a small fraction of systemic type II collagen. Articular
cartilage was 8% of the total cartilage in young dogs22.
The respiratory tract, discs, ribs and other tissues are also
major sources. Although respiratory tract cartilages conceivably may be stimulated by high aerobic activity, any
such effect might be expected to be similar in all three
sports.
Studies quantifying bone-loading patterns during rowing
and running are lacking but qualitative differences between
the sports are clear. Runners load one leg at a time resulting in axial stresses with high amplitude and frequency.
Rowers push off both legs simultaneously so the stress amplitude to each leg is lower than in running. Stride cadence
is generally higher than stroke rate so the frequency of
loading is higher in running. Non-axial skeletal loads, particularly above the pelvis, are higher in rowing. These biomechanical differences may explain biomarker findings
suggesting that rowing is more osteo-stimulatory, but less
damaging to joints. Studies in an avian ulna model in vivo
75
Osteoarthritis and Cartilage Vol. 14, No. 1
suggest that lower frequency loading may be more osteogenic than higher frequency loading23. Clearly, this animal
experiment is not directly comparable to rowing, but the
findings call into question the traditional prescription of
high frequency, high amplitude loading as the best method
to build bone. If this is true, it has implications for exercise
counseling in elderly individuals interested in gaining bone
density but wanting to minimize progression of osteoarthritis of weight-bearing joints. A longitudinal study has shown
that walking exercise in post-menopausal women decreased NTx long term after 3 months and increased spinal
BMD24.
In a previous report on urinary NTx in college-age male
and female track athletes monitored over 12 months, no differences in NTx were seen between those who developed
stress fractures and those who did not25. In another study,
bone markers and bone mineral density scans by DEXA
were compared between groups of female college athletes
entered in high-impact (basketball and volleyball), mediumimpact (soccer and track), and non-impact (swimming)
sports vs sedentary controls14. Higher BMD levels were
found in the high-impact group but no differences in NTx.
The low subject numbers (7e14 in each group) and other
differences in study design prevent meaningful comparison
with the present study.
One limitation of the present study is that the molecular
and cellular origins of CTx-II in urine, though specific to
type II collagen, are less well understood than for NTx as
a bone resorption marker. Also, it is known that urinary
NTx can show a significant circadian variation with higher
levels at night and early morning and lower levels in the
afternoon26. The markers might also be influenced by
hormonal status, dietary intake and genetic effects25.
Short-term effects on the markers from the last period of exercise and time before sample collection are also possible.
However, in a study of the acute effects on bone biomarkers
of moderate exercise in untrained young men (22 G 1 year),
serum NTx levels fell in the hours after exercise indicating
an acute decrease in bone resorption activity, but 24-h urine
NTx values did not differ from pre-exercise or control values27. In conclusion, we interpret the observed differences
between groups as being primarily due to the effect on skeletal metabolism of chronic training in the individual sports.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Acknowledgements
This research was funded in part by NIH Grants AR37318
and AR36794 and the Burgess Chair program of the University of Washington.
16.
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