Download Grant/Funding Support: None

Letters to the Editor
* Address correspondence to this author at:
St. Savas Oncology Hospital
Nuclear Medicine Department
171 Alexandras Ave.
Athens 115 22, Greece
e-mail [email protected]
DOI: 10.1373/clinchem.2007.102194
Acute Variation of Osteocalcin
and Parathyroid Hormone in
Athletes after Running a HalfMarathon
To the Editor:
Fig. 1. Box and whisker plots of the derived PTH 95% CIs for patients on
L-thyroxine therapy.
(A), Patients (n ⫽ 64) with excessively suppressed TSH (⬍0.4 mIU/L) concentrations [mean (SD) TSH ⫽ 0.29 (0.14) mIU/L]. The true calcium concentration
(albumin-corrected) for this group of patients was 9.61 (0.26) mg/dL. (B), Patients
(n ⫽ 31) with TSH concentrations within reference limits [mean (SD) TSH ⫽ 0.87
(0.36) mIU/L). Means of the PTH concentrations for each group were compared
with the application of the t-test for independent samples, with logarithmic
transformation of data, assuming unequal variances (Welch-test, P ⫽ 0.0197).
Data for the healthy blood donors (n ⫽ 39) are not shown.
needed to confirm our observation
and to clarify its underlying mechanism. Knowledge of this effect
may help clinicians to avoid unnecessary and expensive work-ups
for suspected hyperparathyroidism.
Grant/Funding Support: None
declared.
Financial Disclosures: None declared.
References
1. Aloia JF, Feuerman M, Yeh JK. Reference range
for serum parathyroid hormone. Endocr Pract
2006;12:137– 44.
2. Pantazi H, Papapetrou PD. Changes in parameters of bone and mineral metabolism during
therapy for hyperthyroidism. J Clin Endocrinol
Metab 2000;85:1099 –106.
3. Kumeda Y, Inaba M, Tahara H, Kurioka Y, Ishikawa T, Morii H, Nishizawa Y. Persistent increase in bone turnover in Graves’ patients with
subclinical hyperthyroidism. J Clin Endocrinol
Metab 2000;85:4157– 61.
4. Corbetta S, Lania A, Filopanti M, Vicentini L,
Ballaré E, Spada A. Mitogen-activated protein
kinase cascade in human normal and tumoral
parathyroid cells. J Clin Endocrinol Metab 2002;
87:2201–5.
5. D’Amour P, Räkel A, Brossard JH, Rousseau L,
Albert C, Cantor T. Acute regulation of circulating parathyroid hormone (PTH) molecular forms
by calcium: utility of PTH fragments/PTH(1– 84)
ratios derived from three generations of PTH
assays. J Clin Endocrinol Metab 2006;91:283–9.
Adonios Zanglis*
Demetrios Andreopoulos
Nikolaos Baziotis
St. Savas Oncology Hospital
Nuclear Medicine Department
Athens, Greece
Parathyroid hormone (PTH) and
osteocalcin (OC) play important
roles in bone remodeling and bone
metabolism. Although the physiological functions and clinical significance of these markers are well
established, the influence of other
biological variables aside from diurnal and seasonal variability has
been less well investigated (1 ). Because little information is available
on the kinetics of such markers after physical exercise, we measured
PTH and OC in 15 athletes performing a half-marathon run. The
study population consisted in 15
healthy trained white males, (mean
age, 47 years) who had been engaged in specific endurance training for at least 5 years. Participants
performed a 21-km, half-marathon run under competition conditions, while equipped with a
heart-rate monitor [mean (SE)
VO2 max 85% (3%)]. Prior to the
race, preexercise baseline fasting
blood samples were collected from
the volunteers after a 48-h rest
from the last training, 30 min before they warmed up for the race.
Post exercise samples were collected immediately after the race,
and 3 h, 6 h, and 24 h thereafter. All
study participants gave informed
consent for being tested, and the
study was approved by the ethics
Clinical Chemistry 54:6 (2008) 1093
Letters to the Editor
committee. Blood was collected in
vacuum tubes containing no additives (Becton Dickinson) and centrifuged at 1500g for 10 min at
room temperature, and the serum
was immediately analyzed. OC and
intact PTH were measured on an
Elecsys 2010 by electrochemiluminescence immunoassays (Roche
Diagnostics GmbH). We used the
N-MID Osteocalcin assay that is
specific for intact OC (amino acids
1– 49) and the main N-terminal
fragment (amino acids 1– 43) resulting from proteolytic cleavage
after blood collection. Among
markers of bone turnover, OC was
selected to verify the effects of
physical training on osteoblast
function, because it may indirectly
reflect acute changes in cortisol,
and it is currently considered an indirect marker of growth hormone
abuse in sports (2 ). The total assay
imprecision (CV) was 1.1%–5.9%
for OC and 1.7%–5.5% for intact
PTH, respectively (3 ). The ageand sex-specific reference ranges
were 1.6 – 6.9 pmol/L for PTH and
14 – 42 ␮g/L for OC, respectively.
Hematocrit and hemoglobin were
measured on an Advia 120 (Bayer
Diagnostics). Because the estimated decrease in total body water
does not differ from the percentage
plasma volume change (%PVC),
results were adjusted for the
%PVC, calculated from pre- and
postexercise levels of hematocrit
and hemoglobin (4 ). The Wilcoxon signed-rank test was used to
evaluate the significance of exercise-induced variations during the
study period. Data with a nongaussian distribution were normalized
using a logarithmic transformation
before analysis, and the level of statistical significance was set at P ⬍
0.05. Data are presented as geometric mean (SE).
The concentrations of both
markers increased significantly by
nearly 1.2-fold (OC) and 2.1-fold
(PTH) immediately after the run,
but 3 h thereafter rapidly returned
to values comparable to those measured before the run (Table 1). The
number of participants with values
above the respective reference
ranges remained stable throughout
the study period for OC (n ⫽ 2,
13%), whereas it increased from 0
(0%) to 6 (40%) immediately after
the run and returned to 0 (0%) 3 h
thereafter for PTH.
These results support the hypothesis that a 21-km run produces an acute and transitory increase of both PTH and OC, which
is completely reversed 3 h thereafter. Although earlier studies have
investigated PTH and OC after
strenuous aerobic exercise, especially marathon and ultramarathon running (5 ), no information
is available on the kinetics of these
markers following moderately
strenuous aerobic physical exer-
Table 1. Variation of osteocalcin and parathyroid hormone before (pre),
immediately after (post), and 3, 6 and 24 h after a 21-km, halfmarathon run, in 15 male recreational athletes.a
Pre
Plasma volume change, %
Weight loss, %
Osteocalcin, ␮g/L
Parathyroid hormone, pmol/L
3h
6h
24 h
–8.0 (1.0)
b
–0.4 (1.0)
1.2 (1.1)
3.8 (1.0)b
—
8.8 (0.7)
b
1.6 (0.7)
–0.1 (0.8)
–1.7 (0.7)b
22.0 (2.5)
27.3 (3.0)b
21.6 (2.6)
20.2 (2.9)
22.3 (2.5)
3.1 (0.3)
b
3.1 (0.3)
3.1 (0.5)
3.2 (0.3)
—
Post
a
6.4 (0.7)
Values are presented as geometric mean (SE). Differences from the premarathon values were evaluated
by the Wilcoxon signed-rank test.
b
P ⬍ 0.01, vs the pre-run sample.
1094 Clinical Chemistry 54:6 (2008)
cise, such as a 21-km half-marathon run, a distance that is more
typically accessible for a general
population of active individuals.
Although Mouzopoulos et al. observed that PTH concentrations
increase by nearly 1.3-fold immediately after 245 km of marathon
running, these investigators also
reported a significant decrease in
OC concentrations. Such differences may be due to different preanalytical conditions (sample matrix and storage) or intensity and
duration of the run. The transient
suppression in osteoblast function
occurring during an ultramarathon run, which has been attributed to increases in cortisol and
PTH concentrations (5 ), might
not occur during shorter-distance
runs. Perhaps athletes should be
advised to run a half or a fraction of
a marathon to avoid potential
harmful effects of strenuous endurance exercise. Because OC is a
marker of growth-hormone abuse
in sports (2 ), such variations
should be acknowledged when
evaluating the athletes for clinical
or antidoping purposes, because
these measurement results should
be interpreted as physiological responses to exercise rather than pathology or consequences of unfair
practices.
Grant/Funding Support: None
declared.
Financial Disclosures: None declared.
References
1. Scharnhorst V, Valkenburg J, Vosters C, Vader H.
Influence of preanalytical factors on the immulite intact parathyroid hormone assay. Clin Chem
2004;50:974 –5.
2. McHugh CM, Park RT, Sönksen PH, Holt RI.
Challenges in detecting the abuse of growth
hormone in sport. Clin Chem 2005;51:1587–93.
3. Schmidt-Gayk H, Spanuth E, Kötting J, Bartl R,
Felsenberg D, Pfeilschifter J, et al. Performance
evaluation of automated assays for beta-CrossLaps, N-MID-Osteocalcin and intact parathyroid
hormone (BIOROSE Multicenter Study). Clin
Chem Lab Med 2004;42:90 –5.
4. Maughan RJ, Whiting PH, Davidson RJ. Estima-
Letters to the Editor
tion of plasma volume changes during marathon
running. Br J Sports Med 1985;19:138 – 41.
5. Mouzopoulos G, Stamatakos M, Tzurbakis M,
Tsembeli A, Manti C, Safioleas M, Skandalakis P.
Changes of bone turnover markers after marathon running over 245 km. Int J Sports Med
2007;28:576 –9.
Giuseppe Lippi1*
Federico Schena2
Martina Montagnana1
Gian Luca Salvagno1
Giuseppe Banfi3
Gian Cesare Guidi1
1
Sezione di Chimica Clinica
Dipartimento di Scienze
Morfologico-Biomediche
Università di Verona, Italy
2
Sezione di Scienze Motorie
Dipartimento di Scienze
Neurologiche e della Visione
Università di Verona, Italy
3
Istituto Galeazzi e Università di Milano
Italy
* Address correspondence to this author at:
Sezione di Chimica Clinica
Dipartimento di Scienze
Morfologico-Biomediche
Università degli Studi di Verona
Ospedale Policlinico G.B. Rossi
Piazzale Scuro, 10
37134, Verona, Italy
Fax 0039-045-8201889
e-mail [email protected]
DOI: 10.1373/clinchem.2007.102657
Rapid Real-Time PCR
Detection of HP del Directly
from Diluted Blood Samples
To the Editor:
Anhaptoglobinemic patients have
been reported to experience severe
anaphylactic reactions to transfusions due to the production of
antihaptoglobin (anti-HP) antibodies (1, 2 ). Anhaptoglobinemia
in patients homozygous for HP del,
which is a deletion of an approximately 28-kb segment of chro-
mosome 16 extending from the
promoter region of the HP (haptoglobin) gene to exon 5 of HPR
(haptoglobin-related protein), has
been adequately characterized only
recently (1 ). Use of a simple duplex PCR method has detected
the HP del allele in East and Southeast Asian populations at frequencies of 1%–3% but this allele
has not been detected in African,
West and South Asian, and European populations (1, 3, 4 ). Thus,
diagnosing HP del homozygosity
prior to blood transfusion or the
infusion of blood components
into individuals from East and
Southeast Asian populations is effective for preventing anaphylactoid shock due to anti-HP antibodies. We have developed a simple
method that uses a 5⬘ nuclease
real-time PCR assay (TaqMan;
Applied Biosystems) to detect the
HP del allele without having to isolate genomic DNA. The ethics
committee of Kurume University
School of Medicine approved this
study protocol.
To distinguish alleles, we performed real-time PCR assays that
detect the 2 regions that encompass the HP del breakpoint and the
5⬘ region of HP exon 1, which is
deleted in HP del. The 20-␮L PCR
reaction contained 200 ␮mol/L deoxynucleoside triphosphates, 1 ␮L
of template (diluted blood or
genomic DNA), 0.5 U of Ex TaqHS
with its buffer (Takara), and the
following primers and TaqMan
probes (see Fig. 1 legend for sequences): Hp5⬘-F and -R primers
(450 nmol/L); Hp5⬘–TaqMan probe
(125 nmol/L) for detecting the
5⬘ region of HP; Hpdel-F and -R
primers (900 nmol/L); and Hpdel–
TaqMan probe (250 nmol/L) for
detecting HP del. The PCR temperature profile was 95 °C for 30 s, followed by 50 cycles of denaturing at
95 °C for 5 s and annealing and extension at 60 °C for 30 s. All oligonucleotides were synthesized by
Biosearch Technologies. We mon-
itored amplification progress by
monitoring the fluorescence at the
end of each cycle with an Mx3000P
instrument (Stratagene) with excitation and emission wavelengths
of 492 and 516 nm (FAM), and
585 and 610 nm (CAL Fluor Red
610).
With genomic DNA (5 ng/␮L)
as a template, we used dual-color
scatter plots to distinguish individuals previously determined to
have the HP/HP, HP/HP del, and
HP del/HP del genotypes. Samples
with the HP/HP genotype had little
FAM fluorescence and plotted along
the x-axis, HP del/HPdel samples had
little CAL Fluor Red 610 fluorescence and plotted along the y-axis,
and HP/HP del samples were located between the homozygote
samples in the plot (Fig. 1). To the
TaqMan real-time PCR mixture,
we directly added 1 ␮L of samples
diluted 100-fold with PCR-grade
water (previously frozen samples
of buffy coat from 47 Indonesians
from Surabaya or a freshly drawn
blood sample from 1 Japanese individual from Fukuoka). Blood
was collected in EDTA-containing
tubes (Indonesian and Japanese
samples) and in a heparin-containing tube (the Japanese sample).
The results from 2 independent
experiments showed no discrepancies. In addition, the results obtained with the present TaqMan
real-time PCR method were fully
concordant with those obtained
with a previously described PCR
method for the same individuals
(i.e., 46 HP/HP individuals and
2 Indonesians with HP/HP del;
Fig. 1) (1 ). We previously had
collected blood samples from
105 Indonesian individuals from
Surabaya and had not found the
HP del allele in 58 of these individuals (3 ); however, in the
present study we did find 2 HP del
alleles among the remaining 47 individuals in this population sample. Thus, the HP del allele is also
present in Southeast Asian populaClinical Chemistry 54:6 (2008) 1095