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6-1-2007
³H-tetracycline as a proxy for ⁴¹Ca for measuring
dietary perturbations of bone resorption
Connie Weaver
Purdue University - Main Campus
Jennifer Cheong
Purdue University - Main Campus
George Jackson
Purdue University - Main Campus
David Elmore
Purdue University - Main Campus
George McCabe
Purdue University - Main Campus
See next page for additional authors
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Weaver, C.M., Cheong, J., Jackson, G., Elmore, D., McCabe, G., Martin, B. ³H-tetracycline as a proxy for ⁴¹Ca for measuring dietary
perturbations of bone resorption. Nuclear Instruments and Methods in Physics Research 259:1, 790-795, 2007.
3
H-tetracycline as a proxy for
41
Ca for measuring dietary
perturbations of bone resorption
Connie Weaver
a,
*
, Jennifer Cheong
a
, George Jackson
b
, David Elmore
b
,
George McCabe
c
, Berdine Martin
a
a
Department of Foods and Nutrition, Purdue University, 700 W. State Street, West Lafayette, IN 47906-2059, USA
b
PRIME Lab, Purdue University, West Lafayette, IN, USA
c
Statistics, Purdue University, West Lafayette, IN, USA
Available online 8 February 2007
Abstract
Our group is interested in evaluating early effects of dietary interventions on bone loss. Postmenopausal women lose bone following
reduction in estrogen which leads to increased risk of fracture. Traditional means of monitoring bone loss and effectiveness of treatments
include changes in bone density, which takes 6 months to years to observe effects, and changes in biochemical markers of bone turnover,
which are highly variable and lack specificity. Prelabeling bone with
41
Ca and measuring urinary
41
Ca excretion with accelerator mass
spectrometry provides a sensitive, specific, and rapid approach to evaluating effectiveness of treatment. To better understand
41
Ca tech-
nology as a tool for measuring effective treatments on reducing bone resorption, we perturbed bone resorption by manipulating dietary
calcium in rats. We used
3
H-tetracycline (
3
H-TC) as a proxy for
41
Ca and found that a single dose is feasible to study bone resorption.
Suppression of bone resorption, as measured by urinary
3
H-TC, by dietary calcium was observed in rats stabilized after ovariectomy, but
not in recently ovariectomized rats.
Ó 2007 Elsevier B.V. All rights reserved.
PACS: 82.80.Ms; 87.58.Xs; 82.39.Rt
Keywords: Calcium; Bone; Rats;
41
Ca
1. Introduction
Osteoporosis, a disease that is characterized by low bone
mass, warrants attention. In 2002, the estimated national
direct expenditure for osteoporotic hip fractures was
$18 billion. It is projected that the number of people with
osteoporosis will increase from 10,100,000 in 2002 to
12,000,000 an d 13,900,000 in 2010 and 2020, respectively
[1]. Consequences of osteoporosis are potentially fatal as
an average of 24% of hip fracture in patients aged 50 and
over usually die within a year following fracture [1]. Man-
agement of osteoporosis is crucial because there are treat-
ments, but no cure, for the disease.
There are various ways to manage osteoporosis, includ-
ing medications such as bisphosphonates and hormone
therapy, as well as dietary means such as calcium and vita-
min D supplementation. There is a large body of evidence
to indicate that calcium supplementation has beneficial
effects on bone during the entire life span. During child-
hood and adolescence, calcium increases bone acquisition
that may ultimately protect against low bone mass an d
fracture later in life. It has been calculated from a meta-
bolic balance study that an increase of calcium intake from
an average of 920 mg/d to 1300 mg/d during adolescence
would increase net calcium retention by 112 mg/d [2]. This
translates to an additional 4% of skeletal mass accrual over
1 year if the increase in dietary calcium were maintained.
During adulthood, dietary calcium supplementatio n
reduces fractures and slows down age-related bone loss,
0168-583X/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.nimb.2007.02.004
*
Corresponding author. Tel.: +1 765 494 8237; fax: +1 765 494 0674.
E-mail address: weavercm@purdue.edu (C. Weaver).
www.elsevier.com/locate/nimb
Nuclear Instruments and Methods in Physics Research B 259 (2007) 790–795
NIM
B
Beam Interactions
with Materials & Atoms
possibly by replacing the daily excretory and cutaneous
losses that would otherwise have drained skeletal calcium.
The importance of adeq uate dietary calcium during adult-
hood has also been shown through an increase in bone
mineral density when hormone therapy was administered
in the presence of sufficient calcium [3].
The mechanism by which calcium exerts a positive
effect on bone has been examined by kinetic studies. In a
randomized, cross over, study of calcium kinetics in adoles-
cent girls, the increased retention seen on a high calcium
diet (47.5 ± 3.9 mmol/day) versus a low calcium diet
(21.5 ± 2.0 mmol/day) was due to an increase in absorbed
calcium and suppression of bone resorption without affect-
ing bone formation [4]. This is in contrast to another kinet-
ics study in normal adults that assessed the response in
calcium metabolism to dietary calcium perturbation [5].
In this study, a high dietary calcium intake did not result
in an increase in absorbed calcium for all the subjects.
However, when absorbed calcium increased in some
subjects, bone resorption was decreased, while renal and
gastrointestinal calcium clearance were increa sed. A low
calcium intake (0.2 g/day) resulted in a reduction of
absorbed calcium, an increase in bone resorption rate,
and a decrease in renal and gastrointestinal rate constants,
possibly suggesting that an increase in parathyroid hor-
mone (PTH) is the common mediating factor since PTH
exerts its action on the kidney, intestine, and bone.
There is emerging evidence that calcium functions as an
indirect regulator of remodeling of the skeleton [6]. During
bone remodeling, the bone mineral that is being released
after bone is resorbed is either recycled or used to counter-
balance excretory losses. When dietary calcium intake is
low for a prolonged period of time, bone remodeling con-
tinues to be high, resulting in an increase in bone fragility.
Therefore, high dietary calcium reduces bone remodeling,
which results in an immediate reduction in fracture risk
before any changes in bone mass or bone balance are
detected [6].
Calcium status is a crucial factor in determining bone
health and fracture risk. Calcium status is difficult to assess
because serum calcium levels are tightly controlled. Bone
mineral content (BMC) measured by dual X-ray absorpt i-
ometry (DXA) is a proxy for cumulative calcium status
because 99% of the body’s calcium is in the bones. Indirect
measures of bone metabolism which impinge on calcium
status include bone biochemical markers of turnover that
measure substances that are relea sed by osteoblasts and
osteoclasts, as well as substances produced during the for-
mation or breakdown of collagen, a main protein found in
bone. Although DXA is considered the gold standar d for
assessing bone status, it is limited by the long lag time that
is required between measurements (6 months to years ) in
order to show changes in bone mineral density (BMD)
and BMC. In addition, biochemical markers of bone turn-
over have huge variation. Therefore, there is a need for a
more sensitive but yet direct method of assessing bone sta-
tus in response to treatments. A direct method for assessing
release of calcium from the skeleton uses a calcium isotopic
tracer to assess bone changes in response to treatments.
However, some calcium isotopic tracers have limitations
for use in humans due to their short half life or radioactive
nature. Fortunately,
41
Ca has a long half life of 100,000
years and is safe for use in humans due to the low level
of radioactivity required. Recently,
41
Ca has been proposed
as a tool in bone research [7]. However, there is much to
learn about designing studies and interpreting data for
optimizing
41
Ca technology as a tool for assessing bone
metabolism. In order to further our understanding of
41
Ca technology, we utilized
3
H-tetracycline (
3
H-TC) as a
proxy for
41
Ca in an animal study, with the ultimate goal
of developing the use of urinary
41
Ca appearance from
bone to directly measure bone resorption in humans. Previ-
ous studies have shown that upon injection of
3
H-TC in
animals, it chelates with calcium and is incorporated into
the skeleton. During bone resorption,
3
H-TC is released
in a form that cannot be reincorporated into the skeleton
[8]. Thus, urinary
3
H-TC is thought to reflect bone
resorption.
The aim of our study was to use a single
3
H-TC injec-
tion, as a proxy for
41
Ca, to understand timing of label
incorporation and release when bone resorption was
manipulated by dietary calcium.
2. Experimental
Sixty-four ovariectomized (OVX) 6-month-old rats were
purchased from Harlan (Indianapolis, IN). All the rats
were fed an AIN 93 M diet [9] (Dyets Inc., Bethlehem,
PA) during the acclimation period at our animal research
facility. This is a standard semi-purified diet that uses
casein as the protein source and meets nutrient require-
ments except as adjusted for calcium. Each rat was dosed
with 30 uCi of
3
H-TC. The early OVX rats were dosed with
3
H-TC within 1 month after OVX while the OVX stabilized
rats were dosed 3 months after OVX. Rats were random-
ized to receive eithe r 0.2% calcium or 0.5% dietary calcium
treatment. The recommended calcium intake is 0.5% which
is the level in the standard diet and serves as the control.
The 0.2% calcium intake was selected to be marginally defi-
cient without inducing weight loss. Within each dietary cal-
cium level, rats were further randomized according to time
of sacri fice (1 week, 1 month, 3 months, or 6 months).
There was no difference in average baseline weight among
groups. Urine and feces were collected for 2–4 days at 1
week, 1 month, 3 months, and 6 months post dose. Imme-
diately after collection, urine samples were centrifuged at
4000 RPM for 20 min. The supernatants were stored in a
20 °C freezer. After all the metabolic collections for the
entire study were completed, urine samples were thawed
at room temperature and centrifuged again at 3000 RPM
for 20 min. The supernatants were analyzed for calcium
and
3
H-TC content. Feces were ashed for 4 days at
600 °C, diluted with 0.5% Lanthan ium Chloride to 25 mL
and analyzed for calcium. Whole skeletons were recovered
C. Weaver et al. / Nucl. Instr. and Meth. in Phys. Res. B 259 (2007) 790–795 791
by using Dermastid beetles (provided by Dr. Alan York,
Entomology Department, Purdue University) as described
by Hefti et al. [10] after the rats were sacrificed. The skele-
tons were separated into cortical-rich midshaft femur, tra-
becular-rich proximal tibia and L1-4, and the rest of the
skeleton. Bones were digested with nitric acid and brought
up to volume with nitric acid for the assessment of percent
3
H-TC present and
3
H-TC labeling efficiency. Total cal-
cium in bone, urine, and feces was measured by atomic
absorption spectrophotometry as previously described [4]
(A Analyst 300, Perkin Elmer). Urinary and skeletal
3
H-
TC content were measured by scintillation counting (LS
6500, Beckman Coulter Inc., Fullerton, CA) using a single
labeled disintegration per minute (DPM) program.
Calcium balance was determined as follows:
Calcium balance ¼ Dietary calcium intake ðurinary
þ fecal calcium excretionÞ
Percent calcium retention was determined as:
100% Calcium balance=Dietary calcium intake
Percent
3
H-TC present in bone was calculated as:
100%
3
H-TC in boneðdpmÞ=
3
H-TC dose given to ratðdpmÞ
3
H-TC labeling efficiency in bone was calculated as:
Percent
3
H-TC in bone=Calcium content in boneðgramsÞ
Bone mineral density (BMD) was determined by dual
energy X-ray absorpt iometry (Lunar DPX IQ 5455, Mad-
ison, WI) at the start of the study and again before sacrifice
at 1 week, 1 month, 3 months, and 6 months post dose to
assess change in BMD and total bone calcium. This study
was approved by the Purdue Animal Care and Use
Committee.
SAS statistical software (version 8.0, SAS Institute,
Cary, NC) was used for all analyses. Data were report ed
as mean ± standard deviation, unless otherwise indicated.
Urinary
3
H-TC, calcium balance, and percent calcium
retention were compared at all time points by repeated
measures analysis of va riance with multiple comparisons
of the means using Tukey’s test. Constant variance and
normality assumptions were improved when
3
H-TC in
bone and urine was transformed by taking the log of the
original data. Therefore, all analyses for
3
H-TC in bone
and urine were based on log transformed data. Tukey’s test
was used for multiple comparisons when the differences in
mean were significant. p values less than 0.05 were consid-
ered statistically significant.
3. Results and discussion
The effect of calcium intake on calcium balance over
time is shown in Fig. 1. In an overall statistical model, rats
that were fed the 0.5% calcium diet had significantly higher
calcium balance (p < 0.005) and percent calcium retention
(p = 0.052) up to 6 months post dose, but this was driven
by the rats stabilized to ovariectomy, as no time point
was statistically significant in the early OVX rats. Rats
fed adequate calcium were in net positive balance, whereas
rats fed low calcium intakes were in negative balance at
1 month and in positive balance by 3 months. Rats fed
the 0.5% calcium diet excreted more calcium in the urine
and feces than rats fed the 0.2% calcium diet. Calcium
excretion over time is shown in Fig. 2. Rats fed the 0.5%
calcium diet consumed 2.5 times more calcium and
excreted approximately 2.5 times more calcium in the feces
and 1.4 times more calcium in the urine than rats fed the
0.2% calcium. Urinary calcium loss accounted for 3–6%
of calcium intake in rats.
Bone resorption, as measured by urinary
3
H-TC, is
shown in Fig. 3. At 1 month post dose, higher calcium
intakes significantly (p = 0.013) suppressed bone resorp-
tion in the OVX stabilized rats. The lack of a significant
difference in urinary
3
H-TC due to calcium intake at 6
months (p = 0.2) despite a greater difference in means than
at 1 month, which was statistically significant, was
undoubtedly due to loss of power as rats were sacrificed
Early OVX rats
-5
0
0123456
0123456
5
10
15
20
25
30
Months post dose
0.2% Ca
0.5% Ca
Calcium balance (mg/d)
P= 0.37 81
P= 0.20 13
P=0.2013
OVX stabilized rats
*
*
-5
0
5
10
15
20
25
30
35
40
Months
p
ost dose
Calcium balance (mg/d)
0.2% Ca
0.5% Ca
P=0.1683
P= 0.00 57
P=0.5 96 3
Fig. 1. Effects of the level of dietary calcium on calcium balance (mg
calcium/day) in early OVX and OVX stabilized rats. Each line represents
the mean ± SD values of 4–16 rats, depending on the time of urine and
feces collection. Asterisks indicate dietary calcium effect *p < 0.05.
792 C. Weaver et al. / Nucl. Instr. and Meth. in Phys. Res. B 259 (2007) 790–795