Element analysis in femur of diabetic osteoporosis model by SRXRF microprobe

Abstract

Diabetes mellitus affects bone metabolism and leads to osteopenia and osteoporosis, but its pathogenic mechanism remains unknown. To address this problem, mineral element of bone was analyzed in experimental diabetic osteoporosis model. Male Wistar rats were randomly divided into streptozotocin (STZ)-induced diabetic group (n = 5) and control group (n = 5). The experiment lasted 68 days and at the end of the experiment, femoral bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry and element content in femur of animals was determined by synchrotron radiation X-ray fluorescence (SRXRF) microprobe analysis technique. Results showed that femoral BMD in diabetic group was significantly lower than that in control (P < 0.01). Relative mineral content of calcium (Ca), phosphorus (P) and zinc (Zn) in diabetic femurs decreased significantly compared to controls. And strontium (Sr) in diabetics reduced 11% (P = 0.09). Relative content of sulfur (S) in average was statistically higher (P < 0.01) in diabetics than that in controls. But no obvious difference was observed in relative content of chromium (Cr), iron (Fe), copper (Cu), and lead (Pb) between the two groups. Statistical analysis revealed that Ca correlated positively with P (R = 0.85 and P < 0.001), with Sr (R = 0.38 and P < 0.05) and with Zn (R = 0.37 and P < 0.05). Whereas, Zn correlated negatively with S (R = −0.40 and P < 0.05). Our results reveal that loss of minerals accounts for the BMD reduction in diabetics.

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Element analysis in femur of diabetic osteoporosis model by
SRXRF microprobe
Yurong Feia, Min Zhanga, Ming Lia, Yuying Huangb, Wei Heb,
Wenjun Dinga, Jianhong Yanga,*
aDepartment of Biology, Graduate University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, PR China
bBeijing Synchrotron Radiation Facility, Institute of High Energy of Physics, Chinese Academy of Sciences,
19 Yuquan Road, Beijing 100049, PR China
Received 2 August 2006; accepted 11 September 2006
Abstract
Diabetes mellitus affects bone metabolism and leads to osteopenia and osteoporosis, but its pathogenic mechanism remains unknown. To
address this problem, mineral element of bonewas analyzed in experimental diabetic osteoporosis model. Male Wistar rats were randomly divided
intostreptozotocin(STZ)-induceddiabeticgroup(n = 5)andcontrolgroup(n = 5).Theexperimentlasted68daysandattheendoftheexperiment,
femoralbonemineraldensity(BMD)wasmeasuredbydual-energyX-rayabsorptiometryandelementcontentinfemurofanimalswasdetermined
by synchrotron radiation X-ray fluorescence (SRXRF) microprobe analysis technique. Results showed that femoral BMD in diabetic group was
significantly lower than that in control (P < 0.01). Relative mineral content of calcium (Ca), phosphorus (P) and zinc (Zn) in diabetic femurs
decreased significantly comparedtocontrols. Andstrontium(Sr) indiabeticsreduced 11%(P = 0.09).Relativecontentofsulfur (S)inaveragewas
statistically higher (P < 0.01) in diabetics than that in controls. But no obvious difference was observed in relative content of chromium (Cr), iron
(Fe),copper(Cu),andlead(Pb)betweenthetwogroups.StatisticalanalysisrevealedthatCacorrelatedpositivelywithP(R = 0.85andP < 0.001),
with Sr (R = 0.38 and P < 0.05) and with Zn (R = 0.37 and P < 0.05). Whereas, Zn correlated negatively with S (R = ?0.40 and P < 0.05). Our
results reveal that loss of minerals accounts for the BMD reduction in diabetics.
# 2006 Elsevier Ltd. All rights reserved.
Keywords: Osteoporosis; Diabetes; Element analysis; Synchrotron radiation; X-ray fluorescence microbe
1. Introduction
Bone is connective tissue mainly composed of organic
protein collagen, providing soft framework and inorganic
hydroxyapatite, adding strength and hardening the framework.
Moreover, bone is mineral reservoir for calcium, phosphorus,
zinc,strontiumandmanyotherminerals.Abnormalitiesinbone
metabolism could cause osteoporosis, a disease in which bones
become fragile and more likely to break. Osteopenia or
osteoporosis is found in diabetic women, as well as in diabetic
men (Inzerillo and Epstein, 2004; Thrailkill et al., 2005;
Bridges et al., 2005). And studies have proved that diabetes can
bring about abnormalities in element homeostasis (Cooper
etal.,2005;Zargaretal.,1998),which arelikelyrelated tobone
metabolism. However, the exact role of bone minerals in BMD
of diabetics is still not clear right now.
SRXRFmicroprobeisavaluabletechniqueforboneelement
analysis due to its advantages of high sensitivity, non-
destructing, multi-elemental data and relative easy procedure.
Several studies have demonstrated that SRXRF microprobe is a
very appropriate tool for the study of element content and its
distribution in biological samples (Zhang et al., 2005; Shi et al.,
2004; Abraham et al., 2005). Zhang et al. (2005) has studied
element content distribution in femoral head slice with
osteoporosis and found remarkably lower concentration of
Ca, P in spongy and cartilage zones. Therefore, we propose that
loss of minerals probably leads to significant reduction in BMD
of diabetics. To explore the cause of BMD reduction in
diabetics, here we investigate relative element content in femur
of experimental diabetic osteoporosis model by SRXRF
microprobe. Moreover, we will also discuss the correlation
between important elements.
www.elsevier.com/locate/micron
Micron 38 (2007) 637–642
* Corresponding author. Tel.: +86 10 88256348; fax: +86 10 88256080.
E-mail address: yangjh@gucas.ac.cn (J. Yang).
0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2006.09.003

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2. Materials and methods
2.1. Animals
Eight-week old Wistar rats (Department of Laboratory
Animal Science, Peking University Health Science Center,
Beijing, PR China) weighing 240 ? 10 g were selected and
studied in this study. The animals were kept under standard
conditions of temperature, light and were free to standard diet
and water. Replacement of cages, daily replacement of tap
water and other routine hygiene procedures were done by a
licensedtechnicianintheanimallabofInstituteofHighEnergy
Physics, Chinese Academy of Sciences. All animal procedures
in this study were carried out according to the standards of the
guide for the Care and Use of Laboratory Animals.
2.2. Inducement of diabetes
After 1 week of adaptation to the laboratory environment,
rats were randomly separated into two groups: control group
(n = 5) and diabetic group. In the beginning of the experiment,
on day 0, rats received a single intravenous administration of
freshly prepared streptozotocin (STZ) (Sigma, St. Louis, MO,
USA) dissolved in 0.1 M citrate buffer (pH 4.5) at a dose of
55 mg/kg body weight. Equal volume of vehicle was injected
into the control rats. After 1 week of administration, five rats
with fasting blood glucose (12 h) higher than 12 mmol/L were
selected and studied in this research. The experiment lasted 68
days after the onset of diabetes.
2.3. Blood glucose and body weight
Glucose was determined immediately by a glucose analyzer
(ACCU-CHEK, Roche Diagnostic GmbH, D-68298 Man-
nheim,Germany)after blood was collectedfrom acut atthetail
vein of 12 h fasted rats in both diabetic group and control group
on days 0, 13, 22, 30, 40, 50, 60, and 68, respectively.
Body weight was monitored before glucose determination
each time.
2.4. Measurement of bone mineral density
At the end of experiment on day 68, the fasted rats were
anesthetized with barbital. Then right femurs in both diabetic
group and control group were immediately dissected, cleaned
of adherent soft tissue. And bone samples were stored in 10%
formalin until analysis.
The total area of bone mineral density (BMD), bone mineral
content and bone mineral area of the right femurs were
measured by dual-energy X-ray absorptiometry (Excellplus,
Norland, USA) with the small animal total body option in the
osteoporosis measurement.
2.5. Element analysis
The element determination was carried out at the X-ray
fluorescence (XRF) microprobe station at Beijing synchrotron
radiation facility (BSRF), Institute of High Energy Physics,
Chinese Academy of Sciences. The X-ray light source came
from the 4W1B beam line at BSRF. This beam line could
provide multi-chromatic X-rays (white light), which energy
ranged from 4 to 30 keV. The electron energy in the storage
ring is 2.2 GeV, with a current range from 60 to 106 mA. At
present, the beam of the microprobe was focused on
150 mm ? 150 mm. The emission X-ray spectrum was
recorded with the counting live time of 200 s. The femoral
sample was fixed on a micro-stage with 0.5 cm/step by a
computer-controlled motor. Five points in each sample were
selected and scanned as shown in Fig. 1. XRF spectra were
collected by a PGT Si (Li) detector, positioned at 908 to the
beam line, 4.5 cm from the target. The collected spectra were
analyzed by an AXIL program.
Three of five femur samples in each group were randomly
selected and were scanned by SRXRF microprobe for element
analysis. Data were deleted if they were less than 3 times of the
corresponding standard deviation. To correct the effect of
synchrotron radiation beam flux variation, the elemental peak
area was normalized to the peak counts of Ar, which exists only
in air with a constant proportion. The normalized peak areas
were for estimating the relative contents of elements.
2.6. Statistical analysis
Data analysis was performed by SPSS (version 10.0). All
results were presented as means ? S.E.M. Comparisons
between groups were made by independent-samples t-test.
Fig. 1. A sketch of femur sample and five points were selected and scanned by
SRXRF microprobe in this experiment.
Y. Fei et al./Micron 38 (2007) 637–642 638

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Significant difference was defined at either P < 0.05 or
P < 0.01. A linear correlation test was made by origin 6.1.
3. Results
3.1. Changes on blood glucose
Changes on glucose were monitored throughout the study
(Fig.2).Glucoseindiabeticgrouphadbeensignificantlyhigher
than that in control group since the onset of diabetes in the
experiment, indicating successful model of STZ-induced
diabetes mellitus.
3.2. Changes on body weight
Body weights of experimental animals throughout the study
were present in Fig. 3. Average body weight in diabetic group
was remarkably lighter than that in controls. Particularly, body
weight of diabetic rats at the end of the experiment decreased
5% while those in control group increased 49.8% in average
comparedwith initialbodyweights.Besides,highconsumption
of diet and water were also observed in diabetic rats (data not
shown), consisting with the most of common symptoms of
diabetes (e.g., polyphagia, polydipsia and polyuria).
3.3. Femoral BMD of diabetic and control rats
Femoral bone mineral content and bone mineral area of
diabetic rats were statistically lower than those of controls
(Table 1).Consequently,BMD ofdiabeticrats wassignificantly
lower than that of controls after the onset of diabetes (P < 0.01;
Fig. 4).
3.4. Element analysis
The typical energy spectrum of P, S, Ca, Cr, Fe, Cu, Zn, Sr,
and Pb in femur was investigated by SRXRF microprobe in this
experiment (Fig. 5). As shown in Table 2, Ca and P were of the
macro-elements of bone; S, Cr, Fe, Cu, Zn, Sr, and Pb were
among the trace elements of bone (Table 2).
Relative content of element Ca in diabetic group was
significantly lower than that in control group (P < 0.01).
Moreover, P and Zn in diabetics also reduced markedly
(P < 0.05). Strontium in femur of diabetic rats reduced 11%
(P = 0.09). However, sulfur in diabetes was obviously higher
than that in controls (P < 0.01). For Cr, Fe, Cu, or Pb, no
significant difference was observed between the two groups.
Statistical analysis showed that Ca had a strong correlation
with P (R = 0.85 and P < 0.001) (Fig. 6A). Calcium also
Fig. 2. Bloodglucose of diabetic and control rats;**P < 0.01 vs. control group.
Fig. 3. Body weight of diabetic and control rats;**P < 0.01 vs. control group.
Table 1
Femoralbonemineralcontentandbonemineralareain controlanddiabeticrats
Bone mineral content (g) Bone mineral area (cm2)
Control (n = 5)
Diabetes (n = 5)
0.32 ? 0.01
0.22 ? 0.01**
2.45 ? 0.05
2.14 ? 0.05**
**P < 0.01 vs. control group.
Fig. 4. Femoral bone mineral density (BMD, total area) in diabetic (solid bar)
and control (hollow bar) rats;**P < 0.001 vs. control group.
Fig. 5. A typical SRXRF spectra of femur. The Ar peak was from the air.
Y. Fei et al./Micron 38 (2007) 637–642 639

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correlated positively with Sr (R = 0.38 and P < 0.05) (Fig. 6B)
and with Zn (R = 0.37 and P < 0.05) (Fig. 6C). But element Zn
correlated negatively with S (R = ?0.40 and P < 0.01)
(Fig. 6D).
4. Discussion
So far, there has been no report on element analysis with
SRXRF microprobe to study BMD reduction in experimental
diabetic osteoporosis model. Our results showed that abnorm-
alities in bone minerals, especially in Ca, P, Sr, S and Zn,
closely related to BMD reduction in diabetics. Other elements
including Cr, Fe, Cu, and Pb were also analyzed in this study
and no statistic difference was observed in relative content of
these elements between diabetics and controls.
Living and growing tissue, bone is made of organic
component (primarily the collagen protein) and mineral
substance—hydroxyapatite. There are two main functions of
the bone mineral substance. A biomechanical role is the
stability of the skeleton and a metabolic one is reservoir for
many ions, control mineral homeostasis. Approximately 80–
90%ofbonemineralcontentiscomposedofCaandP(Ilichand
Kerstetter, 2000) and more than 99% of the body’s Ca is
contained in the bones and teeth (National Institute of Health
Osteoporosis and Related Bone Diseases, 2005). Extracellular
Ca is one of the main factors regulating the process of bone
remodeling, by means of a multi-organ cross-signaling cascade
(Purroy and Spurr, 2002). And the availability, storage and
disposal of Ca are regulated by a systemic mechanism partly
through PTH and Vitamin D (Purroy and Spurr, 2002).
Table 2
Relative element contents in femur of control and diabetic rats
P*
S**
Ca**
CrFeCu Zn*
SrPb
Controls
Numbera
0.70 ? 0.03
15
0.24 ? 0.01
15
205.75 ? 5.40
15
0.05 ? 0.00
15
0.69 ? 0.04
15
0.21 ? 0.01
15
11.64 ? 0.49
15
1.74 ? 0.09
15
0.20 ? 0.02
7
Diabetes
Numbera
0.61 ? 0.03
15
0.53 ? 0.05
15
171.76 ? 9.20
15
0.06 ? 0.01
15
0.64 ? 0.02
15
0.21 ? 0.02
15
9.67 ? 0.47
15
1.55 ? 0.06
15
0.20 ? 0.02
14
*P < 0.05,**P < 0.01 vs. control group.
aThe valid points scanned in diabetic or control group after data were analyzed.
Fig. 6. (A) Correlation of Ca and P (R = 0.85 and P < 0.001) in femur of control group investigated by SRXRF microprobe. (B) Correlation of Ca and Sr (R = 0.38
and P < 0.05) in femur of control group investigated by SRXRF microprobe. (C) Correlation of Ca and Zn (R = 0.37 and P < 0.05) in femur of control group
investigated by SRXRF microprobe. (D) Negative correlation between S and Zn (R = ?0.40 and P < 0.05) in femur of control group investigated by SRXRF
microprobe.
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Numerous studies have proved that Ca plays a key role in bone
structure (Ilich and Kerstetter, 2000). For example, it has been
reported that calcitriol improves STZ-induced diabetes and
recoversbonemineraldensityindiabeticrats(DelPino-Montes
et al., 2004). Furthermore, statistical analysis in our research
showed that Ca has a strong correlation with P (R = 0.85 and
P < 0.001) (Fig. 6A), consistent with others’ reports (Zhang
et al., 2005). Phosphorus, an inorganic element, is second to Ca
in abundance in the human body with 85% bound to the
skeleton (Ilich and Kerstetter, 2000). Results in this study
showed significant decrease in relative content of Ca and P in
femur of diabetic rats than those in controls, although there
were no changes in Ca mineral concentration between diabetic
and control group by Instrumental Neutron Activation Analysis
(Facchini et al., 2006). Rats in their study were 9- to 12-month
old and were female but rats in ours were 2-month old and were
male. Thus, the experimental animal and analysis method
possibly lead to the different result. However, Einhorn et al.
(1988) found decreased ash content of Ca and P. Furthermore,
also applying the SRXRF microprobe for bone element
scanning analysis, Zhang et al. reported that the concentration
of Ca and P was obviously low in both spongy and cartilage
zones for the osteoporosis patient slice of the femoral head, but
there was no marked difference in the compact zone (Zhang
et al., 2005). Therefore, we believe that significant loss of
mineral Ca and P in bone accounts for the marked reduction in
BMD of diabetics.
Trace elements also influence the process of bone
remodeling by affecting bone mineral crystal size, density,
and solubility, through their roles as metallo-enzymes in the
synthesis of collagen and other proteins that form the
structure of bone (Reinhold, 1975; Saltman and Strause,
1993). Results in our investigation showed that relative
concentrations of Sr and Zn were obviously low in diabetics
than in controls. Besides, Ca correlated positively with Sr
(R = 0.38 and P < 0.05) (Fig. 6B) and with Zn (R = 0.37 and
P < 0.05) (Fig. 6C). Intensive studies have been done on Sr
for biological interest lately. Strontium ranelate, a novel
agent containing two strontium atoms, has been licensed in
United Kingdom for the treatment of osteoporosis (Fogelman
and Blake, 2005). Recent studies in vitro showed that
strontium ranelate acts as an effective anti-osteoporotic drug
by inhibiting bone resorption by osteoclasts and promoting
osteoblast replication and bone formation (Marie, 2005).
Studies have proved that Sr is an agonist of the calcium-
sensing receptor with a lower affinity than Ca and that the
reported anti-osteoporotic effects of the drug is due to the
modulation of he calcium-sensing receptor in bone cells by
strontium ranelate (Coulombe et al., 2004), and other
mechanisms are to be identified. Here, in the present
experiment, we found that relative content of Sr in femur
of diabetics reduced 11% (P = 0.09) than that of controls and
statistical analysis showed that Sr had a good correlation with
Ca. The decrease in Sr in diabetics is assumed to be one of the
causes leading to diabetic osteoporosis.
An essential element in bone metabolism, Zn acts as a
cofactor for several enzymes, such as ALP – necessary for bone
mineralization and collagenase – essential for development of
the collagenous structure of bone (Beattie and Avenell, 1992).
Beneficial effect of Zn supplementation in bone formation is
well reported by Gonzalez-Reimers (Gonzalez-Reimers et al.,
2005) and other research groups. Besides a positive correlation
with Ca, Zn also had a negative correlation with S (Fig. 6D),
which increased significantly in diabetics compared to controls
(Table 2). Sulfur exists in many substances that are essential to
bone metabolism. For example, heparin sulfate is a major co-
factor in common sharing with the majority of osteoblast
growth promoting elements (Cool and Nurcombe, 2005). And
estrone sulfate, which may be a better marker for estrogen
status, is the predominant circular estrogen in normal
physiological situations (Castracane et al., 2006). The
dehydroepiandrosterone sulfate binds strongly to albumin
and seems to be a storage form of testosterone (Keles et al.,
2006). However, the specific mechanism of S in bone is not
outlined right now. Therefore, obvious decrease of Zn in the
current study probably contributed to BMD reduction of
diabetic animals and increase of S is possibly not beneficial to
bone remodeling.
In summary, investigated by SRXRF microprobe in the
experimental diabetic osteoporosis, significant decrease was
observed in bone mineral content of Ca, P, Zn, Sr and marked
increase in relative content of S in diabetic femur than those in
controls. These results show that loss of minerals, detrimental
to bone metabolism, is an important reason for BMD
reduction in diabetics. And our study on element analysis
will help to clarify the mechanism of BMD reduction in
diabetics.
Acknowledgements
This work is supported by the Scientific Research
Foundation of Graduate University of Chinese Academy of
Sciences (no. 055101FM03) and China National Natural
Sciences Foundation (grant no. 20571084). We thank Yanbin
Hao and Maocai Zhang for help in statistical analysis.
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