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Osteoporosis is a syndrome characterized by
a reduced bone mass such that there is an
increased risk of bone fracture. The hip, wrist
and spine are the most vulnerable.
Osteoporosis is due to due to low peak bone
mass and/or excessive bone loss.
Osteoporosis
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Type II or Senile Osteoporosis
It affects people 75 years old or more, with
women twice as likely to be affected. It ischaracterized by both cortical and trabecular
bone loss. Fractures may occur in all the
skeletal sites, but the pathognomonic
fracture for type II osteoporosis is the hip
fracture.
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Type III Osteoporosis or Secondary Osteoporosis
In 10% of women and 50% of men osteoporosis issecondary to other diseases or treatments.
* Amenorrhoeic athletes * Pregnancy
* Hyperprolactinemia * Lactation
* Hyperthyroidism * Alcohol intake
* Long-term corticosteroids therapy * Smoking
* Immobilization * Anorexia nervosa
* Malabsorption * Osteogenesis Imperfecta (Familial)
* Hypothyroidism (Iry) * Hypogonadism
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Calcium Regulating Hormones: PTH, l,25 dihydroxy Vit. D,
CT.
Other Hormones: Thyroid, Pituitary, Adrenal and Gonadal
Steroids.
Cytokines and Growth Factors: M-CSF, IL-1, IL-6, IL-11
control osteoclasts development.
TGF : local mediator of osteoblast - osteoclast coupling. IGFs I & II, FGFs, PDGFs: Influence osteoblastic activity.
PTH-RP, FGFs: Involved in cartilage differentiation.
Regulatory Mechanisms of Skeletal
Remodelling and Calcium Metabolism
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1. Measurement of BMD: assess risk of fracture.
2. Histomorphometry of bone biopsies: provide
information on the pathogenesis but it is invasive
method.
3. Calcium kinetic studies: provide information on the
rate of bone turnover and Ca++ absorption but it is
time consuming.
Radiological Assessment
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Provides Information on:
1) Pathogenesis.
2) Rate of bone turnover.
3) Monitoring antiresorptive therapy.
4) Identify fast bone losers at risk of hip fracture.
Biochemical Assessment
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Blood level correlates with histomorphometric and 47 Ca
measurements of bone formation.
Biomarkers of Bone Formation
B-ALP Isoenzyme present on osteoblast membrane,
its synthesis increases during osteoblastic
differentiation.
OC Synthesized by osteoblasts.
PICP and P1NP Present in bone and skin and other tissues
containing collagen.
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* Released only during bone resorption
Biomarkers of Bone Resorption
Bone tartarate resistant acidphosphatase (TRACP)
Present in osteoclast and other macrophages. Serumlevel correlates with histomorphometry.
OHPr Present in all collagen molecules, released during
both bone formation and bone resorption.
Galactosy1 hydroxylysine
(GHYL)*
Present only in collagen; 5/7 times more abundant in
type I collagen of bone than in type 1 collagen ofskin.
Pyridinium cross-links (Pyr. &
DPyr)*
Non-reducible cross-links that form among 2
hydroxylysine residues on the collagen telopeptides
and one lysine or hydroxylysine residue on the helix protion of an adjacent molecule after deposition
of collagen in matrix DPyr is present mainly in bone.
ICTP and INTP (NTX)* Present in all tissues containing type 1 collagen
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Serum IR BSP
Bone Sialoprotein (BSP) is a heavily glycosylated and
phosphorylated protein that accounts for 5-10% of the non-
collagenous proteins of the bone extracellular matrix. BSP
plays an important role in cell-matrix adhesion processes
and in the supramolecular organization of the extracellular
matrix of mineralized tissues. BSP appears to be a sensitive marker of bone turnover and
its serum levels reflects processes related to bone
resorption (Seible et al., 1996).
Biomarkers of Bone Resorption Cont'd
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Closer concordance of BMD in monozygotic as
opposed to dizygotic twins (Smith 1993).
Reduced BMD in daughters of osteoporoticwomen when compared with control subjects
(Fotino et al., 1977)
Family history of osteoporosis predicts BMD(Soroko et al., 1994).
Evidence In Favour of A Genetic Contribution
to Osteoporosis
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An association between VDR genotypes and
serum Vit. D response gene Osteocalcin
(Lander and Schork, 1994).
A strong association between VDR
polymorphisms and BMD (Morrison et al., 1994).
Candidate Genes for Osteoporosis
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Estrogen Receptor Gene ER Gene
An osteoporotic phenotype has been noted in a
man with a coding region mutation of the ER
gene (Smith et al., 1994).
Intronic RELP and T/A repeat polymorphism of
the ER gene has been associated with BMD
(Kobayashi et al., 1996).
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Mutations affecting coding unions of the genes give
rise to a severe osteoporotic phenotype (osteogenesis
imperfecta).
G/T polymorphism at COLI Al predicts bone
mass (Granl et al., 1996).
G/T heterozygotes (Ss) have significantly lower BMD at
the lumber spine than G/G homozygotes (SS) and BMD
is still lower in T/T homozygotes (ss).
Collagen Type I Genes (COL1) Al, COLI A2)
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Genomic DNA extraction can be performed on a venous blood
sample to study genetic polymorphism which are likely to be of
value as indicators of osteoporotic fracture risk in clinical
practice.
1) VDR polymorphism.
2) COLI Al SP1 polymorphism.
These genetic markers may be used - in combination with
existing techniques - to improve the identifications of patients
at risk of osteoporosis before the condition has became
established.
Genetic Biomarkers of Bone Turnover
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Unfavorable Ss and ss genotypes are over
represented in osteoporotic vertebral fracture
patients with a calculated relative risk of fracture of
about 3 in individuals who carry the "s" allele as
compared with "SS" homozygotes. The "s" allele
may act as a marker for increased age related bone
loss rather than peak bone mass.
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Choice of Reliable Biochemical Markersfor the Assessment of Bone Turnover
in Postmenopausal Osteoporosis
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Aim of Work
This study was undertaken to asses bone
turnover in PMO by a battery of 15
biochemical markers of bone formation and
of bone resorption in order to select the most
reliable markers.
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Markers of Bone Formation
Serum biomarker Method Company Controls
Mean +SD
PMO mean
+SD
Significant
changes
Total OC (ng/ml) IRMA CIS 20.6+5.2 32.3+8.5 + 57%
B-ALP (ng/ml) IRMA Hybritech 9.6+4 18.3+5.7 + 91%
PICP (ng/ml) RIA Orion Diag 100+20 120+26 NS
CT (ng/ml) RIA DPC 15.2+12.9 12+6 NS
PTH-C (ng/ml) RIA Immunonuclear 1.8+4.5 0.46+0.2 NS
PTH-M (pmol/L) RIA Immunonuclear 80.9+50.1 64.5+26.8 NS
VIT D3 (mg/ml) RIA Immunonuclear 16.3+4.7 15.4+4.8 NS
Ca++ (mg/dl) Colorimetric Bioanalytics 8.7+0.8 9.2+1.7 NS
P (mg/dl) Colorimetric Bioanalytics 4.1+0.7 4.2+1.4 NS
Intraassay CV : 1.6-7.8%, Interassay CV: 1.1-9.2%
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Markers of Bone Resorption
Biomarker Method Company Controls
Mean +SD
PMO mean
+SD
Significant
changes
u Pyr (nmol/mmol Cr) ELISA Metra 35.6+10 48+11.2 + 35%
u D Pyr (nmol/mmol Cr) ELISA Metra 5.8+1 10+4 + 72%
u. NTX (nmol/mmol Cr) ELISA Ostex 30.8+14.3 90.3+36.4 + 193%
s. ICTP (ng/ml) RIA Orion 4.2+1 4+0.9 NS
u. cAMP (nmol/mg Cr) RIA Immunonuclear 25+22.3 21.7+19.2 NS
u. OHPr (mg/mg Cr) Colorimetric ----- 0.02+0.02 0.02+0.01 NS
Intraassay CV : 2.8-8.6%, Interassay CV: 7.1-10%
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T-score = X-M / SD
Calculated Uncoupling Status = 4.19-9.25 =
-5.06
T* (z) Score of Biomarkers of Bone
Turnover in PMO
Bone formation T-score Bone resorption T-score
OC 2.25+2 Pyr 1.2+1.1
B-ALP 2.2+1.9 D Pyr 4.2+3.2
PICP 0.98+1.0 NTX 4.2+2.6
Vit D3 -0.2+0.3 ICTP -0.2+0.2
PTH-M -0.33+0.3 cAMP -0.15+0.2
PTH-C -0.3+0.4 OHPr 0.0
CT -0.4+0.5 - -
Net T-score 4.19 Net T-score 9.25
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T-Score of Biomarkers of Bone
Turnover in PMO
The calculated uncoupling status of 12
biomarkers of bone formation and bone
resorption equals to - 5.0. On the other hand, thecalculated value of the 4 most reliable
biomarkers of bone formation (OC & B-ALP) and
bone resorption (NTX and DPyr) equals to -4.0.This difference does not necessitate the assay
of 12 instead of 4 biomarkers.
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Bone Turnover in PMO was significantly
higher than in healthy PM women.
The most reliable markers which are able to
discriminate between slow and fast bone losersin PM women were B-ALP and OC for
assessment of bone formation and NTX and
DPyr for assessment of bone resorption. These
4 combined assays may be able to augment or
replace bone densitometry for monitoring
antiresorptive in PMO.
CONCLUSION
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Markers of Bone Formation (Osteoblastic Activity):
Ostase (B-ALP): ............ ng/ml (NV-1 -15)
Osteocalcin (GLA-Protein): ....... ng/ml (NV-8-28).
Markers of Bone Resorption (Osteoclastic activity):
NTX (Osteomark) ............ mmol/mmol Cr (NV-up to 58)
Deoxypyridmollns (Pyr) mmol/mmol Cr (N-0.4-6.4)
Calculated uncoupling statues of T-scores:
- Normal : below-0.8
- Slow bone losers : 0.8 to 3.0
- Fast bone losers : above -3.0
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Early detection of Osteoporosls
Selection of suitable patients for HRT
Monitoring anti-resorptive therapy
Diagnosis of fast bone loser at high risk of bone fracture.
Monitoring bone metabolism in patient with:
- Hypothyroidism.
- Hyperthyroidism.
- CRF.- GH deficiency.
Monitoring bone metabolism in patients on long term
corticosteroids or NSAID therapy.
Indications of Bone Markers Assays
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Activated T Cells Mediated Bone Loss
through the triangle
RANKL / RANK/OPG
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Activated T-cells can increase the number
of osteoclasts and reduce BMD both in
vitro and in vivo (Kong, et al, 1999). This
may explain the association of bone loss
with leukemia or infections.
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TNF Ligand family member: Receptor
Activator of Neuclear Factor-kB Ligand
(RANKL) or Osteoprotegerin Ligand (OPGL)
and its receptor (RANK and soluble receptor
Osteoprotegerin (OPG) has established a
novel paradigm of osteclast biology.
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RANKL, RANK and OPG constitute the 3 essential
regulatory components of ostecldast formation, fusion,
survival, activation and apoptosis (Hofbouer et al,
2000).
- RANK-ODF (Osteoclast differentiation factor).
- RANK-ODAR (Osteoclast differentiation activation
receptor).
- OPG-OCIF (Osteoclastogenesis Inhibitory factor).
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RANKL increases the pool of active
osteoclast by activating its specific receptor
RANK located on osteoclastic cells, thus,
increasing bone resorption.
OPG neutralizes RANKL and has opposite
effects.
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RANKL and OPG are produced by bone marrow
derived stromal cells and osteoblasts and are
regulated by various calcitropic cytokinei,
hormones and drugs. Abnormalities in RANKL/OPG
ratio have been implicated in different metabolic
bone diseases characterized by increased
osteoclastic differentiation, activation and
enhanced bone resorption.
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Among the factors that increases RANKL mRNA
expression in osteoblastic cells are the
prolnflammatory cytokines IL-1, IL-6, IL-11 and
TNF- (Yasuda et al, 1998 and Hofbauer et al,1999). Inflammatory lesions of bone are
characterized by abundant osteoclast
proliferation.
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TNF- is a potent osteoclastogenic agent andappears to mediate orthopedic implant loosening
(Merkel et al, 1999). TNF- stimulates osteoblasticcells to express RANKL which in turn prompt
macrophages to become osteoclasts. Consistent
with this hypothesis, systemic adminstration of OPG
blocks osteoclastogenesis in experimental arthritis,
a situation in which there are abundant amounts of
TNF- (Kong et al, 1999).
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Products of breast cancer cells (PTH rP, IL-6, IL-
11 and cyclooxygenase-2 generated prostanoids)
promote RANKL formation by acting on residentosteoblasts and/or stromal cells (Thomas et al,
1999) The release of growth factors especially
TGFp can influence the growth of tumor cells andtheir production of bone-resorbing cytokines (Vin
et al, 1999).
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Rheumatoid arthritis is characterized by destruction
of articular cartilage and by excessive subchondrial
osteoclastic bone resorption (Romas et al, 2000). In
the inflammatory state, macrophages, which
differentiate into osteoclasts, accumulate in the
rheumatoid synovial membrane where there are
many osteoclastogenic cytokines including IL, IL-6,
IL-11, IL-13, IL-17 (Kotake et al, 1999) and PTHrP.
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Rheumatoid synovial fibroblasts produce
RANKL (Romas et al. 2000) and T-cells
producing RANKL have been shown to promote
osteoclast formation without the participation of
other cells (Norwood et al, 1999).
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On the other hand OPG production is stimulated by
E2 (Saika et al, 1999), calcium cation (Yasuda et al,
1998) and bone morphogenetic protein-2 (Hofbauer
et al, 1998).
OPG production is decreased by PGE2 (Brandstrom
et al, 1998), glucocorticoids (Vidal et al, 1998), 1 ,25 (OH)2 D3 (Norwood et al, 1998), estrogen receptor
antagonist (Hofbauer et al, 1999) and PTH (Lee &
Lorenzo, 1999).
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Therefore RANKL/OPG, are the key agonist /
antagonist cytokine system that regulate osteoclast
biology including differentiation, fusion, survival,activation and apoptosis. RANKL increases the pool
of active osteoclasts by activating its specific
receptor RANKL located on osteoclastic cells, thus
increasing bone resorption, whereas OPG which
neutralizes RANKL, has opposite effects.
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PTH Glucocorticoids Vit D3
E2PRL
Hypothyroidism
Decreased OPG
Abnormal RANKL/OPG Ratio
Decreased Osteoblastic Activity
Increased Bone Resorption
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Infections / cancer
T-cell activation Increased osteoclastogenic
Cytokines IL-6, IL-11, TNF-
PE
Increased osteoclast
Activating growth factors(TGF- )
PGE2 Increased RANKL PTH, PTHrP Abnormal RANKL/OPG ratio
Increased osteoclastic activity
Increased bone resorption
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Shaarawy M, Zaki S, Shehata H. Circulating levels
of osteoclast activating cytokines, interleukin-11
and transforming growth factor B2 as valuable
biomarkers for the assessment of bone turnover
and monitoring antiresorptive therapy in
postmenopausal osteoporosis. Clin. Lab
(Germany) 2003; 40: 625-636.
Further Readings
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Shaarawy M, Abassi AF, Hassan H, Salem ME.
The relationship between serum leptin
concentrations, bone mineral density and
biochemical markers of bone turnover in
postmenopausal osteoporosis. Fertil Steril, 2003;
79: 919-924.
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Shaarawy M, Hasan M. Serum bone slaloprotein,
a reliable biomarker of bone resorptlon In
diagnosis and monitoring treatment of
postmenopausal osteoporosis. The Scandinivian
Journal of Clinical and Laboratory Investigation
2001; 61: 513-522.
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Shaarawy M, Shaltout F, Idris O, Sheiba M, Wali
M. Circulating levels of insulin-like growth factor-
1 and insulin-like growth factor binding protein -3
as valuable markers in the assessment of bone
remodeling and monitoring antiresorptive
therapies in postmenopausal osteoporosis. Arab
J. Lab Med 2002; 28(1): 79-104.
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Shaarawy M, EI-Mallah SY, Hassan HE, Aref
At. Choice of reliable biochemical markers for
assessment of bone remodeling in
postmenopausal osteoporosis. J. North
American Menopause Society 1997; 4: 212-218.
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Shaarawy M, EI-Dawakhly AS, Mosaad M, El-
Sadek M.M Biomarkers of bone turnover and
bone mineral density in hyperprolactinemic
amenorrheic women. Clin. Chem Lab Med
(Eur) 1999; 37 (4): 433-438.
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Darwish NA, Shaarawy M, Ezzat R, EI-Kafas H.
Hormonal and biochemical changes in
postmenopausal age related bone loss. A
biochemical profile for the differential diagnosis
of senile and postmenopausal osteoporosis and
assessment of the severity of osteoporotic
changes. Egypt Soc Obstet Gynecol 1988; 14 (1):
103-111.
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Shaarawy M, EI-Mallah SY, Shaltout F, Abbasy A.
Serum osteocalcin Gla-protein levels in senile
and postmenopausal osteoporosis. Arab J. Lab
Med. 1990; 16(1): 1-10.
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