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Osteoporosis

• 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.
Type II or Senile Osteoporosis
• It affects people 75 years old or more, with
women twice as likely to be affected. It is
characterized 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.
Type III Osteoporosis or Secondary Osteoporosis
In 10% of women and 50% of men osteoporosis is
secondary 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


Regulatory Mechanisms of Skeletal
Remodelling and Calcium Metabolism

• 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.
• TGFβ : local mediator of osteoblast - osteoclast coupling.
• IGFs I & II, FGFs, PDGFs: Influence osteoblastic activity.
• PTH-RP, FGFs: Involved in cartilage differentiation.
Radiological Assessment

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.
Biochemical Assessment

Provides Information on:

1) Pathogenesis.

2) Rate of bone turnover.

3) Monitoring antiresorptive therapy.

4) Identify fast bone losers at risk of hip fracture.


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.

Blood level correlates with histomorphometric and 47


Ca
measurements of bone formation.
Biomarkers of Bone Resorption

Bone tartarate resistant acid Present in osteoclast and other macrophages. Serum
phosphatase (TRACP) level correlates with histomorphometry.
OHPr Present in all collagen molecules, released during
both bone formation and bone resorption.
Galactosy1 hydroxylysine Present only in collagen; 5/7 times more abundant in
(GHYL)* type I collagen of bone than in type 1 collagen of
skin.
Pyridinium cross-links (Pyr. & Non-reducible cross-links that form among 2
DPyr)* hydroxylysine residues on the collagen telopeptides
and one lysine or hydroxylysine residue on the α
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

* Released only during bone resorption


Biomarkers of Bone Resorption “Cont'd”

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).
Evidence In Favour of A Genetic Contribution
to Osteoporosis

• Closer concordance of BMD in monozygotic as


opposed to dizygotic twins (Smith 1993).
• Reduced BMD in daughters of osteoporotic
women when compared with control subjects
(Fotino et al., 1977)
• Family history of osteoporosis predicts BMD
(Soroko et al., 1994).
Candidate Genes for Osteoporosis

• 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).


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).
Collagen Type I Genes (COL1) Al, COLI A2)

• 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).
Genetic Biomarkers of Bone Turnover

• 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.
• 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.
Choice of Reliable Biochemical Markers
for the Assessment of Bone Turnover
in Postmenopausal Osteoporosis
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.
Markers of Bone Formation
Serum biomarker Method Company Controls PMO mean Significant
Mean +SD +SD 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%


Markers of Bone Resorption

Biomarker Method Company Controls PMO mean Significant


Mean +SD +SD 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%


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

T-score = X-M / SD
Calculated Uncoupling Status = 4.19-9.25 =
-5.06
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, the
calculated 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.
CONCLUSION

• 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 losers
in 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.
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
Indications of Bone Markers Assays

• 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.
Activated T Cells Mediated Bone Loss
through the triangle
RANKL / RANK/OPG
• 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.


• 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.
• 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).


• 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.
• 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.
• Among the factors that increases RANKL mRNA
expression in osteoblastic cells are the
prolnflammatory cytokines IL-1, IL-6, IL-11 and
TNF-α (Yasuda et al, 1998 and Hofbauer et al,
1999). Inflammatory lesions of bone are
characterized by abundant osteoclast
proliferation.
• TNF-α is a potent osteoclastogenic agent and
appears to mediate orthopedic implant loosening
(Merkel et al, 1999). TNF-α stimulates osteoblastic
cells 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-α (Kong et al, 1999).
• Products of breast cancer cells (PTH rP, IL-6, IL-
11 and cyclooxygenase-2 generated prostanoids)
promote RANKL formation by acting on resident
osteoblasts and/or stromal cells (Thomas et al,
1999) The release of growth factors especially
TGFp can influence the growth of tumor cells and
their production of bone-resorbing cytokines (Vin
et al, 1999).
• 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.
• 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).
• 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), 1α ,
25 (OH)2 D3 (Norwood et al, 1998), estrogen receptor
antagonist (Hofbauer et al, 1999) and PTH (Lee &
Lorenzo, 1999).
• 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.


↑ PTH Glucocorticoids Vit D3 ↓

↓ E2 Hypothyroidism
↑ PRL

Decreased OPG

Abnormal RANKL/OPG Ratio

Decreased Osteoblastic Activity

Increased Bone Resorption


Infections / cancer PE

T-cell activation Increased osteoclastogenic Increased osteoclast


Cytokines IL-6, IL-11, TNF- Activating growth factors
α (TGF-β )

↑ PGE2 Increased RANKL PTH, PTHrP ↑

Abnormal RANKL/OPG ratio

Increased osteoclastic activity

Increased bone resorption


Further Readings

• 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.
• 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.
• 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.


• 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.


• 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.
• 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.


• 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.
• 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.