Metabolic Bone Disease & Bone

Markers
Felicity Stokes
Senior Clinical Biochemist
Royal Liverpool & Broadgreen University Hospital Trust
Talk Outline
Introduction to bone
Metabolic bone disease
Osteoporosis
Osteomalacia/Rickets
Pagets disease
Renal Osteodystrophy
Bone disease of malignancy
Assessment of bone Bone Markers
Analysis
Clinical Utility

Bone: Structure and Function
Function

Structural support to body, mechanical
Protection of organs and bone marrow
Reservoir of calcium and phosphate for
homeostasis
Bone: Structure and Function
Microscopic Structure:
Extracellular organic matrix (~35%)
collagen (~90%) & proteins

Hydroxyapatite crystals (~60%)
(Ca
10
(PO
4
)
6
(OH)
2
)

Cells (<5%)
osteoblasts and osteoclasts, osteocytes, bone lining cells
Macroscopic Structure:
Cortical bone
(80% of skeleton) 80 90% mineralised
Mechanical/ protective
Trabecular bone (spongy bone)
(20% of skeleton) 15 25% mineralised
Metabolically active
Hydroxyapatite crystals become
incorporated into extracellular matrix
Bone goes through a period of resting.
Recruitment of osteoblasts, which produce structural and
enzymatic proteins to form new extracellular matrix
Multi-nucleated osteoclasts excavating
resorption pit. Release H
+
and enzymes
to degrade extracellular bone matrix.
Resting
Resorption
Formation
Mineralisation
osteoclasts
osteocytes
osteoblasts
Many growth factors, cytokines & hormones determine whether formation or resorption take
place
RANK, RANKL and Osteoprotegerin (OPG)
RANK
RANKL
OPG
RANKL > OPG
OPG > RANKL
RANKL
Receptor activator of nuclear
factor B ligand
RANK
Receptor activator of nuclear
factor B
Bone Mineral Density (BMD)
Bone mass (BMD) with age up to 20 30 years (peak bone mass)
After this bone mass gradually decreases

Adult BMD is determined by peak bone mass AND rate of loss

Genetic Environmental
Factors that affect BMD
Decreased sex hormones (eg.
During menopause) causes
increased loss
May determine
peak bone mass
Age
Diet
Physical activity
Calcium intake
Sex hormones
Drugs steroids
cyclosporin
anticonvulsants
Metabolic Bone Disease
Osteoporosis
Pagets disease
Osteomalacia
Renal Osteodystrophy
Metabolic bone disease of malignancy
Osteoporosis
Most prevalent metabolic bone disease in developed
countries
Ageing population reaching epidemic levels
Major cause of morbidity and mortality
Low bone mass
Increases the risk of fragility fractures
(esp. hip, wrist & spine)
Diagnosis
Dual-energy X-ray Absorptiometry (DEXA scan)
to measure spine or hip BMD

T score < -2.5 Osteoporosis
T score -1 to -2.5 Osteopaenia

T score = number of SDs below BMD of a
young adult
Clinical Manifestations
May be silent until fracture
Height loss
Pain
Fracture
Causes
Primary causes genetic
Secondary causes
Secondary causes of Osteoporosis
Endocrine disorders Drugs
1 and 2 hypogonadism Alcohol
Thyrotoxicosis Glucocorticoids
Hyperparathyroidism Heparin
Cushings syndrome Aromatase inhibitors
Hyperprolactinoma GnRH analogs

Malignant disease Other Causes
Myelomatosis Malabsorption
Leukaemia Gastrectomy
Lymphoma Chronic liver disease
Mastocytosis CKD
Transplantation
Connective Tissue Disorders Rheumtoid arthritis
Osteogenesis imperfecta Immobilisation
Marfans syndrome
Homocysteinura
Investigation of 2 causes
FBC, ESR and SPE Multiple Myeloma
Calcium profile Ca
2+
/PO
4
-
/Vit D deficiency
PTH if high Ca
2+
Hyperparathyroidism
TFTs Hyperthyroidism
LFTs Alcohol excess
Anti-TTG ab Coeliac disease

Risk Factors
Aim is to identify patients at high risk of
osteoporotic fracture

Assess BMD + risk factors

Age (> 65 years)
Previous fracture
Strong family history of fracture
Glucocorticoid therapy
Smoking
Alcohol abuse
Secondary Osteoporosis, eg. RA
The FRAX

algorithms give the 10-year probability of fracture. The output is a 10-

year probability of hip fracture and the 10-year probability of a major
osteoporotic fracture (clinical spine, forearm, hip or shoulder fracture).
Management
Aim to protect against fractures Prevent further loss of BMD
Increase BMD (if possible)
Dual action
Strontium ranelate
Anabolic therapy
Teriparatide (1-34 recombinant PTH)
Anti-resorptive therapy
Bisphosphonates
Estragens HRT
SERMs (Raloxifene)
Calcitonin
Denosomaub
1
st
line treatment
Incorporated into bone & poison osteoclasts
Long lasting effects
Adverse effects
Hypocalcaemia (ensure Vitamin D replete)
ONJ
Atypical fracture
Calcium and vitamin D
PLUS
Subcutaneous injections
Human monoclonal ab to RANKL (mimics OPG)
Self-administered daily injections
Stimulates increased bone turnover
including formation
High cost therefore only high-risk patients
Mechanism of action
Bisphosphonates
Structure similar to pyrophosphate
(contains 2 phosphates)
Incorporated into bone high affinity for
calcium.
Ingested by osteoclasts trying to resorb
the bone.
Stimulates osteoclast apoptosis.
Denusomaub
Strontium ranelate
Teriparatide
Increases bone formation AND decreases
resorption.
Similar structure to Ca
2+
. Incorporated into bone
in place of Ca
2+
.

Continued raised levels of PTH causes
increased resorption (as seen in
hyperparathyroidism)
But small intermittent doses given in daily
injections activates osteoblasts more than
osteoclasts, causing overall increased bone
formation
Stimulates the CaSR differentiation of pre-
osteoblasts to osteoblasts.
Stimulates osteoblasts to secrete OPG
inhibiting osteoclast formation.
Antibody to RANKL (mimics OPG)
Osteomalacia & Rickets
More common in developed countries
Defective mineralisation of bone matrix

Rickets children
Osteomalacia adults

Phosphate or vitamin D deficiency
1 or 2 causes
Vitamin D-dependant rickets
Primary Vitamin D deficiency
VDDR type I
Mutation in 1 hydroxylase therefore cant activate 25-OH Vit D 1,25-OH Vit D

VDDR type II
Mutation in Vitamin D Receptor (VDR) end organ insensitivity to 1,25 OH Vit D
Secondary Vitamin D deficiency
Malabsorption Decreased synthesis
Liver disease Elderly
Coeliac disease Dark skin

Increased metabolism Decreased activation
Anticonvulsants Renal failure
Hypophosphataemic rickets
Primary Causes

X linked hypophosphataemic rickets
Autosomal Dominant Hypophosphataemic
rickets
Oncogenic Osteomalacia

Clinical Manifestations
Rickets
Bowing of legs
Short stature
Indentation of lower ribs
Flattening of skull

Osteomalacia
Bone pain
Stress fractures
Biochemistry
Raised ALP
Increased osteoblastic activity associated with producing unmineralised
osteoid
Vitamin D dependant
Hypophosphataemic
Low Ca
2+
Low/normal phosphate
Low phosphate
High FGF-23
Low 1,25-OH vitamin D
Management
Oral Ca
2+
& vitamin D supplements
Alfacalcidol or calcitriol
Oral Phosphate
Alfacalcidol/ calcitriol
Hypophosphataemic Vitamin D dependant
Pagets Disease
Localised disorder of one or two bones
Increased osteclastic resorption formation increases
to try to match, but in a chaotic fashion
Produces woven bone weak, thickened

Cause unknown
?viral
?genetic component

Clinical Manifestations
Depend on the bone affected
Advanced disease can produce deformities, such as
skull enlargement, bowing of weight-bearing bones
Increased risk of fracture

Biochemistry
Increased markers of bone turnover
Abnormal bone scans
Increased ALP (up to 10 x ULN)
Management
Decrease osteoclastic bone resorption
Bisphosphonates
Calcitonin
Surgery to correct skeletal deformities
Renal Osteodystrophy
Mechanism
Bone disease associated with chronic kidney disease (CKD)
Calcium deficiency
Phosphate accumulation
due to decreased excretion
Activation of FGF-23
renal function
1-hydroxylase to
activate 25-OH vitamin D
to 1,25-OH vitamin D
Activation of PTH
resorption of bone
releasing Ca
2+
and PO
4
-
Loss of BMD
Treatment with phosphate binders,
Calcium and 1,25 OH vitamin D
Suppressed PTH
adynamic bone disease
Ca
2+

soft tissue calcification
Treatment with non-calcium
containing phosphate-binders
Selevamer and calcimemetics
cinicalcet
Bone disease of malignancy
Bone is a common site for metastases
Serious complication of cancer severe pain, fractures, high/low Ca
2+
Increased bone resorption by
osteoclasts
PTHrp
Humoral hypercalcaemia of malignancy
Osteolytic metastases
Osteoblastic metastases Multiple myeloma
Breast Ca, squamous cell lung Ca,
other solid tumours
Prostate Ca, some Breast Ca
Only metastases that
are purely osteolytic
Osteoblastic activity stimulated
Mechanisms unknown
Low serum Ca
2+
Tumour cells produce factors
stimulate proliferation & action of
osteoclasts
cytokines &
growth factors
Markers of bone resorption also
increased. Therefore patients
may benefit from
bisphosphonates
Myeloma cells stimulate
osteoclasts by producing:
IL-6 RANKL
+
Osteoblastic activity inhibited by:
Myeloma cells express Dickkopf
1 (DKK1) inhibits Wnt signalling
pathway
High serum Ca
2+
High serum Ca
2+
Metabolic Bone Disease - Summary
Bone Mass/BMD peaks at ~20-30 years and decreases after this
BMD in adulthood depends on peak bone mass AND rate of bone loss
Genetic
Environmental
Metabolic Bone Disease
Osteoporosis Most common low BMD. Increased fracture risk.
Rickets/Osteomalacia Phosphate or Vitamin D deficiency. Genetic or secondary
Pagets Affects 1 or more bones. Increased resorption chaotic formation
Renal Osteodystrophy Low Ca
2+
and High PO
4
-
PTH Increased resorption
Bone Disease of Malignancy Osteolytic, Osteoblastic, Multiple Myeloma
Diagnosis DEXA scan, bone markers, Calcium profile, Vitamin D
Monitoring Bone markers & DEXA scan
Management Ca
2+
& vitamin D, bisphosphonates, strontium ranelate, denosamaub
Bone Markers
They may be:
proteins that are secreted directly by osteoclasts or osteoblasts
during their activity
a by-product of bone turnover that is released either during the
formation of new bone or the breakdown of old bone

Usually classified according to the metabolic process their presence is
thought to indicate formation or resorption

Aid in diagnosis, monitoring efficacy of treatment and compliance
Biochemical substances released into blood during bone
remodelling that can be measured in blood or urine to give an
indication of the dynamic state of bone
Bone Markers
Bone specific ALP
Formation Resorption
P1NP
TRAP5b
CTX
Pyridinolines
Osteocalcin
OPG
RANKL
Advantages of Bone Markers:
Non-invasive
Early monitoring of therapy 18 months vs 3 months
Easy to repeat
Easy to check compliance of treatment
Comparatively inexpensive
Over BMD assessed by DEXA scan
Analytical
Storage & stability of samples
Lack of standardisation of assays
No reference methods available
Inter-assay variation many different assays available
High analytical imprecision

Clinical
High biological variation
Circadian rhythm
Diet
Age/ gender
Pregnancy/ lactation
Disadvantages of Bone Markers:
Difficult to distinguish if [bone marker] clinically significant or due to biological &
analytical variation
Research into optimal specimen type
and storage conditions
Eg. CTX collect into EDTA & separate
immediately & store at -20C

Automated assays
greatly increased precision
MANY manual assays
replaced with a few
automated assays
Research into affects of timing
& diet standardised
sampling with morning fasting
samples
Analytical & Biological variation minimised results more likely to be clinically
significant




Clinical Utility of Bone Markers
Monitoring treatment efficacy & compliance
Easier to use in monitoring treatment efficacy at earlier time
points than BMD changes
Significant reduction in markers of resorption on anti-resorptive
may be seen after only 1 month
Decrease in CTX by 50 70% after 3 months has been reported with
bisphosphonates

Increase in markers of formation on anabolic agents
May start to increase after only 1 month with teriparatide and up to
>200% increase
Assessment of fracture risk (adds information to FRAX and BMD)

PROCOLLAGEN TYPE 1 PROPEPTIDE (P1NP)
Type 1 collagen is a trimeric helical protein that comprises ~ 90% of the bone matrix.
Synthesized by osteoblasts as procollagen
Post-translational modification
N-terminal (P1NP) and C-terminal
(P1CP) domains are enzymatically
cleaved off.
P1NP
P1CP
Type I collagen assembles into fibrils and crosslinked
layers which are incorporated into the surface of the
bone matrix.
Pro-peptides released into circulation
P1NP Analysis & Clinical Utility
Analysis

Immunoassay
P1NP is initially released as an intact trimeric structure from the procollagen, but
rapidly degrades at 37C to a stable monomeric structure

Very stable (in serum or plasma). Not much difference between different sample
types

Clinical Utility
The automated assay for serum total P1NP is precise and sensitive
enough to detect changes that exceed the LSC in a majority of
postmenopausal women after 3 months of treatment with PTH 1-84 or
alendronate. Because of its convenience and high throughput, this bone
formation marker may be useful for the monitoring of patients with
osteoporosis. Garnero, P., Vergnaud, P., and Hoyle, N. (2008) Clinical Chemistry 54, 188-196

Osteocalcin
49 residue polypeptide
Most abundant non-collagenous protein in bone
Produced specifically by osteoblasts in a Vitamin D and Vitamin K dependent
manner

Vitamin K acts as a co-factor for the post-
translational -carboxylation of specific
glutamyl residues - 17, 21 and 24
Conformational change to a more stable secondary structure which has a higher
affinity for binding the calcium and hydroxyapatite crystals
Osteocalcin
Function not fully elucidated
?role in regulating osteoblastic function
?involved in the process of mineralisation

Majority of OC forms part of extracellular bone matrix
A smaller fraction is released into the circulation
Circulating forms of osteocalcin
Osteocalcin analysis & Clinical Utility
Analysis

Immunoassay
Large number of assays detecting different fragments - ?which are
clinically relevant

Large analytical variation between assays and between different labs
using same assay

Very unstable needs to be collected into EDTA, separated and
frozen prior to analysis

Clinical Utility
As a marker of formation, P1NP has much more favourable
characteristics, therefore mainly research use
Bone ALP
Bone ALP

Produced by osteoblasts during the formation phase of the bone cycle
Function thought to de-phosphorylate proteins to provide phosphate for
mineralisation

Therefore a marker of formation
Encoded by same gene, differ in post-translational glycosylation
Alkaline Phosphatase
Intestinal
Liver/Bone Placental
Liver ~50% of serum ALP Bone ~50% of serum ALP
Bone ALP analysis and Clinical Utility
Immunoassays for bone ALP may exhibit a degree of cross-reactivity with liver ALP (3 20%)

Therefore the concentration of bone ALP may be affected by liver diseases as well
as changes in bone turnover.

Activity Assay (U/L)
Monoclonal bone ALP ab coated onto 96 well plate. Add p-NPP and
measure abs at 405nm
Mass Assays (ug/L)
Sandwich IA with 2 monoclonal ab directed against Bone ALP
Advantages and Disadvantages of Bone ALP

Expensive
Manual assay longer TAT
Stable
Analysis
-CTX (-isomerized C-terminal Telopeptides)
-CTX analysis & Clinical Utility
Analysis

Immunoassay
Unstable needs to be collected into EDTA and separated immediately (but
more stable than osteocalcin)

Usually very low levels (reference range 0.1 0.5 g/L)

High biological variability circadian rhythm (increased overnight) also
affected by post prandial/fasting (decreased following food intake)
Clinical Utility
Has been shown in a number of studies to exhibit a relationship with BMD
Not used to diagnose OP at the moment, but used to monitor
Several studies have shown a relationship with fracture risk
Less commonly used bone markers
OPG (Osteoprotegerin)
Produced by osteoblasts. A decoy receptor for RANKL. OPG binding to RANKL inhibits
differentiation of osteoclast precursors to mature osteoclasts.
ELISA assay measures free OPG, OPG-RANKL complex, monomeric OPG and dimeric
OPG

RANKL
RANK=receptor, RANKL=ligand
RANKL = key factor for osteoclast differentiation and activation

TRAP5b
Tartrate-resistant Acid Phosphatase isoform
Specifically secreted by osteoclasts


Manual, expensive kits, some are very unstable mainly used for research only
Summary of Bone Markers
Released into circulation as part of bone formation or
resorption
Can be measured in serum/urine
CTX = best marker for resorption
P1NP = best marker for formation
Advantages & Disadvantages
Cost-effective, non-invasive, change quickly in response to Rx
High biological & analytical variation

Clinical utility
Aid in initial assessment of patients
Monitoring treatment
Thank you

Any Questions?