MSC Clinical Biochemistry University of Westminster 2013

Clinical Use of Bone Markers

February 2013

Dr. Alan Courtney

Senior Clinical Scientist
Deputy Director Bone SAS Service
&
Dr Paul Holloway
Consultant Chemical Pathologist and Honorary Senior Lecturer in Metabolic
Medicine
Director Bone SAS Service
St Marys Hospital, Imperial College Healthcare Trust

Objectives: you should be able to

1. Describe the bone remodelling cycle and
control of bone turnover.
2. Know the major biochemical bone markers
of formation and resorption, their
limitations and sources of variability.
3. Describe their clinical use in metabolic
bone disease.

Structure of bone
Skeleton is
80% cortical bone: compact, dense, lamellar
eg long bones
20% trabecular bone: spongy, metabolically very active
eg vertebrae
Both types comprise identical cells (osteoclasts, osteoblasts and
osteoctyes) and matrix
protein matrix: type 1 collagen, layered with osteocalcin
bone mineral: (calcium hydroxyapatite)

Bone Remodelling
Essential part of bone health
healthy bone

osteoporotic bone

Bone remodelling : cells

Regulation of bone remodelling

Remodelling is tightly controlled
(resorption=formation)
Involves coordination between osteoblast &
osteoclast via array of cell signalling molecules:
RANK
RANKL - endogenous activators of
osteoclastogenesis and osteoclast activity
Osteoprotegerin (OPG) -inhibitor of

OPG/RANK/RANKL & regulation

of bone remodelling

Osteoclasts
Pre-osteoclast activation via Ob by binding of
RANK to RANKL, M-CSF to M-CSF receptor
Regulated by OPG soluble decoy receptor
for RANKL
Release resorption bone markers : collagen
crosslinks (Dpd, NTx, CTx) and serum TRAP

Osteoblasts
Receptors for systemic hormones PTH,
1,25(OH)2D3, oestrogen, glucocorticoids, IGFs,
thyroid hormones
Express RANKL, OPG, M-CSF
Release formation bone markers: bone ALP,
osteocalcin and propeptides of type 1 collagen
P1CP, P1NP.

What causes changes in bone

turnover?

Growth
Aging
Drugs
Disease: metabolic causes (e.g.
hyperthyroidism, hypogonadism,
hyperparathyroidism) renal, metastatic
tumours/sarcomas, inherited, others

Metabolic Bone Disease

Osteoporosis
Pagets Disease

Osteoporosis
Definition
a systemic skeletal
disease characterised
by low bone mass and
microarchitectural
deterioration of bone
tissue, with
consequent increase in
bone fragility and
susceptibility to
fracture

Common sites of fracture

Spine

Neck of femur

Wrist

Diagnosis of Osteoporosis - Bone

Density
DEXA scans of:
Lumbar spine (L1-4 - mean density of L2-4)
Right and Left Neck of Femur (mean of both)

Check density values (g/cm2)

T-score

0
= young adult average
-1 to -2.5
= osteopenia
> -2.5
= osteoporosis
> -2.5 + fragility fractures = severe osteoporosis

 Osteoporosis:
- Resorption > Formation Bone Loss
- BMD (T-Score < 2.5), Fracture Risk
- >40yrs there is a generalised loss of
bone: bone formation is no longer as
efficient
- Exclude 2ry causes: renal, EtOH abuse,
myeloma, tumours/metastatic,
osteomalacia, hyper/hypothyroidism

Osteoporosis - Fractures and Age

Bone Mineral Density (DEXA)

LUMBAR and FEMORAL NECK OSTEOPOROSIS

Osteoporosis: treatment

Patients Must be calcium & vit D replete

Antiresorption osteoclast activity

Bisphosphonates: Alendronate, Risedronate, Ibandronate, Zoledronate
SERMs: Raloxifene
RANK Ligand inhihitors: Denosumab

Anabolic
eg Teriparatide (rhPTH 1-34) daily injection for 18 months
Intermittent PTH : major osteoblast activity

Combined Antiresorptive & Anabolic

Strontium ranelate: mildly osteoblast activity plus antiresorptive effects

Action of Vitamin D on Bone

In rats:
1. Given 1% Ca2+, VitD(+): N Bone Mineralisation
2. Given 0.1% Ca2+, VitD(+): N Bone Mineralisation,
altered bone turnover (risk osteoporosis)
3. Given 1% Ca2+, VitD(-): N Bone Mineralisation,
altered bone turnover (risk osteoporosis)
4. Given 0.1% Ca2+, VitD(-): Abnormal Bone
Mineralisation; Osteomalacia/Rickets

Vitamin D can signal via RANK/RANKL,

osteoclasts, osteoblasts and osteocytes.

Action of Vitamin D on Bone

Osteoclasts/Osteoblasts/Osteocytes all
locally (paracrine/autocrine) cause
1hydroxylation of 25-OH VitD
This stimulates both formation/resorption
and can enhance mineralisation by
modulation of bone cell function
Increased circulating 1,25-OH VitD does
not does not protect against bone loss

Action of Vitamin D on Bone

When serum Ca2+ is inadequate bone
turnover, 1,25-OH VitD/PTH levels are
increased: 25-OH VitD acts to an increase
in circulating Ca2+
When serum Ca2+ is adequate bone
turnover, 1,25-OH VitD/PTH levels are
normal: 25-OH VitD modulates
autocrine/paracrine bone cell signalling
enhanced mineral retention, preservation of
bone strength, fracture risk

Many Factors Stimulate Osteoblast

Expression of RANK Ligand
Glucocorticoids
Vitamin D

PTH

CFU-M

PGE2

RANKL

Pre-Fusion Osteoclast

RANK

IL-11
Multinucleated
Osteoclast

IL-6
IL-1
PTHrP
TNF-

Activated
Osteoclast

Osteoblast
CFU-M = colony forming unit macrophage
Adapted from Boyle WJ, et al. Nature. 2003;423:337-342.
Hofbauer LC, Schoppet Ml. JAMA. 2004;292:490-495.

RANK Ligand is an Essential Mediator of

Osteoclast Formation, Function, and
Survival
RANKL

Pre-Fusion
Osteoclast

CFU-M

RANK
Multinucleated
Osteoclast

Hormones
Growth factors
Cytokines

Osteoblasts

Bone Formation
In the presence of M-CSF
CFU-M=colony forming unit macrophage
M-CSF=macrophage colony stimulating factor
Adapted from Boyle WJ, et al. Nature. 2003;423:337-342.

Bone Resorption

Activated
Osteoclast

Osteoprotegerin (OPG) is a Decoy Receptor that

Prevents RANK Ligand Binding to RANK and Inhibits
Osteoclast Formation, Function, and Survival
RANKL

Pre-Fusion
Osteoclast

CFU-M

RANK
OPG

Hormones
Growth factors
Cytokines

Osteoclast Formation, Function,

and Survival Inhibited
Osteoblasts

Bone Formation
In the presence of M-CSF
CFU-M=colony forming unit macrophage
M-CSF=macrophage colony stimulating factor
Adapted from Boyle WJ, et al. Nature. 2003;423:337-342.

Bone Resorption
Inhibited

When RANK Ligand Overwhelms OPG, Bone

Resorption Can Become Excessive Leading to
Osteoporosis
RANKL

Pre-Fusion
Osteoclast

CFU-M

RANK
Multinucleated
Osteoclast

OPG

Hormones
Growth factors
Cytokines
Activated
Osteoclast
Osteoblasts

Bone Formation
In the presence of M-CSF
CFU-M=colony forming unit macrophage
M-CSF=macrophage colony stimulating factor
Adapted from Boyle WJ, et al. Nature. 2003;423:337-342.

Bone Resorption

Osteoporosis: Treatment
OPG-Fc: OPG-like antibodies/fragments that act to
bind RANKL inhibit osteoclast
activation/maturation
Preliminary study: in 52 PM women up to 3 mg/kg
OPG (IV) were given resulting in a NTx by 80%
within 5 days (remained suppressed for ~ 1 month)
OPG also has a potential therapeutic role in
rheumatoid arthritis in rats with adjuvant arthritis
it was shown to be effective in blocking bone and
cartilage destruction

Pagets
Thought to be relatively common in western
countries (>50yrs, prevalence 2 8%; few
develop symptoms)
Causes: genetic, viral, environment
Overactive osteoclasts
Resorption & Formation
New bone is disorganised: fracture risk &
bone deformation
Complications: arthritis, CVD, neurological,
stones, sarcoma (rare)

Mixed osteolytic and osteoblastic

phases of Pagets disease

Pagets: bowing of the leg

Biochemistry of Bone Disorders

Alk Phos Ca

PO4

PTH

1o PTH
Osteomalacia
Osteoporosis N

Pagets

Bony Mets

What Are Bone Markers?

Osteoclast activity results in the release of bone
breakdown products: specifically collagen cross linking
molecules (NTx, CTx and Dpd)
Osteoblast activity results in the production of biproducts of bone formation: BALP, P1NP, Osteocalcin
The measurement of these analytes reflect the overall
bone turnover of the skeleton

Use of bone resorption

markers

Utility of Bone Markers

Provide information over the entire skeleton
Are non-invasive, repeatable test, that is
sensitive to (early) changes in turnover and are
relatively cheap
Bone markers are NOT used for the diagnosis of
osteoporosis. However they can prove useful to
detect patients that are losing bone ( fracture
risk)
Main use: monitoring of therapy and response to
treatment assess compliance

The Ideal Bone Marker

Bone specific
Biological variability
Sensitive to changes in turnover
Independent of GFR/renal & liver function
Validated by clinical trials
Standardised methodology & automation
THERE IS NO SUCH THING AS AN IDEAL BONE MARKER!

Least significant change of

serial measurements
LSC= 2.77 ( CVa2 + CVi2)
-where

CV a = analytical, CV i= intra-individual variation

LSC for serum BALP: 30%

LSC for serum P1NP: 25%
LSC for urine NTx: 40%

Urine NTx response to risedronate in PM

osteoporosis Eastell R & Delmas PD 2005
ULN

Baseline

3-6 months
risedronate

Can we detect which patients

are likely to lose bone?
Baseline bone marker can predict subsequent
rate of bone loss in osteoporosis
If bone density is borderline but bone turnover
high, early treatment to prevent further bone loss
is appropriate

Predict fracture risk ?

Bone resorption (highest quartile) predicts 2fold RR of vertebral fractures in PM women
(OFELY study) and hip fractures in elderly
women (EPIDOS and Rotterdam studies)
This increased risk may be independent of BMD
(OFELY study Garnero et al JBMR 2000;15:1526-36)
May become a major use of bone markers (with
or without BMD) in future

Hip fracture prediction

The EPIDOS study Garnero et al J Bone Miner Res 1996

New data
A recent study evaluated whether baseline bone
turnover markers are associated with long-term
incidence of fracture in a population-based sample
of 1040 Swedish women who were 75 years old.
Results:
levels of serum TRAP5b, serum CTX and urinary
osteocalcin are associated with increased fracture risk
for up to a decade in elderly women.

Conclusion:
The predictive value of these biomarkers for fracture in
elderly women makes these markers useful tools for
public health policies.

Is the patient responding to treatment?

Major use of bone markers
Use in osteoporosis, Pagets, Osteogenesis Imperfecta
Early response (3-6 months) : BMD change takes 2 years

Good response: marker falls by more than LSC and into

the bottom half of premenopausal ref range
Identifies poor response to therapy
Encourages patient compliance with long-term therapy

Potential Utility of Bone Markers

BMD vs. BMs: bone markers can indicate the
quality of bone present (micro fractures) which
BMD does not necessarily provide
Fracture Prediction: levels above
premenopausal ref. range predict fractures (may
eventually be included in the FRAX score)
Bone Metastatic Tumours: identify growing bone
metastases/response to therapy
Prediction Risk of ONj: ?detection of adynamic
bone

Bone Markers at St-Marys

SAS Service: ~35-40% external requests
What do we measure?
Resorption Markers:
N-telopeptide (NTx)

Formation Markers:
Bone Specific Alkaline Phosphatase (BALP) and
Total Procollagen Type-1 N-Terminal Propeptide (P1NP)

Patients:
Osteoporosis, Pagets, Secondary Metabolic Bone
Disease, Childhood Diseases (Osteogenesis
Imperfecta), Metastatic Cancers/Sarcomas.

Urine N-Telopeptide (NTX)

Bone resorption is initiated by osteoclasts via an acid
phosphatase & other hydrolytic enzymes
Resorption releases: bone minerals & collagen
fragments
Type-I collagen: cleavage sites for cathepsin K lead
to the production of peptide fragments NTx
NTx: some degraded further (liver & kidney), most is
incompletely digested and excreted in the urine

Action of Cathepsin K on Type-I

Collagen
NTx

ICTP

CTx

Type-I Collagen

Cat K

Cat K

Cat K

Serum CTx (not routinely measured

at SMH)
Crosslaps: antibodies recognise EKAHD-GGR octapeptide with -isomerized Asp
Serum CTx : automated ( Roche Elecsys)
Variability: requires fasting sample
Unstable so needs early processing (collection
must be in EDTA tubes)
Good response to antiresorptive therapy
Predictive for fracture risk

Diurnal Variation in Serum CTx

Mean serum CTx in 11 pre-menopausal women Christgau et al 1998

Serum CTx in PMO: individual response to alendronate therapy

Kyd & Fairney Ann Clin Biochem1999

mean

mean

Choose a marker sensitive to changes in bone turnover:

women with osteoporosis treated with Alendronate 10mg or
placebo

months

Greenspan et al 1998

Urine N-Telopeptide
Bisphosphonates: treatment of choice in
postmenopausal osteoporosis, anti-catabolic
action that suppresses osteoclast activity
Decrease in markers of bone resorption in
response to treatment
Typically:50 70% during 1st 12 weeks of
treatment (significant urNTx within 8 weeks
treatment with alendronate)
Require baseline, 3, 6 & 12 monthly levels

NTx Requirements &

Measurement

Sample Type: Urine (in plain plastic)

Sample Volume: 1ml
Transport: First Class Post
Price: 20
TATs: 3 weeks (although assay runs are
dependent on the number of samples)
Stability: up to 7 days at 2 - 8C, longer term
-20C (up to 5 freeze thaw cycles), protect
from light, collection without preservative is
preferable

 Specific Requirements: Samples should be drawn

at the same time of day. Ideal sample is a 2 nd void
urine collected between 8 11am.
Sources of Variation: significant circadian rhythm
( excretion at night), day to day variation in
excretion (>24%), menstrual cycle ( luteal phase),
growth ( in children), aging ( turnover >40yrs)
Disease & Drugs: turnover in diseases other than
osteoporosis (e.g. thyrotoxicosis), drugs
(corticosteroids), recent fractures
Assay Performance: CV for assay quoted at 7%
across the reference range
EQA: Member of UK NEQAS

Interpretation of NTx Results

Ref Range:
- Premenopausal: 5 65 nM/mmol creat.
- Postmenopausal: 5 131 nM/mmol creat.
- Males: up to 51 nM/mmol creat.
- Children: up to 5xULN (325 nM/mmol
creat.)
Monitoring therapy: LSC is 40%
Monitoring therapy & bone marker targets:

Significant uNTx
Return bone markers to lower half of the reference
range (premenopausal range)

Serum Bone Specific Alkaline

Phosphatase
ALP: liver, bone, placenta, intestine
Routine: LFT profile
Specialist: isoenzymes (electrophoresis, ELISA)
BALP: 35 40% of total circulating ALP,
hydrolyses phosphate from its esters during bone
formation (allows phosphate to bind to
hydroxyapaptite)
BALP Assays: ELISA & automated immunoassay

Serum Bone Specific Alkaline

Phosphatase
BALP: quantitative marker of bone turnover,
formation marker, provides information on
bone remodelling in osteoporosis & Pagets
disease, monitoring changes due to therapy
Marker of choice for monitoring Pagets
Disease
Useful marker for CRF patients (especially
those on haemodialysis

BALP Requirements &

Measurement

Sample Type: Serum (in plain plastic)

Sample Volume: 1ml (min >0.100ul)
Transport: First Class Post
Price: 24
TATs: 3 weeks (although assay runs are
dependent on the number of samples)
Stability: stable at 2 8C for >1 week, -20C
indefinitely
Useful information: serum Ca2+, total ALP, LFTs
(ALT + bilirubin)
EQA: UK NEQAS

Interpretation of BALP Results

Ref Range:
- Premenopausal: 11.6 30.6 U/L
- Postmenopausal: 14.8 43.4 U/L
- Males: up to 15.0 43.1 U/L
- Children: up to 5xULN (~200 U/L)
Monitoring therapy: LSC is 30%
Beware liver disease: cross reactivity of ~10%

Serum Total Procollagen Type-1 NTerminal Peptide (P1NP)

Type 1 Collagen: accounts for >90% of the organic
matrix, derived from type 1 procollagen, synthesised by
osteoblast/fibroblasts
Cleavage of type 1 procollagens amino and carboxy
terminal regions by specific proteases during its
conversion to collagen
P1NP is released into the blood stream during type 1
collagen formation
P1NP reflects the deposition of collagen and can hence
bone formation

Serum Total Procollagen Type-1 N-Terminal

Peptide (P1NP)

P1NP Requirements &

Measurement
Sample Type: Serum/Plasma (Lithium
Heparin/EDTA/SST)
Sample Volume: 1ml (250l min for
assay)
Transport: P1NP samples should sent by
first class post
Price: 20
TATs: 3 - 4 weeks

Why measure P1NP?

Normal
Bone

Osteoporotic
Bone

Interpretation of P1NP Results

Formation marker of choice for measuring
response to treatment by teriparatide (best
predictor of lumbar BMD at 18 months)
Least significant increase 25%
However: reasonable response is an increase of
at least 40%; a very good response an increase
of 100 200%
Considerations: Preanalytical variability of
formation markers is lower, bone formation
markers due to recent fractures (12 months)

Bone Markers & Response to Teriparatide

(Rao, Kyd, Fairney & Holloway ECTS 2008)

2. Osteoporosis

1. In Health
Resorption

Resorption

Formation

Formation

Time

Time

3. BP. Therapy
Normal

4. hPTH Therapy
Resorption
Resorption

Formation

Formation
Normal
Time

Time

Other existing bone markers

Other Bone Markers

Routine Biochemistry Tests: total ALP,
Ca2+, Pi, PTH, VitD, Creatinine
Specialist Tests: osteocalcin, serum NTx,
FGF-23, 1,25 VitD, OPG, RANKL,
TRAP5b, cathespin K
Biochemical Markers of Cartilage: type-II
collagen (PIICP, PIINP, CTx-II, Helix-II),
aggrecan

Osteocalcin (Bone Gla Protein)

Tissue specific: bone, dentine, calcified cartilage

Clearance:
renal ( increased in renal failure)
Sample: very labile, keep at -20oC
Assays: wide variation in results from different
kits: circulating intact protein and major fragments.

NOT WIDELY USED

BALP shows a significantly better and quicker
response to treatment

Metabolism of Osteocalcin

Serum TRAP 5b
5b isoform of tartrate-resistant acid
phosphatase is specific for osteoclast
Unaffected by renal or liver failure
Specific ELISA method available (Quidel)
Storage requires -70 freezer: research
only

Bone markers have poor sensitivity

for metastases - TRAP 5b in Ca prostate
R Maddison et al 2002

New Markers of Bone Turnover

RANKL and OPG - RANKL: activates
osteoclasts - OPG: mops-up RANKL
inhibits
osteoclasts
Assays: tend to be commercially
available ELISAs (? Reliable)
Assay Issues: assay sensitivity, sample
type, stability, ?exactly what assay is
measuring

New Markers of Bone Turnover (2)

Biological Variation:
OPG: diurnal, age ( from 50 60yrs) - RANKL:
binds rapidly to OPG
Weak correlation of OPG to BMD; no correlation
of RANKL with BMD
Paget's: OPG & RANKL
Response to treatment for Pagets: OPG,
RANKL (both respond to osteoporosis
treatment, modest response of OPG to hPTH)

New Markers of Bone Turnover (3)

FGF23: fibroblast growth factor 23
251a.a. protein, unique C-terminal region,
expressed in many tissues, largest molecule
in FGF family
Increases phosphate excretion in the
kidneys: inhibits tubular reabsorption
Acts on the kidney to inhibit production of
1,25 VitD (?also acts on PTHr)
FGF family is also involved in cell
growth/signalling and morphogenesis

New Markers of Bone Turnover (4)

Klotho: -Klotho, single-pass transmembrane
protein, expressed by kidney tubular cells,
parathyroid gland and choroid plexus
Circulating levels: via secretion or extracellular
cleavage
Acts directly as a paracrine/endocrine factor:
interacting with FGF23
Paracrine: regulation of surface Ca2+ channel
TRPV5 and K+ channel ROMK abundance,
inhibits NaPi cotransporters in the absence of
FGF23, can induce Pi excretion in the urine

New Markers of Bone Turnover (5)

Endocrine: anti-oxidative properties
(inhibits insulin and IGF1, enhances
epithelial NO2 production), inhibits vascular
calcification and decreases abundance of
Ca2+ channel TRPC6
Reliable measurement of circulating
Klotho has become available only
recently: ELISA
R&D Only

Klotho FGF23 Dependent Actions

Kidneys

Parathyroid Gland

Calcitriol Synthesis

PTH

Phosphate Resorption

Klotho FGF23 Independent Actions

Kidneys
TRPV5
ROMK
NaPi-2a

Intestine
NaPi-2b

Vascular System
TRPC6
Vascular Calcification

Genetics of Bone Disease

Why study DNA variation?
- Disease pathogenesis
- Treatment/drug developments
- Diagnostic developments
Enviromental factors: diet, exercise, sun exposure
etc (vary a lot between populations)
Several study designs to find risk alleles
- Rare, large effects, monogenic (linkage)
studies
- Rare/more common, subtle effects, high
throughput studies

Population Size

Genetic Determinates of
Osteoporosis
Mutations
with serious
effects
(rare:
LRP5,
COLIA1)

Mutations
with serious
effects
(rare:
LRP5,
SOST,
CICN7)

SNPs that have modest

BMD effects can
explain osteoporosis
Low BMD

BMD

High BMD

Genetic Determinates of
Osteoporosis
Studies: microarray
Issues: small sample sizes,
standardisation, reproducibility, cost
GENOMOS March 2008: ESR1, COLI,
VDR, TGFb, LRP5, LRP6
Phenome Scans: pleiotrophic effects of
a risk gene