Metabolic Bone Disease & Bone Markers

Felicity StokesSenior Clinical Biochemist

Royal Liverpool & Broadgreen University Hospital Trust

Talk Outline• Introduction to bone• Metabolic bone disease

– Osteoporosis– Osteomalacia/Rickets– Paget’s 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%)• (Ca10(PO4)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

osteoclastsosteocytes

osteoblasts

Many growth factors, cytokines & hormones determine whether formation or resorption take place

RANK, RANKL and Osteoprotegerin (OPG)

RANK

RANKL

OPG

RANKL > OPGOPG > RANKL

RANKLReceptor activator of nuclear factor κB ligand

RANKReceptor activator of nuclear factor κB

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

AgeDietPhysical activityCalcium intakeSex hormonesDrugs – steroids

cyclosporinanticonvulsants

Metabolic Bone Disease

• Osteoporosis• Paget’s 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 OsteoporosisEndocrine disorders Drugs 1° and 2° hypogonadism AlcoholThyrotoxicosis GlucocorticoidsHyperparathyroidism HeparinCushing’s syndrome Aromatase inhibitorsHyperprolactinoma GnRH analogs

Malignant disease Other CausesMyelomatosis MalabsorptionLeukaemia GastrectomyLymphoma Chronic liver diseaseMastocytosis CKD

TransplantationConnective Tissue Disorders Rheumtoid arthritisOsteogenesis imperfecta Immobilisation Marfan’s syndrome Homocysteinura

Investigation of 2° causes

• FBC, ESR and SPE Multiple Myeloma• Calcium profile Ca2+/PO4

-/Vit D deficiency

• PTH – if high Ca2+ 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 factorsAge (> 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).

ManagementAim – to protect against fractures Prevent further loss of BMD

Increase BMD (if possible)

Dual actionStrontium ranelate

Anabolic therapy

Teriparatide (1-34 recombinant PTH)

Anti-resorptive therapyBisphosphonatesEstragens – HRTSERMs (Raloxifene)CalcitoninDenosomaub

1st line treatmentIncorporated into bone & ‘poison’ osteoclastsLong lasting effectsAdverse effectsHypocalcaemia (ensure Vitamin D replete)ONJAtypical fracture

Calcium and vitamin D

PLUS

Subcutaneous injectionsHuman monoclonal ab to RANKL (mimics OPG)

Self-administered daily injectionsStimulates increased bone turnover – including formationHigh cost – therefore only high-risk patients

Mechanism of actionBisphosphonates

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 Ca2+. Incorporated into bone in place of Ca2+.

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 ricketsPrimary Vitamin D deficiency•VDDR type IMutation in 1α hydroxylase – therefore can’t activate 25-OH Vit D 1,25-OH Vit D

•VDDR type IIMutation in Vitamin D Receptor (VDR) – end organ insensitivity to 1,25 – OH Vit D

Secondary Vitamin D deficiency

Malabsorption Decreased synthesisLiver disease ElderlyCoeliac disease Dark skin

Increased metabolism Decreased activationAnticonvulsants Renal failure

Hypophosphataemic rickets

Primary Causes

• X linked hypophosphataemic rickets• Autosomal Dominant Hypophosphataemic rickets• Oncogenic Osteomalacia

Clinical ManifestationsRickets• Bowing of legs• Short stature• Indentation of lower ribs• Flattening of skull

Osteomalacia• Bone pain• Stress fractures

BiochemistryRaised ALP

– Increased osteoblastic activity associated with producing unmineralised osteoid

Vitamin D dependantHypophosphataemic

Low Ca2+

Low/normal phosphate

Low phosphateHigh FGF-23Low 1,25-OH vitamin D

Management

Oral Ca2+ & vitamin D supplementsAlfacalcidol or calcitriol

Oral PhosphateAlfacalcidol/ calcitriol

Hypophosphataemic Vitamin D dependant

Paget’s 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

1α-hydroxylase to activate 25-OH vitamin D to 1,25-OH vitamin D

Activation of PTH

resorption of bone releasing Ca2+ and PO4

-Loss of BMD

Treatment with phosphate binders, Calcium and 1,25 OH vitamin D

Suppressed PTH adynamic bone disease

Ca2+ soft tissue calcification

Treatment with non-calcium containing phosphate-binders

‘Selevamer’ and calcimemetics ‘cinicalcet’

Bone disease of malignancyBone is a common site for metastasesSerious complication of cancer – severe pain, fractures, high/low Ca2+

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 Ca2+

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 Ca2+High serum Ca2+

Metabolic Bone Disease - SummaryBone 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 DiseaseOsteoporosis – Most common – low BMD. Increased fracture risk.

Rickets/Osteomalacia – Phosphate or Vitamin D deficiency. Genetic or secondary

Paget’s – Affects 1 or more bones. Increased resorption – chaotic formation

Renal Osteodystrophy – Low Ca2+ and High PO4- 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 – Ca2+ & 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

PyridinolinesOsteocalcin

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 Δ [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 -20°C

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 – Δ 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 procollagenPost-translational modification N-terminal (P1NP) and C-terminal (P1CP) domains are enzymatically cleaved off.

P1NPP1CPType 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 UtilityAnalysisImmunoassay • P1NP is initially released as an intact trimeric structure from the procollagen, but

rapidly degrades at 37°C 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 γ-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 UtilityAnalysis

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

β-CTX (β-isomerized C-terminal Telopeptides)

β-CTX analysis & Clinical UtilityAnalysis

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 μg/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

• RANKLRANK=receptor, RANKL=ligandRANKL = key factor for osteoclast differentiation and activation

• TRAP5bTartrate-resistant Acid Phosphatase isoformSpecifically secreted by osteoclasts

Manual, expensive kits, some are very unstable – mainly used for research only

Summary of Bone MarkersReleased 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?