Biochemical Markers of Bone Turnover

Dr Jemil Makadia PG 3rd year Biochemistry dept. LHMC Click to edit Master subtitle style

19th August, 2011

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Outline

Structure and function of bone Bone turnover What is metabolic bone diseases? Structure of Collagen-I Bone turnover markers Uses
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Structure and function of bone

Structure Cortical (80%) fun: mechanical protective Trabecular (20%) metabolic

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Composition Organic matrix Type-I collagen (90%) Non collagenous protein (osteocalcin) Cellular
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Minerals Calcium, Phosphate Carbonate, Magnesium Na+, K+

osteoblast

Bone turnover
Osteoclast precursors Proliferate and fuse to Form Osteoclast Hydrogen Ion Remove mineralisation 4/19/12 Lysosomal enzyme digest metrix matrix bone lining cell Osteoblast

RANK ligand and Osteoprotegerin

Osteoblast RANK ligand Osteoprotegerin(OPG) (Receptor activator of Nuclear factor kB) for bind receptor on
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Decoy receptor RANK ligand

Metabolic bone diseases

Result of a partial uncoupling or imbalance between resorption and bone formation

Osteoporosis Osteomalacia / Rickets Renal osteodystrophy Pagets disease

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Structure of collagen-I
Triple helix three polypeptide chains (1000 AA) -- Two 1 and One 2 chains -- amino terminal and carboxy terminal not triple helical (telopeptides) -- covalent cross links intra strand and inter strand by Pyridinoline (PYD) and Deoxypyridinoline (DPD)

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NTELOPEPTID E REGION

HELICAL REGION

CTELOPEPTID E REGION

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NT x P yr Dp d CT x

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Bone turnover markers

Markers of Bone Resorption - Telopeptides - N-telopeptide (NTx) - C-telopeptide (CTx) - Pyridinium cross-links - Free deoxypyridinoline (DPD) - Free pyridinoline (PYD) - Total DPD and PYD

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- Tartrate-resistant acid phosphatase (TRAP)

Markers of Bone Formation - Bone ALP - Osteocalcin (Bone Gla protein, BGP)

- Procollagen type I propeptide (collagen peptides ) - N-terminal (PINP) - C-terminal (PICP)

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Telopeptides

NTx and CTx Serum and Urine ELISA (immunoassay) It does not recognize precursor that is not cross linked DPD cross link with 2 chain (not 1- also present in skin) 2/3 DPD binds to NTx and 1/3 DPD binds to CTx So NTx is more specific

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Pyridinium

PYD and DPD DPD more sensitive and specific because present only in Bone, Dentine, ligament and Aorta whereas PYD is widespread DPD- form during collagen maturation - not metabolize before excreted in urine - mainly from bone - not from diet

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Only in Urine Free and Total Free ELISA Total- HPLC Peak at 5am- 8am Second morning void sample (10 am)

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Clinical significance (NTx, CTx, PYD and DPD)

Increase in - osteoporosis - Pagets ds - metastatic bone disease - hyperparathyroidism - hyperthyroidism - postmenopausal women - infancy and adolescence

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Tartrate-resistant acid phosphatase (TRAP)

Two isoform 5a and 5b Osteoclast produce 5b isoform Enzyme is instable and associated with 2 macroglobulin complicated development of methods for detection Monoclonal antibody against TRAP 5b Kinetic method with fluoride inhibition or heparin inhibition
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Hydroxyproline

Mainly found in collagen 10% of hydroxyproline released during collagen catabolism is excreted in urine Assays involved oxidation of hydroxyproline to pyrrole Not specific because

Other tissues like muscle and skin contain it Collagen is degraded during synthesis and maturation

Incresed in pagets ds. Hyperparathyroidsm, 4/19/12 acromegaly, psoriasis, burns and

Bone Alkaline phosphatase

ALP is found in many tissues like bone, liver, intestine, kidney and placenta ALP from liver bone and kidney are isoforms of the same gene product It increases in

Osteoporosis Osteomalacia and rickets Hyperparathyroidsm Renal osteodystrophy Thyrotoxicosis

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Measurement of bone ALP has several advantages

Long half life Unaffected by diurnal variation More stable in vitro Useful in individuals with impaired renal function

Immunoassay is more sensitive and specific for bone ALP

Two monoclonal antibodies Immunoadsorption assay using single monoclonal antibody

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Ref value : men- 15-41 u/l

Osteocalcin

Non collagenous protein 1% of total protein in human bone During bone formation 10-30% of OC synthesized from osteoblast is released in circulation Synthesis stimulated by vit-D Excreted by kidney Half life is 5 min.

Immunoassay targeting N-terminal/ midregion 4/19/12 fragment is used. specimen should be

Clinical significance Increase in

Osteoporosis Osteomalacia and rickets Hyperparathyroidsm Renal osteodystrophy Thyrotoxicosis Acromegaly Hypoparathyroidism

Decrease in

Hypothyroidism 4/19/12

Procollagen peptides

N and C terminal of procollagen type-I Also from several other tissue so non specific Helpful in pt on vit-D as ALP and OC are not reliable in that condition

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Preanalytical and Analytical variables

With in individual variability is 15-60% in urine sample and 5-10% in serum sample Diurnal variation (except ALP)

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Uses

Monitoring the therapy (mc and only use) Selection of patient for therapy Prediction of bone loss Prediction of fracture risk

After bone resorption therapy significant reduction in bone resorption markers with in few weeks and reach to plateau with in 3-6 months. And bone formation markers take 6-12 months
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Limitation

Can not detect site of disease Can not diagnose the disease Methods are not specific cost

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Thank you
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