Metabolic Bone Disorders & Biochemical markers of Bone Turnover

ByDr O.M Akinosun

INTRODUCTION

Metabolic bone disease (MBD) refers to the conditions that produce diffuse decrease in bone density and strength because of an imbalance between bone resorption and bone formation

BONE PHYSIOLOGY

• The skeletal system is a dynamic organ that provides the body with mechanical stability and structural support, in addition to protecting internal organs.

• It produces erythrocytes and other hematopoietic elements, serves as a reservoir of calcium and other life-supporting ions, and can bind toxins and heavy metals along its enormous mineral surface, thereby minimizing their ability to cause cellular damage.

TYPES OF BONE

• Trabecular and

• Cortical bones

• Trabecular or cancellous bone surrounds the marrow and appears as fine, lacelike strands that make up the inner framework of the bones and gives bone its compressive strength.

• Trabecular bone makes up the major portion of the vertebrae, the ends of long bones, andthe pelvis

• Cortical bone is located around the circumference of the bone shaft and is made of thick, densely packed layers of mineralized collagen.

• It is responsible for giving bone its rigidity.

• Bone is a metabolically active tissue that undergoes continuous remodelling by two counteracting processes, namely bone formation and bone resorption. • These processes rely on the activity of

osteoclasts (resorption), osteoblasts (formation) and osteocytes (maintenance)

• Under normal conditions, bone resorption and formation are tightly coupled to each other, so that the amount of bone removed is always equal to the amount of newly formed bone

• This balance is achieved and regulated through the action of various systemic hormones e.g.

• PTH, • vitamin D, • other steroid hormones) and • local mediators (e.g. cytokines, growth

factors)

• In contrast, somatic growth, ageing, metabolic bone diseases, states of increased or decreased mobility, therapeutic interventions and many other conditions are characterised by more or less pronounced imbalances in bone turnover

• Remodeling or bone turnover is the process by which bone is repaired and reinforced to compensate for the mechanical stress placed on the skeleton by repetitive activity.

• This continuous breakdown and renewal of bone consists of the balanced activity of skeletal destruction (or bone resorption) by osteoclasts and skeletal reconstruction (or bone formation) by osteoblasts.

• Biochemical monitoring of bone metabolism depends upon measurement of enzymes and proteins released during bone formation and of degradation products produced during bone resorption.

• Together with clinical and imaging techniques, biochemical tests play an important role in the assessment and differential diagnosis of metabolic bone disease.

Metabolic Bone Disorders

• osteoporosis (the most common MBD)• Paget disease, • rickets and osteomalacia,• primary and secondary

hyperparathyroidism

Osteoporosis

• Osteoporosis is defined by a low total bone mass and disruption of the normal architecture of bone, resulting in increased fragility and enhanced risk of fracture.Histologically,

• the cortices are thinned and porous. • The trabeculae are few in number, thinner,

and less connected.

It can result from a number of clinical conditions including • states of high bone turnover, • endocrine disorders (primary and secondary

hyperparathyroidism and thyrotoxicosis), • osteomalacia, • renal failure, • gastrointestinal diseases, • long-term corticosteroid therapy, • multiple myeloma, and cancer metastatic to the bones.

Osteomalacia /Rickets

• Osteomalacia is a disorder of mineralization of the newly formed organic matrix, leading to soft bone.

• In children, this abnormal calcification of the protein matrix is referred to as rickets.

Biochemical markers of Bone Turnover

• Bone formation markers

• Bone Resorption Markers

Bone Resorption

• Bone resorption consists of the dissolution of bone mineral and catabolism of the bone matrix constituents by osteoclasts, leading to formation of a resorptive cavity and release of bone matrix components

Bone Formation

• During bone formation, osteoblasts synthesise bone matrix, which fills up the resorption cavity and undergoes rapid primary mineralisation followed by the slow long-termsecondary mineralisation.

NOTE!!!

• Markers of bone formation and resorption are high in men aged 20 to 30 years which corresponds to the late phase of formation of peak bone mass.

• Thereafter they decrease reaching their lowest levels between 50 and 60 years

Bone formation markers

• Serum bone–specific alkaline phosphatase• Serum osteocalcin• Serum type 1 procollagen (C-terminal/N-

terminal): C1NP or P1NP• Serum total alkaline phosphatase

Osteocalcin (OC)

• OC is a vitamin K-dependent protein synthesised by osteoblasts odontoblasts and hypertrophic chondrocytes

• vitamin-K dependent, gamma carboxyglutamic acid (Gla) residuesof OC are responsible for the calcium binding properties of the protein.

• At its carboxy-terminus, OC can also interact with other proteins, including cell surface receptors.

• These functions predispose OC as a molecule active in the organisation of the extracellular matrix.

• OC is considered a specific marker of osteoblast function.

• It is estimated that, directly after its release from osteoblasts, the largest part of the newly synthesised protein is incorporated into the extracellular bone matrix where it constitutes approximately 15% of the non-collagenous protein fraction

• A smaller fraction is released into the circulation where it can be detected by immunoassays.

• Serum levels of immunoreactive OC have been shown to correlate well with the bone formation rate as assessed by histomorphometry

• OC is incorporated into the bone matrix, hence some investigators have suggested that OC fragments may be released even during bone resorption.

• This may be particularly true for some smaller N-terminal fragments of OC, which are found in individuals with high bone turnover.

• Quick processing of the blood sample after drawing is essential for most assays since a loss of reactivity is noted within a few hours at room temperature

Procollagen Type I Propeptides

• Type I collagen is the most abundant protein of bone matrix. Other sources include

• skin, • dentin, • cornea, • vessels, • fibrocartilage, and tendons

• N-terminal and C-terminal propeptides of type I procollagen (P1NP, P1CP) are cleaved during the extracellular metabolism of procollagen and released into the blood, whereas

• the central part of the molecule is

incorporated into the bone matrix.

• PICP has a MW of 115 kDa,

• is stabilised by disulphide bonds,

• cleared by liver endothelial cells via the mannose receptor and therefore

• has a short serum half-life of 6–8 minutes

• PINP has a MW of only 70 kDa,

• is rich in proline and hydroxyproline and

• is eliminated from the circulation by liver endothelial cells by the scavenger receptor.

• Since both PICP and PINP are generated from newly synthesised collagen in a stoichiometric fashion, the propeptides are considered quantitative measures of newly formed type I collagen.

• Although type I collagen propeptides may also arise from other sources, most of the non-skeletal tissues exhibit a slower turnover than bone, and contribute very little to the circulating propeptide pool.

Serum Total Alkaline Phosphatase (AP)

• Serum total AP is the most widely used marker of bone metabolism due to the wide availability of inexpensive and simple assay methods.

• Once liver disease is ruled out, serum levels of total AP provide a good impression of the extent of new bone formation and osteoblast activity

• AP is a ubiquitous, membrane-bound tetrameric enzyme attached to glycosyl-phosphatidylinositol moieties located on the outer cell surface.

• The precise function of the enzyme is yet unknown, but it obviously plays an important role in osteoid formation and mineralisation

• The total AP serum pool (TAP) consists of several dimeric isoforms, which originate from various tissues:

• liver, • bone, • intestine, • spleen, • kidney, • Placenta and• certain tumours may express macromolecular forms of AP

• The physiological isoforms of AP are coded by four gene loci, including three tissue-specific and one non-tissue-specific gene on chromosome 1.

• The non-tissue-specific gene on chromosome 1 encodes for the most abundant isoforms, namely bone, liver, and kidney AP

• Many techniques have been developed to differentiate between the two main isoforms of circulating AP, including

• heat denaturation, • electrophoresis, • precipitation, • selective inhibition and, • immunoassays

• In healthy adults, most methods show a good correlation between bone specific and total AP but these assays show a certain degree of cross-reactivity between bone and liver AP (15–20%) leading to false positive results

Bone-specific AP

• Bone alkaline phosphatase (BAP) is the bone-specific isoform of alkaline phosphatase

• A glycoprotein enzyme located on the outer surface of osteoblasts,

• most probably involved in the regulation ofthe mineralisation of osteid.

• BAP has been shown to be a sensitive and reliable indicator of bone metabolism

• From a clinical perspective, however, detection of the bone-specific AP (BAP) isoenzyme is increasingly preferred because of its higher specificity.

• Bone alkaline phosphatase (BAP) concentration is high in Paget disease and osteomalacia.

• Antiresorptive therapies lower BAP from baseline measurements in Paget disease, osteomalacia, and osteoporosis

• When used as a marker for monitoring purposes, it is important to determine the critical difference (or least significant change).

• The critical difference is defined as the difference between 2 determinations that may be considered to have clinical significance.

Bone Resorption Markers

• Urinary total pyridinoline (PYD)• Urinary free deoxypyridinoline (DPD)• Urinary collagen type 1 cross-linked N-telopeptide

(NTX)• Urinary or serum collagen type 1 cross-linked C-

telopeptide (CTX) • Urinary hydroxyproline• Bone sialoprotein (BSP)• Tartrate-resistant acid phosphatase 5b

3-Hydroxypyridinium Crosslinks of Collagen Pyridinoline (PYD) and Deoxypyridinoline

(DPD)• formed during the extracellular maturation of

fibrillar collagens• bridge several collagen peptides and

mechanically stabilise the collagen molecule • During bone resorption, crosslinked collagens

are proteolytically broken down and the crosslink components are released into the circulation and the urine

• The measurement of hydroxypyridinium crosslinks is not influenced by the degradation of newly synthesised collagens and their levels strictly reflect the degradation of mature i.e. crosslinked collagens.

• In addition, the urinary excretion of pyridinium crosslinks is independent of dietary sources since neither PYD nor DPD are taken up from food.

• Finally, the two hydroxypyridinium components show a high specificity for skeletal tissues

• While PYD is found in cartilage, bone, ligaments and vessels, DPD is almost exclusively found in bone and dentin.

• Neither derivative is present in the collagen of skin or elastin

• Since bone has a much higher turnover than cartilage, ligaments, vessels or tendons, the amounts of PYD and DPD in serum or urine are mainly derived from the skeleton.

• Thus, the pyridinium crosslinks are currently viewed the best indices for assessing bone resorption

Crosslinked Telopeptides of Type I Collagen

• derived from specific regions of the collagen type I molecule, namely the aminoterminal (NTP) and the carboxyterminal (CTP) telopeptide

• RIA for the carboxyterminal type I collagen telopeptide (ICTP) in serum appears to be sensitive for pathological bone resorption as seen in multiple myeloma, metastatic bone disease and other degradation processes involving hasty breakdown of skeletal and non-skeletal type I collagen

• Another group of immunoassays involve the carboxyterminal telopeptide of type I collagen, abbreviated CTX

• Employing a polyclonal antiserum against a synthetic octapeptide containing the crosslinking site, a first ELISA (termed β-CTX) recognised the C-terminal type I collagen telopeptide containing an isoaspartyl (=β-aspartyl) peptide bond in its L-enantiomeric form.

• The β–L-aspartyl is considered to result mainly from the ageing of extracellular proteins.

• Simultaneous measurement of both monoclonal-based RIA for the non-isomerised octapeptide (EKAH- αD-GGR) in urine (“α-CTX”) and β-CTX forms may be used to calculate the ratio of α-CTX/β-CTX as an index of bone turnover.

• This ratio has been shown to be elevated in the urine of patients with untreated Paget's disease of bone, where rapid bone formation and resorption result in an increase of α-CTx (non-isomerised octapeptide)

Bone Sialoprotein (BSP)

• BSP is a phosphorylated glycoprotein with an apparent MW of 70–80 kDa,

• accounts for 5–10% of the non-collagenous matrix of bone

• major synthetic product of active osteoblasts and odontoblasts.

• Found also in osteoclast-like and malignant cell lines.

• BSP or its mRNA is detected mainly in mineralised tissue such as bone, dentin and at the interface of calcifying cartilage

• serum BSP reflects processes mainly related to bone resorption

Tartrate-Resistant Acid Phosphatase (TRAP, TRAcP)

• family of the ubiquitous acid phosphatases• five different isoforms known• expressed by different tissues and cells such

as prostate, bone, spleen, platelets, erythrocytes, and macrophages

• inhibited by L(+)-tartrate, except band 5 termed tartrate-resistant acid phosphatase (TRAP or TRAcP).

• 2 subforms, 5a and 5b are known of TRAP or TRAcP

• 5a contains sialic acid, whereas 5b does not• TRAP-5b is characteristic of osteoclasts• Specific immunoassays for TRAP 5b have been

described and clinical results indicate that this marker may be useful to assess osteoclast activity

• Serum TRACP 5b is a reliable osteoclast-specific andsensitive marker of bone resorption

• ► TRACP 5b is proportional to the number of osteoclasts, may be used as a marker of osteoclast number (may be of interest in novel treatments inhibiting bone resorption without affecting OC number, i.e. CIC-7 inhibitors).

• Serum TRACP 5b activity has low technical and biological variability, does not accumulate in renal and hepatic failure, but has low storage stability above -70°C

• ► Serum TRACP 5b has a favorable signal-to-noise ratio, hence may be a useful marker in monitoring anti resorptive therapy

Cathepsin K

• member of the cysteine protease family • has the unique ability to cleave both helical

and telopeptide regions of collagen • clinical relevance was appreciated with the

discovery that pycnodysostosis, an autosomal recessive disease characterised by osteopetrosis, was the result of mutations in the cathepsin K gene

• Pycnodysostosis, autosomal recessive bone sclerosing disorder, is caused by a deficiency in cathepsin K activity characterised by decreased bone turnover and an accumulation of undigested collagen fibrils in OC (osteopetrosis and short-stature)

• cathepsin K is located intracellularly in vesicles, granules and vacuoles throughout the cytoplasm of osteoclasts

• it is secreted into bone resorption lacunae for extracellular collagen degradation hence

• useful and specific biochemical marker of osteoclastic activity.

Studies Measuring Serum Cathepsin K

• Cathepsin K decrease with age in women and men (Kershan-Schindl et al, Experimental Gerontology 2005)

• ► Cathepsin K correlates with BMD and fracture history (Holzer et al, J Lab Clin Med 2005)

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