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No.29
Biochemical Bone
Marker ELISA kits
Sclerostin
Sclerostin
Bone Sialoprotein
Cathepsin K
DKK-1
Osteoprotegerin (OPG)
sRANKL
Tartrate-resistant acid phosphatase
or acid phosphatase 5, tartrate
resistant (ACP5 or TRAP)
Vitamin D
N-telopeptide (NTx)
C-telopeptide (ICTP and CTx)
Propeptide of type I procollagen N
terminal (PINP)
Deoxypyridinoline (Dpd)
Canonical Wnt-signalling plays an
important role in the regulaton of
bone homeostasis by promoting the
development of osteoblasts. Negative
regulators of the Wnt pathway are
potential therapeutic targets regarding
diseases with enhanced bone resorption
like osteoporosis or myeloma bone
disease. One of these molecules is
Sclerostin, which is of special interest,
because of its high bone specificity.
Sclerostin, a 190 residue, secreted
glycoprotein of the cystine-knot family
acts by binding to the Wnt-coreceptor
LRP5 thus preventing the binding of
Wnt molecules. Because of its high bone
specificity, antibodies against Sclerostin
are found to be very promising
therapeutic agents in disease with
elevated bone resorption.
Osteocalcin
Bone Sialoprotein
Bone Specific Alkaline Phosphates
Bone Sialoprotein (BSP) is a
glycoprotein, which accounts for
approximately 10% of the noncollagenous matrix protein of bones.
BSP (as well as other glycoproteins such
as osteonectin and osteopontin) plays
a regulatory role in bone metabolism,
e.g. the control of mineralisation in
the formation of new bones and bone
resorption. BSP contains an Arg-GlyAsp (RGD)-sequence, which is essential
for the attachment of cells to bone
surfaces. BSP has important functions
in the initiation of hydroxyapatite-
Parathyroid hormone (PTH)
Calcitonin
Glycosaminoglycans (GAGs)
Cartilage Oligomeric Matrix Protein
(COMP)
Fibroblast growth factor 23 (FGF-23)
crystallisation and in the interaction
between bone cells and the mineralised
bone matrix. BSP can be detected in
mineralising connective tissue, where
the osteoblasts are primarily formed.
It has however also been detected in
trophoblasts and to a lesser extent in
decidua cells. Tumors, which result in
calcification and formation of metastases
in the bones, can also express BSP. It
has been shown that BSP expression in
human breast cancer is associated with
skeletal metastases.
BSP expression has also been detected
in small cell bronchial carcinomas.
Cathepsin K
Cathepsin K is a highly expressed
cysteine protease in osteoclasts that
plays an essential role in the degradation
of protein components of bone matrix.
This protease cleaves both helicoidal
and telopeptide regions of collagen type
1, the major type of collagen in bone
remodelling and resorption. It seems to
be a valuable marker for various bone
diseases, such as Rheumatoid Arthritis,
Postmenopausal Osteoporosis and
Paget’s Disease.
Continued
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Biochemical Bone Marker ELISA kits
DKK-1
Dickkopf-1 (DKK-1) is a 28,672 Da
secreted protein that acts as soluble
inhibitor of the Wnt-signalling pathway.
This pathway contains lipid-modified
glycoproteins that activate cell surface
receptor-mediated signal transduction
to regulate cell activities like: cell fate,
proliferation, migration, polarity and
gene expression. DKK-1 regulates
developmental processes of all kinds.
Thus, DKK-1 is also involved in the
regulation of bone metabolism as it
effects osteoblast differentiation and in
regulation of tumourgenetic activity.
Osteoprotegerin (OPG)
Osteoprotegerin (OPG), also known
as osteoclastogenesis inhibitory factor
(OCIF), is a cytokine, which can
inhibit the production of osteoclasts.
It is a member of the tumour necrosis
factor (TNF) receptor superfamily.
It is a basic glycoprotein comprising
401 amino acid residues arranged
into 7 structural domains. It is found
as either a 60 kDa monomer or
120 kDa dimer linked by disulfide
bonds. Osteoprotegerin inhibits the
differentiation of osteoclast precursors
(osteoclasts are related to monocyte/
macrophage cells and are derived from
granulocyte/macrophage-forming colony
units [CFU-GM]) into osteoclasts
and also regulates the resorption
of osteoclasts in vitro and in vivo.
Osteoprotegerin is a RANK homolog,
and works by binding to RANKL on
osteoblast/stromal cells, thus blocking
the RANKL-RANK ligand interaction
between osteoblast/stromal cells and
osteoclast precursors. This has the
effect of inhibiting the differentiation
of the osteoclast precursor into a
mature osteoclast. Recombinant human
osteoprotegerin specifically acts on
bone, increasing bone mineral density
and bone volume. Osteoprotegerin has
been used experimentally to decrease
bone resorption in women with
postmenopausal osteoporosis and in
patients with lytic bone metastases.
Osteoprotegerin production is
stimulated in vivo by the female sex
hormone estrogen, as well as the
osteoporosis drug, strontium ranelate.
sRANKL
RANKL (Receptor Activator for
Nuclear Factor kB Ligand), also known
as TNF-related activation-induced
cytokine (TRANCE), osteoprotegerin
ligand (OPGL), and ODF (osteoclast
differentiation factor), is a molecule
important in bone metabolism.
This natural and necessary surfacebound molecule activates osteoclasts,
cells involved in bone resorption.
Overproduction of RANKL is
implicated in a variety of degenerative
bone diseases, such as rheumatoid
arthritis and psoriatic arthritis. RANKL
also has a function in the immune
system, where it is expressed by T helper
cells and is thought to be involved in
dendritic cell maturation. This protein
was shown to be a dentritic cell survival
factor and is involved in the regulation
of T cell-dependent immune response.
T cell activation was reported to induce
expression of the RANKL gene and lead
to an increase of osteoclastogenesis and
bone loss. This protein was shown to
activate antiapoptotic kinase AKT/PkB
through a signalling complex involving
SRC kinase and tumor necrosis factor
receptor-associated factor (TRAF) 6,
which indicated this protein may have a
role in the regulation of cell apoptosis.
Targeted disruption of the related gene
in mice led to severe osteopetrosis and
a lack of osteoclasts. The deficient mice
exhibited defects in early differentiation
of T and B lymphocytes, and failed
to form lobulo-alveolar mammary
structures during pregnancy. Two
alternatively spliced transcript variants
have been found.
Tartrate-resistant acid
phosphatase or acid
phosphatase 5, tartrate
resistant (ACP5 or TRAP)
Tartrate-resistant acid phosphatase or
acid phosphatase 5, tartrate resistant
(ACP5 or TRAP) is a glycosylated
monomeric metalloenzyme expressed in
mammals. It has a molecular weight of
approximately 35kDa, a basic isoelectric
point (7.6-9.5), and optimal activity in
acidic conditions. TRAP is synthesized
as latent proenzyme and activated by
proteolytic cleavage and reduction. It
is differentiated by other mammalian
acid phosphatases by its resistance to
inhibition by tartrate, molecular weights
and characteristic purple colour. The
mechanism of phosphate ester hydrolysis
by TRAP is through a nucleophilic
attack mechanism, whereby, catalysis
occurs with the binding of a phosphatesubstrate to the Fe2+ in the active site
of TRAP. This is then followed by a
nucleophilic attack by a hydroxide
ligand on the bound phosphorus atom,
resulting in cleavage of the phosphate
ester bond and production of an alcohol.
The exact identity and mechanism of
the hydroxide ligand is unclear, but it
is thought to be either a hydroxide that
bridges the metal ions within the active
site or a terminal hydroxide bound to
Fe3+, with confl icting reports for both
mechanisms.
Vitamin D
Vitamin D is a steroid hormone
involved in the intestinal absorption
of calcium and the regulation of
calcium homeostasis. There are two
different forms of Vitamin D, named
D3 and D2, which are very similar in
structure. The D2 is a synthetic product,
which is predominantly absorbed by
fortified food. Physiological Vitamin
D3 levels result not only from dietary
uptake but also by biosynthesis of
7-dehydrocholesterol and UV-light in
skin because of sun exposure. Vitamin
D deficiency can result from inadequate
intake coupled with inadequate
sunlight exposure, disorders that limit
its absorption, conditions that impair
conversion of vitamin D into active
metabolites, such as liver or kidney
disorders, or, rarely, by a number of
hereditary disorders. Deficiency results
in impaired bone mineralization, and
leads to bone softening diseases, rickets
in children and osteomalacia in adults,
and possibly contributes to osteoporosis.
Research has indicated that vitamin D
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Biochemical Bone Marker ELISA kits
deficiency is linked to colon cancer;
confl icting evidence links vitamin D
deficiency to other forms of cancer. In
the liver, the vitamin is hydroxylated to
25-hydroxyvitamin D (25-OH Vitamin
D), the major circulating metabolite
of Vitamin D. Although 1,25-(OH)2
Vitamin D portrays the biological active
form of Vitamin D, which is synthesized
in the kidney, it is widely accepted that
the measurement of circulating 25-OH
Vitamin D provides better information
with respect to patients’ Vitamin D
status and allows its use in diagnose
hypovitaminosis. The concentration
of 25-OH Vitamin D decreases with
age and a deficiency is common among
elderly persons. Clinical applications of
25-OH Vitamin D measurements are
the diagnosis and therapy control of
postmenopausal osteoporosis, rickets,
osteomalacia, renal osteodystrophy,
pregnancy, neonatal hypocalcemia and
hyperparathyroidism. In addition, a
prevalence of subclinical Vitamin D
deficiency has been discussed lately in
different European countries. Vitamin
D intoxication mostly occurs during
a large intake of pharmaceutical
preparations of Vitamin D and may lead
to hypercalcemia, hypercalcuria and
nephrocalcinosis in susceptible infants.
N-telopeptide (NTx)
NTx provides the measurement of the
cross-linked N-telopeptides of bone
type I collagen (NTx). NTx is a specific
biochemical indicator of bone resorption
that is generated as the result of osteoclast
activity on bone. The NTx molecule is
specific to bone due to the unique amino
acid sequence and orientation of the crosslinked alpha-2 (I) N telopeptide.
C-telopeptide (ICTP)
ICTP is a quantitative measurement
of a cross-linked carboxyterminal
telopeptide of type I collagen in human
serum. ICTP indicates pathological
bone degradation specifically without
being influenced by physiological bone
turnover. It also provides fi rst-line
detection of bone involvement in cancer
and multiple myeloma and helps to
predict disease severity in rheumatoid
arthritis.
C-telopeptide (CTx)
Urine CrossLaps® detects fragments
of collagen type I generated during
osteoclastic bone resorption. The assay
can be used for quantification of bone
resorption and assessment of bone loss.
The test is specific for an epitope in the
amino acid sequence Glu-Lys-Ala-HisAsp-ß-Gly-Gly-Arg derived from the
C-telopeptide of the a1 chain of type I
collage.
Propeptide of type I
procollagen N terminal (PINP)
PINP is a quantitative measurement of
intact aminoterminal propeptide of type
I procollagen in serum, an indicator
of osteoblastic activity in bone. PINP
is intended to be used as an aid in
the management of postmenopausal
osteoporosis. PINP detects increased
bone turnover, and can be used to
identify patients at risk of fracture.
Also helps to monitor antiresorptive
and anabolic therapy in patients with
osteoporosis.
Deoxypyridinoline (Dpd)
Deoxypyridinoline (Dpd) occurs
mainly in Type I collagen of bone. After
the formation of the Type I collagen
matrix, the action of lysyl oxidase on
two hydroxylysine and a lysine residue
result in the formation of a Dpd cross
link between two triple helical strands
of collagen. In the process of bone
degradation, Dpd is released into the
blood circulation and cleared by the
kidneys. Hence Dpd has been shown
to be a biochemical indicator of bone
resorption.
Osteocalcin
Osteocalcin or bone Gla protein (BGP)
is the major non-collagen protein of
the bone matrix. It has a molecular
weight of 5800 Da and contains 49
amino-acids, including 3 residues
of gamma carboxyl glutamic acid.
Osteocalcin is synthesized in the bone
by the osteoblasts. After production,
it is partly incorporated in the bone
matrix and the rest is found in the blood
circulation. The exact physiological
function of osteocalcin is still unclear.
A large number of studies show that the
circulating levels of osteocalcin reflect
the rate of bone formation.
Bone Specific Alkaline
Phosphates
The skeletal, or bone-specific, isoform
of alkaline phosphatase is a tetrameric
glycoprotein found on the surface of
osteoblast cells. Osteoblast are a type
of cell responsible for lying down the
protein matrix of bone, on which
calcium salts (particularly phosphates)
are deposited. The function of bonespecific alkaline phosphatase (BAP)
is not clearly understood, although
it’s been shown to be a biochemical
indicator of bone turnover.
Parathyroid hormone (PTH)
Parathyroid hormone (PTH) is
secreted by the parathyroid glands as a
polypeptide containing 84 amino acids.
It acts to increase the concentration of
calcium in the blood by acting upon
parathyroid hormone receptor in three
parts of the body. Regulation of serum
calcium, serum phosphate and vitamin
D synthesis.
Calcitonin
Calcitonin is a 32-amino acid linear
polypeptide hormone that is produced
primarily by the Para follicular
cells of the thyroid. It acts to reduce
blood calcium, opposing the effect of
parathyroid hormone.
The hormone participates in
calcium and phosphorus metabolism
by inhibiting Ca++ absorption by
the intestines, inhibiting osteoclast
activity in bones, inhibiting phosphate
reabsorption by kidney tubules and
increasing absolute Ca++ and Mg++
reabsorption by the kidney tubules.
Continued
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Biochemical Bone Marker ELISA kits
Glycosaminoglycans (GAGs)
The most abundant
heteropolysaccharides in the body are
the glycosaminoglycans (GAGs). They
are located primarily on the surface of
the cells or in the extra cellular matrix.
As an example, cartilage is composed
to a large extent of GAGs, which are
the dominant part of the proteoglycan
Aggrecan.
Cartilage Oligomeric Matrix
Protein (COMP)
Cartilage Oligomeric Matrix Protein
(COMP) is a high molecular weight,
multi-submit protein originally isolated
from cartilage. The protein is abundant
in cartilage but also found in tendon and
other tissues. The protein belongs to the
thrombospondin family. Measurement
of intact COMP and fragments in
synovial fluid or serum have been shown
to correlate to cartilage destruction in
rheumatoid arthritis and osteoarthritis
patient studies.
Fibroblast growth factor 23
(FGF-23)
Fibroblast growth factor 23 (FGF-23)
is a recently discovered, novel member
of a large family of related proteins.
Its gene encodes a 251 amino acid
protein. The amino-terminal portion
of FGF-23 (aa 1-24) is hydrophobic
and is likely to serve as a signal peptide
allowing its secretion into the blood
circulation. Its carboxyl-terminal
portion (aa180-251) shares only limited
amino acid homology with other
members of the FGF family proteins.
The measurement of human FGF-23
in the blood circulation is likely to
provide an important diagnostic tool
for patients with a variety of different
hypophosphatemic disorders, including
oncongenic ostemalacia, X-linked
hypophosphatemic rickets, and
autosomal dominant hypophospatemic
rickets. Furthermore, the sensitive
measurement of FGF-23 is likely to
provide novel insights into the regulation
of bone and mineral homeostasis. $
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