Clinical and Technical Review - Tecomedical

Author:
Peter
Haima, Ph
Clinical and
Technical Review
Bone & Cartilage Metabolism
Cell culture
Diagnostic and therapy control
Animal models

2
Bone turnover Biomarker in
Osteoporosis
Diagnosis of metabolic bone diseases can be established
by measuring bone mineral density (BMD) at the hips or spine.
However, BMD is a static measure of bone composition,
reflecting its history. A baseline BMD value does not offer
any prediction of future bone loss or response to therapy.
Moreover, since biochemical bone marker reflect the wholebody
rates of bone turnover, the combined measurement of
bone marker and BMD provides more information on the
overall bone loss than BMD measurement at specific skeletal
sites alone.
The increasing number of drugs available for treatment of
osteoporosis and other bone diseases requires the use of
more rapid and predictive methods to assess therapy efficacy.
While detectable and significant changes in BMD take 18 to
24 months to develop, bone turnover marker have been
shown to detect changes in bone tissue within 3-6 months
after starting anti-resorptive therapy. Therefore, measurement
of bone turnover marker is increasingly recommended
as a key component of therapy management: to rapidly
identify therapy responders and non-responders, to assess
therapy efficacy and to determine the optimal therapy and
dose of treatment.
Bone marker are intended for use as an
aid in:
• management of postmenopausal osteoporosis and
Paget’s disease;
• monitoring of postmenopausal women on hormonal
or bisphosphonate therapy;
• prediction of skeletal response to hormonal therapy
in postmenopausal women.
• Detection and monitoring of Bone metastasis.
Biomarker and Cartilage Destruction
Degenerative joint disease such as osteoarthritis (OA) and
rheumatoid arthritis (RA) are major causes of disability and
impaired quality of life among the elderly. Despite better
understanding of the underlying pathomechanisms, current
clinical practice for diagnosing these diseases mainly relies
on symptom assessment, which has limited sensitivity and
specificity. By the time radiological techniques can reveal
specific sign, the disease has advanced to a late phase with
pronounced structural damage, when the only therapeutical
option remaining is joint replacement. As marker provide a
dynamic measure of current degradation rate in contrast to
radiological investigations, they allow a rapid determination
of the therapeutic effects, and they may also be used for
selecting the therapies and drug dose, which are most efficient.
In the lack of established diagnostic methods allowing
the detection of cartilage degradation, early diagnosis
and disease monitoring remain a major challenge of health
care professionals.
Therefore Biochemical marker for Cartilage degradation and
synthesis are helpful for the management of Rheumatoid
Arthritis and Osteoarthritis.
Biomarker for diagnosing skeletal
metastases
Cancers have the ability to metastasize to organs distant
from the site of the primary tumor. Breast, prostate,
and lung cancer are primary tumors that most frequently
metastasize to the skeleton. The chronic presence of bone
metastasis often results in complete pathologic remodeling
of the affected bone compartment, making affected bones
vulnerable to several complications. Such complications
include pathologic fractures, spinal cord compression,
hypocalcemia, and severe bone pain. All reducing quality of
life and worsening prognosis. Therefore, early diagnosis and
adequate treatment of bone metastasis is critically important
for the clinical management of cancer patients.
Emerging evidence suggests that biomarker of bone
turnover carry notable potentials to become useful
diagnostic tools for the diagnosis of bone metastases.
The study by Leeming et al was sought to assess the
relative use of eight biomarker for the detection of bone
metastases in cancer forms frequently spreading to the skeleton.
Participants were 161 patients with either breast, prostate,
or lung cancer. The presence and extent of
bone metastases was assessed by imaging techniques
(computer tomography and/or magnetic resonance
imaging) and Technetium-99m scintigraphy. Number of
bone metastases was recorded, and the skeletal load was
graded, as previously proposed by Soloway. The Serum
or urinary level of the bone resorption marker (alpha-CTX,
beta-CTX, NTX, and ICTP), formation marker (BSAP),
and osteoclastogenesis marker (osteoprotegerin, RANKL,
and TRAP5b) was measured by commercially available
immunoassays. There was a uniform pattern of significantly
increased levels of the resorption marker in patients with
bone metastases compared with those without.
The relative responsiveness of marker was calculated to
obtain insights into the relative sensitivity of the different
marker to signal skeletal involvement. The plot demonstrates
a trend toward greater relative increases in the level of the
urine alpha-CTX and serum BSAP marker compared with
other marker; differences becoming more evident with
increasing Soloway score.

Figure 1:
Soloway 0 = Patients without bone metastasis
Soloway 1 = Patients with < 6 bone metastases
Soloway 2 = Patients with < 20 bone metastases
Soloway 3 = Patients with > 20 bone metastases but less
than a “super scan”
Soloway 4 = Patients with “super scan” that is defined by
a > 75 % involvement of the ribs, vertebrae
and pelvic bones
Relative increases in bone resorption, bone formation, and
osteoclastogenesis marker as a function of the extent of skeletal
involvement assessed in 132 breast and prostate cancer
patients. Relative increases are expressed as percentage of
levels in patients with Soloway score 0 (1) .
Currently, the diagnosis of bone metastasis in cancer
patients relies predominantly on imaging techniques,
such as plain radiography or Technetium-99 scintigraphy.
Although scintigraphy is more sensitive than plain
radiography and even can give quantitative information
regarding skeletal involvement, this examination is also
more expensive, invasive, time-consuming and exposes
cancer patients to irradiation, limiting its use for monitoring
purposes. Thus, these weaknesses of current methodologies
point out an unmet need for establishing supplementary
diagnostic tools like biochemical marker.
Regulators of bone turnover
Bone remodeling is an ongoing dynamic process
consisting of bone resorption (due to osteoclasts digesting
type I collagen) and bone formation (due to osteoblasts).
Normally, these processes are balanced, resulting in 10 %
replacement of the skeleton, each year. However, due to
aging, disease or other conditions, bone turnover may
become imbalanced where bone resorption and formation
occur at different rates.
OPG (Osteoprotegerin), also known as osteoclast inhibiting
factor (OCIF), inhibits the differentiation and activation of
osteoclasts.
On the other hand, sRANKL (soluble receptor activator of
nuclear factor (NF)-κB ligand) is the main stimulator for the
formation of mature osteoclasts. Stimulation occurs through
binding of sRANKL to the osteoclastic membrane receptor
RANK.
OPG inhibits the binding of sRANKL to RANK and thus
the activation of osteoclasts. The OPG/sRANKL system is
therefore a key regulator of bone resorption. Abnormalities
in the balance of the OPG/sRANKL system may be the
cause of bone loss in many metabolic bone diseases as
osteoporosis, Paget’s disease, metastatic cancers and
rheumatic bone degradation.
In normal healthy people, sRANKL levels are generally low
because the majority is bound by OPG. Decreased levels of
OPG and/or increased levels of sRANKL may indicate an
imbalance of the OPG/sRANKL system.
OPG and/or sRANKL measurements can
be used as an aid in determining the cause
of bone loss by assessing imbalances in
the OPG/sRANKL system in:
• Post menopausal osteoporosis
• Paget’s disease
• Metastatic cancer
• Diseases with locally increased resorption activity
• Indicating bone loss in rheumatoid arthritis
• Therapy monitoring after treatment with OPG
• Determining an imbalance in vascular calcification (high
OPG was associated with cardiovascular- and all-cause
mortality in hemodialysis patients; Morena et al. 2006).
• Metastatic Renal Cell Carcinoma (OPG)
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4
Bone Formation
Osteoblast:
The osteoblast is the bone cell responsible for:
1) The formation and organization of the extracellular matrix
(ECM) of bone and its subsequent mineralization;
2) Synthesis of collagen and other bone proteins. Three
periods are distinguished in the osteoblast life cycle.
A) Cell proliferation: genes associated with formation of the
ECM, like Type I Collagen, are expressed and gradually
down regulated.
B) ECM maturation: proteins associated with the osteoblast
phenotype are expressed, like Bone-specific Alkaline
Phosphatase (BAP).
C) ECM mineralization: BAP gene expression declines,
Bone Sialo Protein (BSP), osteopontin and osteocalcin
gene expression increase.
Bone matrix:
Consists of Type 1 collagen (90 % of the protein in bone),
osteocalcin, osteopontin, osteonectin, proteoglycans,
alkaline phosphatase and bone sialo protein.
Proteins
OPG:
OPG (Osteoprotegerin) or OCIF (Osteoclast Inhibiting
Factor) or OBF (Osteoclast Binding Factor) is a key factor in
inhibition of osteoclast differentiation and activity.
It binds and acts as as decoy receptor for s-RANKL.
sRANKL:
Soluble Receptor Activator of Nuclear factor (NF)-κB Ligand.
sRANKL binds to osteoclast receptor: RANK (NF-κB ) and is
the main stimulatory factor for the formation of mature
osteoclasts.
BAP:
Bone-specific Alkaline Phosphatase is an osteoblastic enzyme
involved in bone formation. It is assumed that BAP plays
a role in ECM maturation. BAP is a key biochemical bone
marker used for assessing bone turnover and
monitoring therapy.
Bone Sialoprotein (BSP):
Major structural protein of the bone matrix, expression of
BSP is normally restricted to mineralized connective tissues
of bones and teeth. This role has been associated with
mineral crystal formation.
Osteocalcin:
Major structural protein of the bone matrix, binds calcium
and attracts osteoclasts.
Osteonectin:
Protein that binds calcium and is involved in regulation
of mineralization.
Osteopontin:
Cell-binding protein that anchors osteoclasts to
mineralised matrix.
Proteoglycans:
Monomer looks like test tube brush with keratan and
chondroitin sulphate chains (= GAGs) bound to a protein
core molecule. Monomers are attached via a link protein
to hyaluronic acid.
DKK-1:
Dickkopf-1(DKK-1) is a 28,672 Da secreted protein that acts
as soluble inhibitor of the WNT signalling pathway. DKK-1
regulates different developmental processes and is also
involved in the regulation of bone metabolism as it inhibits
the differentiation of osteoblast.
Collagen metabolites and epitopes
Type I Procollagen:
Secreted precursor of Type I Collagen. Extracellular cleavage
results in N- and C-terminal propeptides.
PINP:
Epitope of N- terminal propeptide, released during cleavage
of Type I Procollagen.
CICP/PICP:
Epitope of C- terminal propeptide, released during cleavage
of Type I Procollagen (PICP = CICP).
Type I Collagen:
Collagen molecules consist of three chains to form a triple
helix. Crosslinks between the chains and the molecules of
collagen give collagen its strength.

Bone Resorption
Osteoclast:
Large motile, multinucleated bone cell located on bone surfaces.
Responsible for the resorption of bone matrix (osteoid).
Osteoclasts have a ruffled border of the cell membrane that
is surrounded by an organelle-free region, or « clear zone ».
Mineral Dissolution:
These processes take place beneath the ruffled border
and depend on lysosomal enzyme secretion and an acid
microenvironment. A pH gradient across the ruffled
membrane is the consequence of active transport
mechanisms by the osteoclasts.
Collagen degradation:
Osteoclasts actively synthesize lysosomal enzymes,
in particular the tartrate resistant isoenzyme of acid
phosphatase (TRAP 5b) and cysteine-proteinases such
as Cathepsin K that are capable of degrading collagen.
Two bone collagenolytic pathways exist:
1. A Cathepsin K collagen degradation pathway is the prevailing
one. Resulting in the following epitopes:
CTX, NTX, DPD and PYD.
2. A Matrix MetalloProteinase (MMP) collagen degradation
pathway resulting in the epitope ICTP. This pathway becomes
significant in some situations, including metastatic
bone diseases and multiple myeloma. ICTP however, has
proven to be a very poor marker in osteoporosis.
Proteins
TRAP 5b:
The active isoform of TRAP5b, serum band 5 tartrateresistant
acid phosphatase, is specifically synthesized
by bone-resorbing osteoclasts. It has been shown that
TRAP5b catalyzes the formation of reactive oxygen
species (ROS). Research results indicate that ROS
generated by TRAP5b are involved in the degradation
of bone matrix products in resorbing osteoclasts.
TRAP5b activity reflects the osteoclast activity and is associated
with the number of osteoclasts. TRAP5b is consideredamarkerfortherateofboneresorptionandmaybeof
particular importance for patients with renal failure because
- in contrast to other marker of resorption -
TRAP5b does not accumulate in the blood.
In tumor patients TRAP5b is an indicator for increased bone
resorption due to bone metastases. Changes in TRAP
activity during therapy monitoring allow the assessment of
the efficiency of antiresorptive therapies in osteoporotic
patients.
RANK:
Osteoclastic receptor for sRANKL, the main stimulatory factor
for the formation of mature osteoclasts.
Cathepsin K:
Main osteoclastic protease, responsible for bulk degradation
of Type I Collagen. Acts both intra- and extra-cellularly.
Lysosomal enzymes:
Enzymes secreted by the osteoclasts, responsible for
mineral dissolution in an acid environment.
MMP:
Matrix metalloproteinases MMP-2, -9, -13, -14 participate in
the degradation of the collagenous bone matrix,
resulting in epitope ICTP.
Calcitonin receptor:
Osteoclastic receptor for calcitonin, an inhibitor of
osteoclasts activity.
Sclerostin:
Sclerostin is the protein product of the SOST gene, which is
located at 17q12-21 and highly conserved across vertebrate
species.
The highest expression of sclerostin throughout the adult
skeleton has been observed in hypertrophic chondrocytes
and osteocytes. Sclerostin belongs to the DAN1 family of
glycoproteins of which multiple family members antagonize
bone morphogenetic protein (BMP) and/or Wnt2 activity.
Sclerostin blocks canonical Wnt signaling by binding to the
Wnt coreceptors LRP5/63 . Thus, it inhibits bone formation
by regulating osteoblast function and promoting osteoblast
apoptosis. By blocking the Wnt-pathway Sclerostin also
antagonises bone morphogenetic protein action e.g. osteoblast
differentiation, but does not inhibit direct BMP-induced
responses.
Sclerostin expression is down-regulated by Parathyroid hormone
(PTH) as well as the mechanical stimulation of bone
reduces the expression of sclerostin.
1 DAN = differential screening-selected gene aberrative in neuroblastomaa
2 Wnt = wingless Proteine
3 LRP = low-density lipoprotein receptor-related protein 5 and 6
5

6
Collagen metabolites and epitopes
DPD:
Deoxypyridinoline. Breakdown product from Type I collagen
degradation.
PYD:
Pyridinoline. Breakdown product from Type I collagen
degradation.
NTX:
Cross-linked N-terminal telopeptide resulting from
Type I collagen degradation. NTX = new synthesized and
agemodified bone matrix.
CTX:
C-terminal telopeptide resulting from Type I collagen
degradation, CTX alpha (New Bone Matrix), CTX beta (Old
Bone Matrix).
ICTP=CTX-MMP:
Carboxy-terminal telopeptide of type I collagen.
Larger epitope containing the smaller CTX epitope.
Helical Peptide:
Peptide derived from the helical region of the α-1 chain of
Type 1 collagen.
Note:
The concentration of the different biomarker are partly
dependent on age, gender, race as well as influenced by
diurnal rhythm, food intake etc. (see table on page 8). Age
dependency is of high importance if biomarker are used for
preclinical testing, especially in juvenile laboratory animals
(rat, mice).
Bone turnover regulators
Calcitonin:
Inhibits bone resorption, directly acts on osteoclasts.
Parathyroid hormone (PTH):
Key factor in the maintenance of calcium and phosphate
homeostasis. Stimulates osteoclasts activity.
1,25(OH) 2D3 :
1,25 dihydroxy vitamin D3 (or 1,25-dihydroxycholecalciferol)
is the biologically active form of vitamin D. It is
synthesized in the kidney from 25-vitamin D (25-hydroxycholecalciferol).
1,25(OH) 2D3 is the principal regulator
of calcium homeostasis in the body. It enhances the
efficiency of calcium.
Fetuin A:
Glycoprotein synthesized by liver, secreted into blood.
Deposited as noncollagenous protein in mineralized bones.
Potent inhibitor of soft tissue (vascular) calcification by binding
excess mineral in serum. Regulates calcium metabolism
and osteogenesis.
FGF-23:
Fibroblast Growth Factor 23. Important regulator of
phosphate homeostasis. Able to “block” renal reabsorption
of Pi. FGF-23 abnormalities are involved in renal phos-phate
wasting disorders leading to “hypophosphatemia”.
Literature:
1. Leeming DJ, Koizumi M, Byrjalsen I, Li B, Qvist P, Tanko LB.
The relative use of eight collagenous and noncollagenous markers
for diagnosis of skeletal metastases in breast, prostate, or
lung cancer patients.
Cancer Epidemiol Biomarkers Prev. 2006; 15(1):32-8.
2. Caulfield MP, Reitz RE. Biochemical markers of bone turnover and
their utility in osteoporosis. MLO-online April 2004.
Schaller S, Henriksen K, Hoegh-Andersen P, Søndergaard BC,
Sumer EU, Tanko LB, Qvist P and Karsdal MA. In vitro, ex vivo, and
in vivo methodological approaches for studying
therapeutic targets of osteoporosis and degenerative joint
diseases: How biomarkers can assist? Assay And Drug
Development Technologies 2005; 3:553-580.
3. Delmas PD, Eastell R, Garnero P, Seibel MJ and Stephan J. The use
of biochemical markers of bone turnover in osteoporosis.
Osteoporosis Int 2000: Supp 6: S2-17.

Bone Turnover – Biochemical Marker
Sclerostin:
inhibits differentiation and
function of osteoblasts
through BMP
DKK-1
regultes osteoblast
differentiation
P *
C
I
P
MMPs
BAP
(bone-specific alkaline phosphatase)
“calcification of the matrix”
ICTP **
ICP
*
P
,
P
N
I
P
Helical Peptide
** Not so relevant in
Osteoporosis
Fetuin A
* CICP = PICP
7

8
Bone marker and characteristics
Biomarker Method Main
Sample
Type
Formation
BAP
Osteocalcin
Intact
Osteocalcin
N-mid
PINP
PICP = CICP
Resorption
ICTP =
CTX-MMP
Others
ELISA
IRMA/
ELISA
ELISA
RIA
ELISA
Serum
Serum
Serum
Serum
Serum
A(a) B C D E F G H
Pre- &
postmenopause
+
+
+
+
NA
Analytical
variation
+
+
+
+
+
Treatment
response
Short
time
change
Within
person
variation
Daily
variation
Food
intake
TRAP 5b ELISA Serum ± + ± ± NA + + ±
DPD ELISA Urine + + + + + - + +
NTx ELISA
CTx ELISA
+
+
+
+
+
Sample
stability
Scores sind: + (yes), - (no), ± (fair/indeterminate), NA (not available)
(a) Letters correspond to following, the marker:
H. preferably demonstrates high stability in the biological
A. shows a difference in the rate of bone turnover pre-and
specimen
post-menopause
(b) In contrast to OPG, sRANKL showed no response to
B. demonstrates minimal analytical variation
bisphosphonate treatment of osteoporotic postmeno-
C. significantly changes in response to treatment
pausal women. However, PTH treatment of glucocorticoid
D. detect changes in short time interval (months)
induced osteoporotic women resulted in a fast (1 month)
E. demonstrates minimal within person (biological) variation and sustained increase of sRANKL levels.
F. preferably demonstrates little variation over the day
(c) Cathepsin K is elevated in patients with established
G. no influence by food intake
rheumatoid arthritis.
+
+
+
+
+
+
±
±
±
+
+
+
+
+
±
+
+
+
+
+
+
-
+
+
+
5 days
2–8 °C
4hours
2–8 °C
5 days
2–8 °C
5 days
2–8 °C
5 days
2–8 °C
2 days
2–8 °C
7 days
2–8 °C
Serum + + + + + - ± ± 1day
2–8 °C
Urine + + + + ± - ± +
3 days
2–8 °C
Serum + + + + + - - + 1day
2–8 °C
Urine + + + + ± - - +
ELISA Serum - + - - NA ± + +
7 days
2–8 °C
5 days
2–8 °C
sRANKL ELISA Plasma NA + ± (b) - NA NA + ± 1day
2–8 °C
Osteoprotegerin
(OPG)
ELISA Plasma NA + + - NA NA + ± 1day
2–8 °C
Cathepsine K ELISA Serum NA (c) + NA NA NA NA + ±
2 days
2–8 °C

Bone marker and their behavior in various bone diseases
Biomarker CTX-I NTX-I Free
DPD
Bone Disease
• ↑ Increased values of the biomarker in this disease were
observed in various studies as compared to a healthy
control group.
• ↓ Decreased values of the biomarker in this disease were
observed in various studies as compared to a healthy
control group.
• ↑ Large arrows: indicate large and significant differences.
↑
Small arrows: indicate small to medium differences
with the control group.
Degradation marker Formation marker
Free
PYD
Helical
peptide
ICTP TRAP
5b
BAP OC PICP/
CICP
↑
Osteoporosis Osteoporose ↑ ↑ ↑ ↑ ↑ ± + ± ↑ ↑ ↑
↑
Hyperparathyroidism Osteoporose ↑ ↑ ↑ ↑ ↑ ± ↑ + ↑ ↑ ↑
↑
Hyperthyroidism ↑ ↑ ↑ ↑ ↑ (↑)
Bone metastases* ↑ ↑ ↑ ↑ ↑ ↑ (↑) ↑ ↑
Multiple myeoloma ↑ ↑ ↑ ↑ ↓ ±
Rickets/
Vitamin-D Deficiency
Osteomalacia
“adult rickets”
↑ ↑ ↑ ↑ ↓ ↑
↑ ↑ ↑ ↑ ↑
Paget´s ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
Anorexia nervosa (↑) (↑) (↓) (↓↑) (↓)
Growth hormone
deficiency
↓ ↓ ↓
Renal failure ↑ ↑ ↑ ↑ ↑ ↑
PINP
• (↓) (↑): arrows between brackets: different studies
suggest inconsistent behavior of this marker in
this disease.
• ± indicates that according to various studies this marker
is not considered as valuable for this disease.
• Empty spaces indicate that no information was identified
on this biomarker for this disease.
* Increased values were observed for urin CTX alpha and
Serum BAP in bone metastases.
9

10
Cartilage Degradation
Chrondocyte:
Chondroblasts trapped in lacunae develop into chondrocytes.
Chondrocytes are important in the control of matrix turnover
through production of collagen, proteoglycans and enzymes
for cartilage metabolism.
Proteins
Matrix metalloproteinases:
Are involved in the cleavage of Type II Collagen and the proteoglycan
aggrecan. Three collagenases (MMP-1, -8,-13) are
mainly responsible for primary cleavage of Type II Collagen.
MMP-1, -8, -13 and 14 are involved in cleavage of the core
protein of aggrecan.
Cathepsin K:
Protease produced by synovial fibroblasts, enzyme
plays critical role in cartilage degradation (together with
matrix metalloproteinases). MMPs perform extracellular
predigestion of collagen, after endocytosis of large
fragments, Cathepsin K degrades collagen and aggrecan in
acidic lysosomes.
Aggrecanases:
Enzymes involved in cleavage of aggrecan.
COMP:
Cartilage Oligomeric Matrix Protein is an abundant cartilage
glycoprotein also found in tendon and other tissues.
Synthesized by chondrocytes, synovial and other skeleton
cells. Intact and fragmented COMP in synovial fluid or
serum is correlating to cartilage degradation in OA and RA.
Cartilage metabolites and epitopes
CTX-II:
A 6 amino acid sequence epitope of the nonhelical C- terminal
telopeptides resulting from Type II collagen degradation.
C2C:
C2C or COL2-3/4CLong epitope that specifically appears
into circulation when Type II collagen degradation occurs.
C1,2C:
C1, 2C or COL2-3/4CShort epitope that appears into circulation
when Type II but also Type I collagen degradation occurs.
CS-GAG:
Chondroitin sulfate glycosaminoglycans are bound at high
densities, to a core protein forming the cartilage proteoglycan
aggrecan molecule.
Cartilage Synthesis
Cartilage metabolites and epitopes
Type II Procollagen:
Secreted precursor of Type II Collagen. Extracellular cleavage
results in N- and C-terminal propeptides.
CP-II:
Epitope of C- terminal propeptide, released during
maturation of Type II Procollagen to Type II Collagen.
Type II Collagen:
The principal structural component of cartilage is an extensive
network of Type II collagen molecules, arranged in fibrils.
Collagen molecules consist of three chains to form a triple
helix. Crosslinks between the chains and the molecules of
collagen give collagen its strength.
Proteoglycan Aggrecan:
Responsible for the compressive strength of cartilage. Serve
to trap and hold water to regulate matrix hydration. Monomer
looks like test tube brush with keratan and chondroitin
sulphate chains (= GAGs) bound to a protein core molecule.
Monomers are attached via a link protein to hyaluronic acid.
CS-GAG:
Chondroitin sulfate glycosaminoglycans are bound at high
densities, to a core protein forming the cartilage proteoglycan
aggrecan molecule.
Aggrecan epitope CS 846:
Chondroitin native epitope, present on intact bioactive
(“fetal-like”) proteoglycan only.
PIINP:
Epitope of Type II N-terminal propeptide, released during
maturation of Type II Procollagen to Type II Collagen. PIINP
has been postulated to play a role in chondrogenesis. It has
been found to be synthesized by osteoarthritic chondrocytes
in diseased cartilage and may serve as a specific arthritis
biomarker that reflects an attempt by the chondrocytes to
repair diseased cartilage.
Proteins
BMP1:
BMP1 (Bone morphogenetic protein) is a protein that is
capable of inducing formation of cartilage in vivo. It cleaves
the C-terminal propeptides of procollagen I, II, and III and
plays an important role in collagen maturation.

Biomarker according to NIH Osteoarthritis Biomarker Network
Synovium
Type III collagen
Noncollagenous proteins
Enzymes
Aggrecan
Type II collagen
Cartilage
Noncollagenous proteins
Enzymes
Proliferation/Formation Degradation
PIIINP
anti-CCP
COMP
Hyaluronan
Pentosidine
YKL-40
MMPs
TIMPs
Chondroitin sulfate
PIICP
PIINP
PIIANP
YKL-40
TIMPs
Detailed information cartilage metabolism
"Biomarker for diagnosis and monitoring of degenerative joint diseases" by Petra Seebeck, PhD,
Glc-Gal-PYD
PYD
Keratan sulfate
GAGs
C1, 2C
C2C
COL2-1
COL2-1NO2
CTX-II
HELIX II
TIINE
COMP
CILP
MMPs
ADAMTSs
Cathepsin K
Inflammation marker – synovial metabolism
Joint inflammation is a key feature of RA but can occur also during OA. Proliferative synovial inflammation leads to the
forced syntheses of different marker. However, most of them are not synovium-specific, since they are also produced in
cartilage and other tissues.
anti-CCP
Citrullin arises from enzymatic modification of the amino-acid arginine for instance during inflammatory processes.
Auto-antibodies against cyclic citrullinated peptides (anti-CCP) were detected in up to 80%of RA patients already during a
very early stage of disease, but only in very little patients with juvenile idiopathic arthritis (JIA). The concentrations of anti-CCP
were not correlated to the disease activity score (DAS28), but to radiographic signs of joint destruction. Anti-rheumatic
immunosuppressive therapy (Infliximab) reduced the sanguineous anti-CCP level in RA patients.
Hyaluronan
Hyaluronan is a main component of the cartilage matrix as well as the synovial fluid. In patients with knee OA the serological
hyaluronan level correlated with the degree of synovial proliferation and the length of osteophytes but not with the femoral
cartilage thickness. Increased hyaluronan levels were observed in OA as well as RA patients with patients with higher initial
values showing a more progressive course of disease. Serological hyaluronan levels correlated with the degree of joint space
narrowing. RA patients with synovial inflammation showed decreasing hyaluronan concentrations after anti-inflammatory therapy.
11

12
Cartilage Degradation – Biochemical Marker

Cartilage Synthesis – Biochemical Marker
BMP-
MMP
13

14
Cartilage - Type II procollagen
Figure 3:
Schematic drawing of type II procollagen and the localization of the different epitopes. Numbering is based on the amino-acid
sequence of an alpha (II) chain of human type IIB collagen (COL2A1_HUMAN, P02458, UniProtKB, Swiss).
* Type IIA procollagen would include an additional sequence of sixty-nine amino-acids at position 29-97, the numbering of the
following sections would be shifted accordingly

Cartilage Biomarker and their behavior in various arthritis clinical situations
Biomarker
(u = urinary;
s = serum;
syn = synovial fluid;
c = cellculture))
Clinical situation
Degradation marker Formation marker
u CTX-II s C2C s C1,2C c C1,2C s COMP
1) Only the ratio of C2C / C1,2C was prognostic.
2) Changes in serum COMP are prognostic,
not baseline levels.
3) Prognostic for rapid joint destruction.
4) Levels reflect the degree of synovial inflammation.
syn
s CPII
COMP
syn
CPII
s
PIINP
s
Aggrecan
Epitop
syn
Aggrecan
Epitop
s
YKL-40
OA ↑ ↑ ↑ ↑ ↑ ↓/N ↑ ↓ ↑ ↑ ↑
OA Early ↑ ↑ ↑ N
OA Late ↑ ↑ ↑
OA Severity
(Baseline levels
indicate severity)
OA Prognosis
(Baseline levels predict
progression)
syn
YKL-40
+ + 2) + ± 4)
+ 5) + 1) + 1) + + 5)
RA ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↑ ↑
RA Early ↑ ↑ ↑
RA Late ↑
RA Progressive
(Levels in progressive RA
resulting in rapid joint
destruction)
↑ ↑ ↑ ↑ ↑ ↓/N
RA Chronic ↑ ↑ ↑
RA Severity
(Baseline levels
indicate severity)
RA Prognosis
(Baseline levels
predict progression)
+ + ± 4)
+ + + + 3) − + +
5) Patients with knee OA are characterized by an uncoupling
of type II collagen synthesis and degradation which
can be detected by assays for serum PIINP and urinary
CTX-II. The combination of CTX-II and PIINP measurements
seems prognostic for identifying knee OA patients
at high risk for rapid progression of joint damage.
Table was adapted from Schaller et al. (2005).
15

16
Bonemarker normal values for children
Bone marker
BAP (serum)
bone specified alkaline
phosphatase
CICP/PICP (serum)
c-terminal propeptid
of type I collagen
NTX (serum)
N-terminal telopeptide
of type I collagen
NTX (urine)
N-terminal telopeptide
of type I collagen
Deoxypyridinolin
(urine)
free DPD
Helicale peptide (urine)
Helicale peptide
620 - 633 from the
alpha-I-chain of
type I collagen
Pyridinolin (urine)
free Pyd & DPD
Cat.
No.
8012
8003
504836
504837
8007
8022
8010
Sex Unit Age group
m U/l ± SD
f U/l ± SD
Tanner
Stage
Tanner
I
104 ± 21
0–2 y.
122 ± 19
0–2 y.
Tanner
II–III
94 ± 17
3–9 y.
106 ± 16
3–8 y.
Tanner
IV–V
m U/l ± SD 95 ± 22 114 ± 35 121 ± 37
f U/l ± SD 84 ± 23 113 ± 43 79 ± 46
m/f ng/ml
m ng/ml
f ng/ml
m/f ng/ml
Tanner
Stage
m
f
m/f
Tanner
Stage
m
f
m/f
m/f
m/f
m/f
BCE
nmol/l
BCE
nmol/l
BCE
nmol/l
Creatinin
nmol/mml
Creatinin
nmol/mmol
Creatinin
nmol/mml
Creatinin
ug/mmol
Creatinin
nmol/mmol
Creatinin
nmol/mmol
Creatinin
Pyridinolin (serum) 8019 m/f nmol/l
Tanner
I
50.6
(37.2–88.9)
53.6
(31.2–90.9)
211–1.310
0–2 y.
230–1.104
0–2 y.
Tanner
II–IV
71.7
(32.7–126.0)
50.2
(12.9–79.8)
295–365
4.0–12.9 y.
147–830
3–9 y.
155–568
3–8 y.
113–426
4–13 y.
Tanner
V
18.0
(11.3–50.4)
12.8
(9.6–22.2)
114 ± 21
10–14 y.
112 ± 16
9–12 y.
444 ± 192
13 y.
123–618
10–14 y.
122–373
9–12 y.
449 ± 123
14 y.
127–189
15–19 y.
74–167
13–17 y.
110–443
14–18 y.
0–1 y. 2–5 y. 6–10 y. 11–15 y. 16–20 y.
1639
(102-4769)
Tanner
I
689
(34-1752)
Tanner
II–III
497
(90-1356)
Tanner
IV–V
23.5 ± 1.9 25.6 ± 4.0 31.1 ± 9.5
21.0 ± 2.1 33.0 ± 5.1 23.2 ± 7.0
22.5 ± 1.4 28.7 ± 3.2 28.0 ± 6.2
804 –
11305
4–12.9 y.
45.7–85.1
4–12.9 y.
55.5 ± 6.5
4–12.9 y.
2.49 ± 0.12
4–12.9 y.
429
(34-2158)
40.8 ± 6.4
13–18 y.
1.6 ± 1.0
13–18 y.
192
(34-780)
59 ± 19
15–18 y.
39 ± 23
13–17 y.
347 ± 145
15–18 y.
77–183
20–50 y.
37–133
20–50 y.
References
[1]/[2]
[3]
[4]
[1]
[5]
[6]
[9]
[5]
[10]
[11]
[11]

Bonemarker normal values for children
Cat.
Bone marker Sex Unit Age group
No.
TRAP5b (Serum)
Tartrate-Resistant Acid
Phosphatase active
isoform 5b
8036
Following references refer to the table
on page 16 and 17.
[1] Tsai et al
Bone Alkaline Phosphatase Isoenzyme and Carboxy-Terminal
Propeptide of Type-I Procollagen in Healthy Chinese Girls and
Boys.
Clinical Chemistry 45, No. 1, 1999
[2] Elmlinger MW et al
Significance of bone specific alkaline phosphatase and
procollagen-I-peptide as diagnostic markers of bone
formation for monitoring growth hormone therapy
at the Congress on Calcium Regulating Hormones and Bone
Metabolism from the German Society for Endocrinology, Giessen,
Germany, September 29-30, 1995
[3] Siu L. Hui et al
Difference in Bone Mass between Black and White American
Children: Attributable to Body, Build, Sex Hormone Levels, or
Bone Turnover?
Journal of Clinical Endocrinology & Metabolism 88 (2), 642 - 649,
2003
[4] Winterbottom et al
An assay for the C-term. Propeptide of type I collagen.
The Endocrine Society, Anaheim, CA, June 15 - 18, 1994.
[5] Quidel In house study
status
f U/l
m U/l
Pre-
Pubertal
8.1±3.8
1–9 y.
6.6±3.6
1–10 y.
[6] Van der Sluis
A Cross-Sectional Study on Biochemical Parameters of bone
Turnover and Vitamin D Metabolites in Healthy Dutch Children
and Young Adults
Hormon Research 2002; 57: 170-179.
Early Adolescence
10.0±2.7
10–13 y.
9.9±3.3
11–13 y.
Late Adolescence
2.3±0.7
14–17 y.
3.4±1.4
14–17 y.
[7] F. Rauch
Urinary immunoreactive dpd in children and adolescents:
variations with age, sex, and growth velocity.
Scan J. Clin Lab Invest 1996:56: 715 - 719 (Metra Ref # 1540)
[8] A. Bourdeau
Age Dependent Variations in Healthy Children of Urinary
Excretion of Pyridinium Crosslinks.
XII International Conf. On Calcium Regulating Hormones, Australia,
Feb. 14-19, 1995.
[9] A. Conti
Urinary free deoxypyridinoline levels during childhood.
J. Endo. Invest. 21: 318 - 322, 1998. Ref#2574
[10] Husain, et al
Urinary excretion of pyridinium crosslinks in healthy
4 - 10 year olds.
Arch. Dis. Child 1999, 80: 370 - 373
[11] A. Colwell
The Renal Clearance of Free and Conjugated Pyridinium
Crosslinks of Collagen.
J. of Bone and Min. Res. Vol. 11, Number 12, 1996
References
[12] Price HE, Langman CB and Brooks ER.
TRAP5b – profiles in children with chronic kidney disease.
Poster presented at ASBMR (2007).
[12]
[12]
17

18
Biomarkers in Cell Culture
If cell culture samples are used in assay systems which have been validated for the determination of biomarkers in serum
and plasma, several aspects have to be taken into consideration.
An assay system validated for serum / plasma may be affected by sample material generated from cell culture.
To examine whether the assay system is influenced by cross-reactions or interferences induced by the components used,
it is recommended to determine both the medium and all further substances in the assay. FCS in particular may crossreact
with antibodies in the test or the media can affect the antibody response.
It is recommended to use a culture medium comprising the following components:
• DMEM or equivalent commercial medium
• Fetal bovine or calf serum or serum substitutes (FCS-free medium)
• L-Glutamine or ascorbate
• Antibiotic
To exclude the influence of FCS, it is recommended to use FCS-free medium, if possible.
Testing of medium, medium additives, and other buffers used
• Buffer: e.g. elution buffers which have been used to elute a substance from a gel or tissue
• Matrix: e.g. serum-free medium, serum substitutes
• Testing of medium combination:
1. Medium
2. Medium including all additives, but no FCS or alternative matrix
3. Medium including all additives and FCS or alternative matrix (complete medium)
4. Only FCS or corresponding alternative matrix
• Recovery:
Add standard material/substance to the complete medium (spiking) to determine recovery;
e.g. at a ratio 1:5, test 20% of the highest kit standard and 80% of complete medium in the assay
• Linearity:
Measure dilutions of the spiked medium to control linearity:
a) by using dilution buffer or zero standard from the kit at a ratio 1:2 and 1:4
b) by using medium at a ratio 1:2 and 1:4
• Standard curve (optional):
Dilute stock standard or the highest standard contained in the kit with complete medium and perform measurement

Specific recommendations
BAP
Osteocalcin
CICP / PICP
DPD
PYD
NTX
Helical Peptide
Assay Analyt Comments to the test procedure
Bone Alkaline Phosphatase
Intact Osteocalcin
C-terminal Propeptide of Type-I-
Collagen (CICP)
Desoxypyridinolin-Crosslinks
Pyridinolin-Crosslinks
alpha-2 (I) N- telopeptide
Helical Peptide
The Alkaline Phosphate is membrane
bound and can be measured in cell
lysate.
The cells can be grown in serum containing
media, but osteocalcin must be
harvested from tissue culture supernatant
that is serum-free.
The cells can be grown in serum containing
media; however, to measure
the parameters serum-free tissue culture
supernatant has to be used.
Bone collagen specific –
direct measurement of bone resorption.
The cells can be grown in serum containing
media; however, to measure the
parameters serum-free tissue culture
supernatant has to be used.
First results show that the ELISA is
suited for the determination of the helical
Peptide for in vitro analyses. This
test method is especially advantageous
because it requires small sample quantities
and the curve covers a large
range of the sample concentrations (30
fold of the CTX-beta in vitro Assays).
The background concentrations
through the medium are very low.
TRAP5b Tartratresistent acid phosphatase 5b Measurement of the osteoclast activity.
Interferences
In order to test if there are any cross reactions or interferences of the media used for the cell culture with the assay system,
it is recommended to measure the media and maybe other substances in the assay. Especially FCS could contain interfering
substances respectively cross react or certain media suppress the antibody reaction.
19

20
Measurement of BAP and Osteocalcin
in cell culture
• DMEM or equivalent commercial medium
• Fetal bovine serum or calf serum or serum substitutes
(FCS free medium)
• L-Glutamine or Ascorbat
• Antibiotic
Additionally, 50 nM Vitamin D3 is necessary to generate
measurable quantities of osteocalcin. The cells can be
grown in serum containing media, but osteocalcin must be
harvested from tissue culture supernatant that is serumfree,
because the antibody will also detect this analyte in
serum.
Bone alkaline phosphatase is membrane-bound and it has
to be measured in culture from cell lysate. Cell culture media
should not be treated with ion chelators like EDTA or citrate
because of an inhibition of the BAP enzyme activity.
After the incubation the cell layers should be washed twice
with saline and scraped into TMN buffer solution (20 mM
Tris-HCl, pH 7.4; 2 mM MgCl2; 150 mM NaCl) uising a stir
stick. The number of cells has to be determined (e.g. Coulter
cell counter Model F) before the cells become solubilized by
the addition of Triton X-100 to a final concentration of 1 %.
Centrifugate the samples at 70,000g for 60 min and examine
the aliquots of the supernatant for alkaline phosphatase
activity.
Sheep and human cells will be fine for this purpose.
References for BAP, PICP, OSTEOCALCIN
in cell culture
Kaspar D, Seidl W, Neidlinger-Wilke C, Ignatius A, Claes L
(2000) Dynamic cell stretching increases human osteoblast
proliferation and CICP synthesis but decreases osteocalcin
synthesis and alkaline phosphatase activity.
J Biomech 33, 45-51.
Martínez ME, Medina S, Del Campo MT, et al. (1998) Effect
of polyethylene on osteocalcin, alkaline phosphatase and
procollagen secretion by human osteoblastic cells.
Calcif Tissue Int 62, 453-456.
Measurement of CICP / PICP
in cell culture
The assay is suitable for the measurement of cell culture
supernatant from cells producing type-I collagen. The
METRA ® CICP Test can be used for the determination of the
collagen production level of skin fibroblasts and bone cells.
It is recommended, that the supernatant of these cultures
contains serum-free medium. Bovine serum in the medium
may contain bovine CICP, which cross-reacts with the antibody
in the kit. This would result in erroneously increased
values.
In a confluating cell culture at an optimal collagen production
level, the amount of CICP in the supernatant is approximately
20 – 50 ng/ml or slightly below a normal human serum
sample. Dilute the cell culture supernatant 1:12 (like a normal
serum sample) and calculate the final concentration according
to the dilution. If the CICP concentration of the 1:12
dilution is below the detection limit it is possible to choose
a 1:6-dilution.
Sell S, Gaissmaier C, Fritz J et al. (1998) Different behavior
of human osteoblast-like cells isolated from normal and
heterotopic bone in vitro. Calcif Tissue Int 62, 51-59.
Siggelkow H, Niedhart C, Kurre W, et al. (1998) In vitro differentiation
potential of a new human osteosarcoma cell line
(HOS 58). Differentiation 63, 81-91.
Siggelkow H, Rebenstorff K, Kurre W, et al. (1999)
Development of the osteoblast phenotype in primary human
osteoblasts in culture: comparison with rat calvarial cells in
osteoblast differentiation. J Cell Biochem 75, 22-35.

Bone marker - Reference values in animals
BAP in Dogs
Serum / Plasma: < 1 year 56.3 ± 9.8 U/L
1 – 2 years 10.7 ± 4.5 U/L
2 – 3 years 7.0 ± 2.5 U/L
3 – 7 years 6.7 ± 3.6 U/L
> 8 years 7.0 ± 2.9 U/L
Reference: Allen LC et al. (2000) A comparison of two techniques for the determination of serum bone-specific
alkaline phosphatase activity in dogs. Res Vet Sci 68, 231-235.
BAP in Cats
Serum: < 2 years 10 – 70 U/L
> 2 years 2 – 15 U/L
Reference: DeLaurier A, Jackson B, Ingham K, Pfeiffer D, Horton MA, Price JS. (2002) Biochemical markers of
bone turnover in the domestic cat: relationships with age and feline osteoclastic resorptive lesions.
J Nutr 132, 1742S-4S.
BAP in Horses
Serum: 12.2 – 25.5 U/L
Plasma: 12.6 – 22.7 U/L
Reference: Hoekstra K et al. (1999) Comparison of bone mineral content and biochemical markers of bone
metabolism in stall vs. pasture-reared horses. Equine Exercise Phys Equine Vet J 30, 601-604.
BAP in goats
Serum: 12 ± 4 U/L
Reference: Liesegang A, Risteli J, Wanner M (2005) The effects of first gestation and lactation on bone
metabolism in dairy goats and milk sheep. Bone. Dec 16; [Epub ahead of print]
BAP in sheep
Serum: 13 ± 4 U/L
Reference: Liesegang A, Risteli J, Wanner M (2005) The effects of first gestation and lactation on bone
metabolism in dairy goats and milk sheep. Bone. Dec 16; [Epub ahead of print]
BAP in porcine
Reference: Liesegang A et al. (2002) Influence of a Vegetarian Diet Versus a Diet with Fishmeal on Bone
in Growing Pigs. J. Vet. Med. A 49, 230-238
21

22
DPD in Dogs
Urine: < 1 year 45 nM/mM Creatinine
1 – 2 years 4 – 5 nM/mM Creatinine
2 – 3 years 4 – 5 nM/mM Creatinine
3 – 7 years 4 – 5 nM/mM Creatinine
Reference: Allen MJ et al. (2000) Urinary markers of type-I collagen degradation in the dog.
Res Vet Sci 69, 123-127.
DPD in Cats
Urine: 1-10 years 11.3 nM/mM Creatinine
Reference: DeLaurier A, Jackson B, Ingham K, Pfeiffer D, Horton MA, Price JS. (2002) Biochemical markers of
bone turnover in the domestic cat: relationships with age and feline osteoclastic resorptive lesions.
J Nutr 132, 1742S-4S.
DPD in Horses
Urine: 6.0 – 95 nM/mM Creatinine
Reference: Hoekstra K et al. (1999) Comparison of bone mineral content and biochemical markers of bone
metabolism in stall vs. pasture-reared horses. Equine Exercise Phys Equine Vet J 30, 601-604.
Osteocalcin intact in Horses
Serum: 15 – 50 ng/ml
Reference: Hoekstra K et al. (1999) Comparison of bone mineral content and biochemical markers of bone
metabolism in stall vs. pasture-reared horses. Equine Exercise Phys Equine Vet J 30, 601-604.
Creatinine for animal species
Reference: Sierra RI, Specker BL, Jimenez F, Cruz C, Pedraza-Chaverri J (1997) Biochemical bone markers,
bone mineral content, and bone mineral density in rats with experimental nephrotic syndrome.
Ren Fail19, 409-424.
PTH in Dogs
Serum / Plasma: 15 – 150 pg/ml
PTH in Cats
Serum / Plasma: 3.3 – 22.5 pg/ml
PTH in Horses
Serum / Plasma: 20 – 120 pg/ml mean 56 pg/ml

Cross reactivity - different species
Produkt
Product
Produit
Zellkultur
Cell culture
Culture cellulaire
Human
Human
Homme
Elefant
Elephant
Eléphant
Eichhörnchen
Squirrel
Ecureuil
Huhn
Chicken
Poulet
Hund
Dog
Chien
Kaninchen
Rabbit
Lapin
Katze
Cat
Chat
Cynomolgus Makake
Cynomolgus Macaque
Macaque
Maus
Mouse
Souris
Meerschweinchen
Guinea pig
Cobaye
Pavian
Baboon
Babouin
Pferd
Horse
Cheval
Rind
Bovine
Bovin
Schwein
Porcine
Porc
Ratte
Rat
Rat
Rhesus Affe
Rhesus macaque
Singe Rhésus
KNOCHEN METABOLISMUS / BONE METABOLISM / MÉTABOLISME DE L'OS
Truthahn
Turkey
Dinde
Schaf
Sheep
Mouton
Schimpanse
Chimpanzee
Chimpanzé
BAP X X X X X X N X X X X N X X X
Cathepsin K X
CICP/PICP X X N X X N N X N X N
Ziege
Goat
Chèvre
Hamster
Hamser
Hamster
Alle Spezies
All species
Toutes espèces
Creatinine X
DKK-1 X X X
Helical Peptide X X X X X X X X X X X X X X X X
NTX Serum (X) X X X X X X X
NTX Urine X X X X X X X
Osteocalcin Intact X X N X X N X X X X N X X
Osteocalcin Mouse X X X
Osteocalcin
N-MID (1-43/49) X X
Osteocalcin Rat X X X
Osteopontin
Human X X
Osteopontin
Mouse X X
Osteopontin
Rat X X
Osteoprotegerin
(OPG) Human X X N N N X N N N X N N
Osteoprotegerin
(OPG) Human X X X
Pyridinoline (Pyd)
Serum X X X X X X X X X X X
Pyrilinks
(Pyd + Dpd) X X X X X X X X X X X X
Pyrilinks D (Dpd) X X X X X N X X X X X X X X X X
Sclerostin TECO X X N N
sRankl Human
High Sensitive X X X
Total Dpd X X X X X X X X X X X X X X X X
TRAP5b Human X X N N N N N N N N N
TRAP5b Rat X X
KNORPEL METABOLISMUS / CARTILAGE METABOLISM / MÉTABOLISME DU CARTILAGE
C1–2C X X X (X) X (X) X X (X) X (X)
C2C, Serum/Urine X X X X (X) X X (X) X X (X)
COMP X X
CP II/PIICP X X X (X) X (X) X X X X (X)
CS-846 X X X X (X) X X X X X (X)
Hyaluronic Acid
TECO X X X X X X
23

24
Cross reactivity - different species
Produkt
Product
Produit
Zellkultur
Cell culture
Culture cellulaire
Human
Human
Homme
sGAG X
Elefant
Elephant
Eléphant
Eichhörnchen
Squirrel
Ecureuil
Huhn
Chicken
Poulet
Hund
Dog
Chien
Kaninchen
Rabbit
Lapin
Katze
Cat
Chat
Cynomolgus Makake
Cynomolgus Macaque
Macaque
Maus
Mouse
Souris
Meerschweinchen
Guinea pig
Cobaye
Pavian
Baboon
Babouin
Pferd
Horse
Cheval
Rind
Bovine
Bovin
Schwein
Porcine
Porc
Ratte
Rat
Rat
Rhesus Affe
Rhesus macaque
Singe Rhésus
KNORPEL METABOLISMUS / CARTILAGE METABOLISM / MÉTABOLISME DU CARTILAGE
YKL-40 X X X X X
Chemerin X X
hs-CRP X
Myeloperoxidase
(MPO)
X
Progranulin X
ENTZÜNDUNG / INFLAMMATORY / INFLAMMATION
YKL-40 X X X X X
Calcitonin Human X
Truthahn
Turkey
Dinde
KALZIUM METABOLISMUS / CALCIUM METABOLISM / MÉTABOLISME DU CALCIUM
Calcitonin Rat X X X
Fetuin A Human X X
Fetuin A Rat X N X
FGF-23 Intact X X N N
Schaf
Sheep
Mouton
Schimpanse
Chimpanzee
Chimpanzé
FGF-23 Intact
(Kainos) X X X
FGF-23 (C-Term)
2nd Generation X X N N
FGF-23 Mouse
(C-Term) X X X
PTH 1-34 Anti-
Human Antibody X X
PTH 1-34 Human
High Sensitive X X
PTH 1-84 Bioactive
Human X X X X X X X
PTH 1-84 Bioactive
Rat X N X N X
PTH 1-84 Bovine X X (X) (X) (X) X (X) (X) (X) (X)
PTH 1-84 Intact
Dog X X
PTH 1-84 Intact
Human
X
PTH 1-84 Intact
Mouse X N X N X
PTH 1-84 Intact
Rat X N (X) X N X
PTH 1-84 Porcine X X X (X) X X (X)
PTH C-Terminal
Human X X
PTH C-Terminal Rat X X
PTH Horse X X X X X
PTH Rat X X (X) X X X X X X
25 OH Vitamin D
direct X X
Ziege
Goat
Chèvre
Hamster
Hamser
Hamster
Alle Spezies
All species
Toutes espèces

Cross reactivity - different species
Produkt
Product
Produit
Zellkultur
Cell culture
Culture cellulaire
Human
Human
Homme
Myostatin X
BMP 7 X
Adiponectin Human
TECO X X
Adiponectin
Mouse
Adiponectin
Rat
Chemerin X X
Fetuin A Human X X
Elefant
Elephant
Eléphant
Eichhörnchen
Squirrel
Ecureuil
Huhn
Chicken
Poulet
Hund
Dog
Chien
Kaninchen
Rabbit
Lapin
Katze
Cat
Chat
Cynomolgus Makake
Cynomolgus Macaque
Macaque
Maus
Mouse
Souris
Meerschweinchen
Guinea pig
Cobaye
Pavian
Baboon
Babouin
Pferd
Horse
Cheval
Rind
Bovine
Bovin
Schwein
Porcine
Porc
Ratte
Rat
Rat
Rhesus Affe
Rhesus macaque
Singe Rhésus
MUSKEL – SKELETT / MUSCLE – SKELETON / MUSCLE - SQUELETTE
DIABETES & ADIPOSITAS / DIABETES & OBESITY / DIABÈTE & OBÉSITÉ
X
X
Truthahn
Turkey
Dinde
Schaf
Sheep
Mouton
Schimpanse
Chimpanzee
Chimpanzé
Ziege
Goat
Chèvre
Hamster
Hamser
Hamster
Alle Spezies
All species
Toutes espèces
Ghrelin X X X X X X X X X X X X X
GLP-1 Active
(7-36) Human X X X X
GLP-1 Active
(7-36) Mouse / Rat X X
GLP-1 Total X
Intact ProInsulin
TECO X X (X) (X) (X)
Leptin Human
TECO X X
Leptin Mouse/Rat X X X
Resistin X X N
YKL-40 X X X X X
LEBERERKRANKUNG / LIVER DISEASE / MALADIES DU FOIE
Hyaluronic Acid
TECO X X X X X X
M30-Apoptosense
Chronic Liver Disease
X
hGH / High Sensitive X X
IGFBP-1 X X
WACHSTUMSSTOFFWECHSEL / GROWTH METABOLISM / MÉTABOLISME DE LA CROISSANCE
IGFBP-2 X X X X X X
IGFBP-2 Mouse / Rat X X X X X
ALS (Acid Labile
Subunit)
X
hGH X N
IGFBP-3 X X
IGFBP-3
Functional X X
IGFBP-3
Mouse/Rat X X
IGF-I (BP blocked) X X X X X X X X X X X X X
IGF-I Mouse/Rat X X
IGF-II X X
IGF-II X X X
25

26
Cross reactivity - different species
Produkt
Product
Produit
Zellkultur
Cell culture
Culture cellulaire
Human
Human
Homme
Elefant
Elephant
Eléphant
Eichhörnchen
Squirrel
Ecureuil
Huhn
Chicken
Poulet
Hund
Dog
Chien
Kaninchen
Rabbit
Lapin
Katze
Cat
Chat
Cynomolgus Makake
Cynomolgus Macaque
Macaque
Maus
Mouse
Souris
Meerschweinchen
Guinea pig
Cobaye
Pavian
Baboon
Babouin
Pferd
Horse
Cheval
Rind
Bovine
Bovin
Schwein
Porcine
Porc
Ratte
Rat
Rat
Rhesus Affe
Rhesus macaque
Singe Rhésus
Truthahn
Turkey
Dinde
Schaf
Sheep
Mouton
Schimpanse
Chimpanzee
Chimpanzé
KARDIOVASKULÄRE MARKER / CARDIOVASCULAR MARKER / MARQUEUR CARDIOVASCULAIRE
Big Endothelin X X N N N
BNP Fragment X X N N N N N N N N N N N N N
Endothelin X X X X N X X X
NT-proANP X X X X
NT-proCNP X X X X X X X X X
OXIDATIVE STRESS MARKER / OXIDATIVE STRESS MARKER / MARQUEUR DU STRESS OXYDATIF
oLAB X N N N N N N N N N N N N N
Oxidized LDL X
OxyStat X
EZ4U X
CELL PROLIFERATION & CYTOTOXICITY ASSAY
APOPTOSE / APOPTOSIS / APOPTOSE
M30-
Apoptosense X X X X X X
M30-CytoDeath X X X X X X
M65-EpiDeath X X X A* X X A* X X
ANDERE PARAMETER / OTHER PARAMETERS / AUTRES PARAMÈTRES
ACTH X X X
Erythropoetin X
Fecal Calprotectin X
Prekallikrein Activator
Assay (PKA)
X
TPMT X
TSH Receptor Antibody
2 Gen. TECO
X
Ziege
Goat
Chèvre
Hamster
Hamser
Hamster
Alle Spezies
All species
Toutes espèces

Cross reactivity - different species
Produkt
Product
Produit
Zellkultur
Cell culture
Culture cellulaire
Human
Human
Homme
AH50 Eq X
Elefant
Elephant
Eléphant
Eichhörnchen
Squirrel
Ecureuil
Huhn
Chicken
Poulet
Hund
Dog
Chien
Kaninchen
Rabbit
Lapin
Katze
Cat
Chat
Cynomolgus Makake
Cynomolgus Macaque
Macaque
Maus
Mouse
Souris
Meerschweinchen
Guinea pig
Cobaye
A* = Humane Xenotransplantate / human xenograft / Hétérogreffe humaine
N = keine Kreuzreaktion / no cross reactivity / pas de réaction croisée
(x) = schwache Kreuzreaktion / low cross reactivity / faible réaction croisée
= Nicht getestet / not tested / pas testé
x = Kreuzreaktion / cross reactivity / réaction croisée
Pavian
Baboon
Babouin
Pferd
Horse
Cheval
Rind
Bovine
Bovin
Schwein
Porcine
Porc
Ratte
Rat
Rat
Rhesus Affe
Rhesus macaque
Singe Rhésus
KOMPLEMENT TEST / COMPLEMENT TEST / TEST DE COMPLÉMENT>
Bb Plus X X X X
C1-Inhibitor X
C3a Plus X X X X
C4d X X X X
C5a X
CH50 Eq X X
CIC-C1q X
CIC-C3d (Raji-
Cell-Replacement)
X
iC3b X
SC5b-9 Plus X X X X
Truthahn
Turkey
Dinde
Schaf
Sheep
Mouton
Schimpanse
Chimpanzee
Chimpanzé
Ziege
Goat
Chèvre
Hamster
Hamser
Hamster
Alle Spezies
All species
Toutes espèces
27

The Specialist for Biochemical Markers
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