Faculty of Biosciences, Division of Biochemistry
Faculty of Medicine
University of Helsinki
Riitta K Tähtelä
Utility of Type I Collagen-Derived Markers as Reflectors of Bone Turnover
in Different Clinical Situations
Academic Dissertation
To be presented, with the permission of the Faculty of Biosciences of the University of
Helsinki, for public criticism in the small Auditorium of Haartman Institute, Haartmaninkatu 3,
on May 25th, 2004, at 12 noon.
Helsinki, Finland
2004
Supervised by
Docent Matti Välimäki, MD
Division of Endocrinology, Department of Medicine
University of Helsinki
Docent Sirkka-Liisa Karonen
Department of Clinical Chemistry
University of Helsinki
Reviewed by
Professor Kalervo Väänänen, MD
Institute of Biomedicine, Department of Anatomy
University of Turku
Docent Jorma Salmi, MD
Department of Endocrinology
University of Tampere
Opponent
Dr Eugene McCloskey, MD
Metabolic Bone Unit
University of Sheffield
ISBN 952-91-7137-4 (nid.)
ISBN 952-10-1821-6 (PDF)
Helsinki 2004
Yliopistopaino
1
Contents
Original publications ..................................................................................................................4
Abbreviations...............................................................................................................................5
1
2
Introduction.........................................................................................................................6
Review of the literature.......................................................................................................8
2.1
2.1.1
2.1.2
2.1.3
2.2
Bone composition.................................................................................................................... 8
Osteoblasts.....................................................................................................................................8
Osteoclasts.....................................................................................................................................8
Osteocytes and lining cells.............................................................................................................9
Bone biochemistry .................................................................................................................. 9
2.2.1
Type I collagen ..............................................................................................................................9
2.2.2
Non-collagen proteins..................................................................................................................11
2.2.2.1
Proteoglycans .....................................................................................................................11
2.2.2.2
Bone glycoproteins.............................................................................................................12
2.2.2.3
Bone RGD proteins ............................................................................................................12
2.2.2.4
Bone gla-proteins ...............................................................................................................13
2.2.3
Growth factors and cytokines.......................................................................................................14
2.2.3.1
Growth factors....................................................................................................................14
2.2.3.2
Cytokines ...........................................................................................................................15
2.2.3.3
Arachidonic acid metabolites .............................................................................................16
2.2.4
Bone proteolytic enzymes............................................................................................................16
2.3
Bone remodeling ................................................................................................................... 17
2.3.1
Regulation of bone remodeling....................................................................................................18
2.3.1.1
PTH....................................................................................................................................18
2.3.1.2
1,25-(OH)2D.......................................................................................................................19
2.3.1.3
Calcitonin ...........................................................................................................................19
2.3.1.4
Other than calciotrophic systemic hormones ......................................................................19
2.3.2
Proposed model for the regulation of bone remodeling................................................................20
2.3.3
Disorders of bone remodeling......................................................................................................22
2.3.3.1
Osteoporosis and other benign bone disorders....................................................................22
2.3.3.2
Malignant bone disorders ...................................................................................................24
2.3.3.3
Treatment of bone remodeling disorders ............................................................................25
2.4
Markers of bone turnover ................................................................................................... 26
2.4.1
Markers reflecting bone formation...............................................................................................26
2.4.1.1
Bone alkaline phosphatase..................................................................................................26
2.4.1.2
Osteocalcin.........................................................................................................................27
2.4.1.3
Type I procollagen propeptides ..........................................................................................28
2.4.2
Markers reflecting bone resorption ..............................................................................................29
2.4.2.1
Type I collagen degradation products .................................................................................30
2.4.2.2
Tartrate resistant acid phosphatase .....................................................................................33
2.4.3
Markers of bone turnover in different clinical situations..............................................................33
3
Aim of the study ................................................................................................................35
4
Materials and methods .....................................................................................................36
4.1
4.1.1
4.1.2
4.1.3
4.1.4
4.1.5
4.1.6
4.1.7
Subjects and study designs .................................................................................................. 36
Study I .........................................................................................................................................36
Study II........................................................................................................................................36
Study III.......................................................................................................................................36
Study IV ......................................................................................................................................37
Study V........................................................................................................................................37
Study VI ......................................................................................................................................37
Study VII .....................................................................................................................................37
2
4.1.8
4.2
Study VIII....................................................................................................................................38
Methods ................................................................................................................................. 40
4.2.1
Assay for carboxy-terminal propeptide of type I procollagen (PICP)...........................................40
4.2.2
Assay for amino-terminal propeptide of type I procollagen (PINP) .............................................40
4.2.3
Assay for carboxy-terminal telopeptide of type I collagen ...........................................................40
4.2.4
Assays for urinary pyridinolines ..................................................................................................41
4.2.4.1
Pyridinoline (Pyr) enzyme immunoassay ...........................................................................41
4.2.4.2
Free deoxypyridinoline (DPD) enzyme immunoassay........................................................41
4.2.4.3
Free deoxypyridinoline (DPD) automated chemiluminescent immunoassay ......................42
4.2.5
Assays for urinary amino-terminal telopeptides of type I collagen (NTX)...................................42
4.2.5.1
NTX enzyme immunoassay................................................................................................42
4.2.5.2
NTX automated chemiluminescent immunoassay ..............................................................43
4.2.6
Other bone marker measurements................................................................................................43
4.2.7
Other laboratory methods ............................................................................................................44
4.2.8
BMD measurements ....................................................................................................................44
4.2.9
Assessment of assay performance................................................................................................45
4.2.10
Assessment of factors effecting results....................................................................................45
5
4.3
Samples .................................................................................................................................. 46
4.4
Statistics................................................................................................................................. 46
Results................................................................................................................................47
5.1
5.1.1
5.1.2
5.2
5.2.1
5.2.2
5.2.3
5.2.4
5.3
5.3.1
5.3.2
Performance characteristics .........................................................................................................47
Correlation of the assays ..............................................................................................................48
Factors effecting the assay results ...................................................................................... 50
Effect of hemolysis ......................................................................................................................50
Effect of sample storage...............................................................................................................51
Timing of the sample ...................................................................................................................51
The least significant change of the markers .................................................................................52
Type I collagen markers in healthy subjects, study I ...................................................... 52
S-PINP and S-PICP .....................................................................................................................52
Type I collagen-derived resorption markers.................................................................................53
5.4
Type I collagen markers during inhaled corticosteroid treatment in children with
asthma, study II .................................................................................................................... 55
5.5
Thyroid hormone effects on the type I collagen markers, study III .............................. 58
5.6
Type I collagen markers in metastatic bone disease ........................................................ 60
5.6.1
5.6.2
5.7
5.7.1
5.7.2
5.7.3
6
Performance of the assays ................................................................................................... 47
S-ICTP, S-PICP and S-PINP as markers of metastatic bone disease in breast cancer, study IV ...60
PINP and PICP in aggressive breast cancer, study V ...................................................................61
Type I collagen markers in postmenopausal osteoporosis .............................................. 63
Effect of smoking on transdermal estrogen replacement therapy in postmenopausal women, study
VI ................................................................................................................................................63
Effect of short-term clodronate treatment on bone turnover, study VII ........................................65
Comparison of S-TRACP 5b with S-PINP and U-NTX as the reflector of the efficacy of oral
clodronate treatment in postmenopausal women with vertebral osteopenia, study VIII ...............67
Discussion .........................................................................................................................70
6.1
Assay performance and factors effecting the results ....................................................... 70
6.2
Markers in healthy subjects ................................................................................................ 72
6.3
Markers in different disease states..................................................................................... 73
6.3.1
6.3.2
6.3.3
6.3.4
In children with asthma, on and off inhaled corticosteroid treatment ...........................................73
Markers in patients with prior thyroid cancer, on and off thyroxine treatment .............................74
Markers in bone disease due to malignancy .................................................................................75
Effect of smoking on markers ......................................................................................................76
3
6.4
Markers in the follow-up of antiresorptive treatment of postmenopausal osteoporosis .
.............................................................................................................................................. 77
6.4.1
6.4.2
6.4.3
Estrogen replacement therapy ....................................................................................................... 77
Short-term clodronate treatment.................................................................................................... 78
S-TRACP 5b in comparison with U-NTX and S-PINP as a reflector of treatment efficacy with
clodronate ...................................................................................................................................... 79
7
Conclusion .......................................................................................................................82
8
Summary ..........................................................................................................................84
9
Acknowledgments ............................................................................................................86
10
References....................................................................................................................88
4
Original publications
I.
Tähtelä R, Turpeinen M, Sorva R, Karonen SL. The aminoterminal propeptide of type
I procollagen: evaluation of a commercial radioimmunoassay kit and values in healthy
subjects. Clin Biochem 1997; 30:35-40. Erratum in: Clin Biochem 1997; 30:241.
II.
Sorva R, Tähtelä R, Turpeinen M, Juntunen-Backman K, Haahtela T, Risteli L, Risteli
J, Sorva A. Changes in bone markers in children with asthma during inhaled
budesonide and nedocromil treatments. Acta Paediatr 1996; 85:1176-80.
III.
Toivonen J, Tähtelä R, Laitinen K, Risteli J, Välimäki MJ. Markers of bone turnover
in patients with differentiated thyroid cancer with and following withdrawal of
thyroxine suppressive therapy. Eur J Endocrinol 1998; 138:667-73.
IV.
Tähtelä R, Thölix E. Serum concentrations of type I collagen carboxyterminal
telopeptide (ICTP) and type I procollagen carboxy-and aminoterminal propeptides
(PICP, PINP) as markers of metastatic bone disease in breast cancer. Anticancer Res
1996; 16:2289-93.
V.
Jukkola A, Tähtelä R, Thölix E, Vuorinen K, Blanco G, Risteli L, Risteli J.
Aggressive breast cancer leads to discrepant serum levels of the type I procollagen
propeptides PINP and PICP. Cancer Res 1997; 57:5517-20.
VI.
Sairanen S, Tähtelä R, Laitinen K, Löyttyniemi E, Välimäki MJ. Effects of short-term
treatment with clodronate on parameters of bone metabolism and their diurnal
variation. Calcif Tissue Int 1997; 60:160-3.
VII.
Välimäki MJ, Laitinen KA, Tähtelä RK, Hirvonen EJ, Risteli JP. The effects of
transdermal estrogen therapy on bone mass and turnover in early postmenopausal
smokers: a prospective, controlled study.Am J Obstet Gynecol. 2003;189:1213-20.
VIII.
Tähtelä R, Seppänen J, Laitinen K, Katajamäki A, Risteli J, Välimäki MJ. Serum
tartrate resistant acid phosphatase 5b in monitoring bisphosphonate treatment with
clodronate: a comparison to urinary N-terminal telopeptide of type I collagen and
serum type I procollagen aminoterminal propeptide. Submitted.
5
Abbreviations
AUC
area under curve
MGP
matrix gla protein
BCE
bone collagen equivalent
MMP
matrix metalloproteinase
BMD
bone mineral density
NTX
cross-linked amino-terminal
telopeptide of type I collagen
BMP
bone morphogenetic protein
OC
osteocalcin
BMU
basic multicellular unit
1,25(OH)2D
calcitriol, dihydroxyvitamin D
Bone ALP
bone specific alkaline phosphatase
OP
osteogenic protein
BRU
bone remodeling unit
OPG
ostoprotegerin
BSP
bone sialoprotein
PDGF
platelet derived growth factor
CIA
chemiluminescent immunoassay
PG
prostaglandin
Crea
creatinine
PICP
carboxy-terminal propeptide of type I
procollagen
CS
chondroitin sulfate
PINP
amino-terminal propeptide of type I
procollagen
CSF
colony stimulating factor
PTH
parathormone
CT
calcitonin
Pyr
pyridinoline
CTX
carboxy-terminal telopeptide of type I
collagen
R
receptor
25-OH-D
25-hydroxy vitamin D
RANK
receptor activator of NF-κB
DPD
deoxypyridinoline
RANKL
receptor activator of NF-κB ligand
E
estrogen
RGD
arginine-glysine-aspartic acid
ECIA
electrochemiluminescent immunoassay
RIA
radioimmunoassay
EIA
enzymeimmunoassay
RNA
ribonucleic acid
ELISA
enzyme linked immunosorbent assay
ROC
receiver operator characteristic
ERT
estrogen replacement therapy
RXR
retinoic acid receptor
FGF
fibroblast growth factor
SD
standard deviation
GC
glucocorticoid
T3
triiodothyronine
GPI
glycosyl-phosphatidyl inositol
T4
thyroxine
HOP
hydroxyproline
TGF
transforming growth factor
HRP
horse radish peroxidase
TIMP
tissue inhibitor of metalloproteinase
HRT
hormone replacement therapy
TNF
tumor necrosis factor
ICTP
cross-linked carboxy-terminal
telopeptide of type I collagen
TRACP
tartrate resistant acid phosphatase
IGF
insulin-like growth factor
TSH
thyroid stimulating hormone
IGFBP
IGF binding protein
TSP
thrombospondin
IL
interleukin
VEGF
vascular endothelial growth factor
LSC
least significant change
VDR
vitamin D receptor
MAP
mitogen activated protein
6
1 Introduction
Bone is a metabolically active tissue being throughout life continuously remodeled.1 During
growth bone mass is accumulated until bone modeling ends with epiphyseal closure. After
that bone remodeling takes place, old bone being removed by osteoclastic resorption and
replaced with new bone by osteoblastic bone formation. These two processes are regulated by
systemic hormones and several systemic or paracrine growth factors and cytokines, and they
are tightly coupled. When the regulation of remodeling detoriates, the processes are no longer
coupled and as a result a disease state develops, more often a state where bone resorption
exceeds bone formation, and bone mass is thus lost.
As early as 1920’s alkaline phosphatase activity was found to be elevated in bone diseases,2
and for decades it was the only laboratory parameter reflecting bone formation.
Electrophoretic and chromatographic methods enabled the assessment of bone specific
isoform of the enzyme, at least semiquantitavely.3, 4. Hydroxyproline has been used until
recently as a resorption marker.5, 6 This amino acid is formed during type I collagen
breakdown, but it can also originate from other tissues and even from the diet.
The increasing knowledge of bone matrix components, the characterization of the bone
specific peptide, osteocalcin,7 and the development of assay techniques, especially sensitive
immunoassays, led to the description of a radioimmunoassay method for measuring serum
osteocalcin concentration in 1980.8 In this assay bovine osteocalcin was used as an antigen to
raise the polyclonal antibody of the assay, later monoclonal antibodies were raised against
both bovine and human osteocalcin, and different immunoreactive forms of osteocalcin were
characterized.9 The early osteocalcin assays measured the intact molecule and to a differing
extent several of its breakdown products. For almost ten years osteocalcin was the only
marker believed to reflect bone formation, and it is still widely used as a marker of bone
formation. Numerous methods have been described and are commercially available, the more
recent ones using human osteocalcin as antigen, monoclonal antibodies raised against specific
fragments of the molecule or against the intact molecule, and immunometric assay
principle.10, 11, 12.
During the last ten years several biochemical markers of both bone formation and resorption
have been introduced. Most of these markers are derived from type I collagen, the most
abundant component of bone organic matrix. Ten years after the first assay for osteocalcin
was published a radioimmunoassay method was described for the carboxy-terminal
propeptide of type I procollagen, PICP.13 This large protein, rather than a peptide, is liberated
into the circulation during type I collagen formation, and thus its serum concentration reflects
bone formation rate. Several methods for measuring the extension peptide from the aminoterminal end of the type I procollagen, PINP, have also been described.14, 15, 16 These assays
give somewhat discordant results, and their clinical value has not been fully elucidated.
Immunoassays for measuring specifically bone isoform of alkaline phosphatase have also
been introduced. One of these assays measures the mass concentration of the enzyme,17 the
other assay measures the activity after immunoextraction of the isoform.18 Both of these
assays crossreact to some extent with the liver isoform of alkaline phosphatase, the other
isoform of the enzyme normally found in the circulation.
7
Markers of bone resorption are almost solely type I collagen breakdown products. In addition
to hydroxyproline measurement, in late 80’s a high performance liquid chromatographic
method was described for quantitating urinary excretion of pyridinium crosslinks, present in
mature collagen.19 Several immunoassays have later been introduced to measure different
type I collagen breakdown products, either their excretion to urine or their concentrations in
serum. The first immunoassay for measuring type I collagen breakdown product was
described in early 90’s for the measurement of serum concentration of the carboxy-terminal
telopeptide of type I collagen (ICTP).20 This antigen contains the pyridinium crosslink, and
attached to it, two alpha-1 peptide chains from the non-helical telopeptide region of one
collagen molecule and one alpha-1 or -2 peptide chain from the helical region of the adjacent
collagen molecule. The molecular weight of the antigen is approximately 10 kDa. Assays for
measuring urinary excretion of smaller breakdown products of type I collagen were
introduced somewhat later, first an ELISA method which measured free pyridinoline and
deoxypyridinoline crosslinks and small peptides containing these crosslinks (Pyr),21 and
shortly after an ELISA method measuring urinary free deoxypyridinolines, the more bone
specific of the two crosslinks (DPD).22 ELISA methods for measuring urinary excretion of
two crosslink peptides, cross-linked amino-terminal telopeptides of type I collagen (NTX)23
and for a peptide from the crosslink region of the carboxy-terminal telopeptide of type I
collagen (CTX),24 were also introduced, and serum assays for both of these telopeptides have
become available recently.25, 26
Tartrate resistant acid phosphatase is produced by the osteoclast, and the osteoclast specific
isoenzyme has been suggested to be used as a marker reflecting osteoclast activity. Most
methods published so far have been, however, disappointing. Only recently an
immunoextraction method for the osteoclast isoform of the enzyme was introduced.27
The markers of bone turnover have been widely used in research and clinical trials, and a
huge and continuously increasing number of articles have been published, dealing with the
use of the markers in diagnosis of metabolic and metastatic bone diseases, in the follow-up of
the response to treatment of these disorders, as well as their use as prognostic factors for both
benign and malignant bone disorders.28, 29, 30, 31, 32 It has been shown that markers of bone
turnover decrease as response to efficient treatment after one to six months on treatment
whereas the change in bone mineral density (BMD) can be seen after one to two years after
the treatment has been started. There is also evidence that high bone turnover correlates with
increased fracture risk. The results have, however, been conflicting, and the use of the
markers in normal clinical practice has not been widely accepted.
The aim of the present study was to evaluate some commercial methods for type I collagenderived markers as to their performance, and to evaluate the value of these marker
measurements in different clinical situations – in healthy persons, in children with asthma and
on corticosteroid treatment, in patients with previous differentiated thyroid carcinoma on and
off thyroxine treatment, in metastatic bone disease, and in the follow-up of treatment response
in postmenopausal osteoporosis. The effect of smoking on the markers in postmenopausal
women was also studied.
8
2 Review of the literature
2.1 Bone composition
Bone is composed of mineral and organic matrix. Two thirds of bone dry weight is mineral,
mostly hydroxyapatite. Other minerals, like magnesium, carbonate and sodium, are absorbed
on the hydroxyapatite crystals. The major part of the body calcium (99 %), of the phosphate
(80 %) and of the other minerals, mentioned above, are stored in the skeleton, from where
they are released when needed. The major component of bone organic matrix is type I
collagen, comprising about 90 % of it. In addition to collagen, bone organic matrix contains a
wide variety of non-collagenous proteins. The most abundant of these proteins are osteocalcin
and bone sialoprotein. In addition, proteins involved in cell adhesion are produced by
osteoblasts. Bone matrix contains several growth factors and cytokines, needed for bone cell
differentiation, and synthesized either by the cells in the bone marrow or by osteoblasts. Cells
take up about two percent of the bone organic matrix.33
2.1.1 Osteoblasts
The bone forming cells, cuboidal osteoblasts are responsible for the formation of the bone
extracellular matrix components, and they have a major role in regulating the mineralization
of the osteoid, the newly formed unmineralized bone matrix. Osteoblasts also produce factors
essential for osteoclast differentiation.34 Osteoblasts originate from multipotent mesenchymal
stem cells, which in addition give rise to adipocytes, myocytes, fibroblasts and cartilage
cells.35 Osteoblasts contain a large oval nucleus, well-developed endoplasmic reticulum and
Golgi apparatus. They are clearly polarized, several cells producing simultaneously on basal
aspect the unmineralized organic matrix. Most osteoblast products are similar to fibroblast
products, due to their similar origin, but the relative abundance and the combined production
are typical to the differentiated cell type.33 Only two osteoblast specific products have been
identified to date, one being a transcription factor36 and the other one osteocalcin.37 Typically
osteoblasts contain bone specific isoform of alkaline phosphatase, anchored on to the outer
side of their cell membrane.38
2.1.2 Osteoclasts
Large multinuclear osteoclasts, the bone resorbing cells, cleave bone matrix by their
proteolytic enzymes in acidic environment, provided by osteoclastic enzyme carbonic
anhydrase II, which produces the protons, and vacuolar ATPase, which pumps the protons to
the extracellular space.39, 40 Osteoclasts originate from hematopoietic mononuclear progenitor
cells, which differentiate and fuse to cells of several unique features.41, 42 Typically
osteoclasts contain several nuclei, the number of which in man is normally under ten but can
be as high as 100 in pathological conditions. In addition, osteoclasts contain lysosomes and
several mitochondria. An iron-containing 35 kDa enzyme tartrate-resistant acid phosphatase
(TRACP), is expressed in osteoclasts early during their development, and has for long been
used as their biomarker.43, 44 Unique to osteoclasts is the ruffled border, a specific area of the
membrane, which comprises of many cytoplasmic extensions, facing the surface of the bone
to be resorbed and allowing close contact to the resorption area. The ruffled border is
surrounded by a clear zone, an organelle free cytoplasmic area containing contractile proteins
like actin, and involved in anchoring the osteoclast to the bone surface undergoing
9
resorption.45 Osteoclast membrane also has several integrins, which can bind to RGD
(arginine-glycine-aspartic acid) sequences of the adhesion proteins in the bone matrix, the
most important one of them being the ανβ3 integrin.46, 47, 48 In addition to resorbing bone,
osteoclasts remove the bone matrix degradation products via endocytosis along the ruffled
border, transfer the degradation products via transcytosis to their basolateral membrane, and
release them to the extracellular space.49, 50
2.1.3 Osteocytes and lining cells
In addition to bone forming and resorbing cells, stellar-shaped osteocytes can be found
imbedded to the bone matrix and flattened lining cells on bone surfaces. Both of these cells
form from mature osteoblasts. Osteocytes are connected to each others and to other bone cells
by gap junctions.51 They function in sensoring mechanical stress and signaling it to other cells
in bone by generating signaling molecules such as nitric oxide and prostaglandins. 52, 53 When
bone forming is completed lining cells are formed from osteoblasts, covering the bone surface
during the quiescence phase. They have a role in starting the remodeling cycle.54
2.2 Bone biochemistry
Osteoblast lineage cells produce the extracellular matrix of bone, which is a unique storage of
various growth factors, in addition to the components which give bone its structure and
strength.
2.2.1 Type I collagen
Collagens are extracellular matrix proteins, which are of crucial importance for the normal
structure and function of vertebrate connective tissues. To date some twenty different
collagens are known. Typical to collagen structure is the repeat triplet amino acid sequence
glycine-X-Y, where X and Y stand often for proline or hydroxyproline. This repetitive
sequence is the basis of collagen structure, a stable semirigid triple helical molecule of three
collagenous polypeptide chains.55, 56
Type I collagen, one of the interstitial fibrillar collagens, is the major organic component of
bone matrix, comprising about 90 percent of it. Although not specific to bone, type I collagen
of bone has specific features, not found in type I collagen of other tissues. Type I collagen is a
heterodimer of two alpha 1(I) and one alpha 2(I) chains, and in addition to the large helical
middle part the molecule has short non-helical telopeptide regions at each end of the
molecule. Osteoblasts start to synthesize type I preproalphachains in an early phase of
osteoblast differentiation.57, 58 COL1A1 gene on chromosome 17 and COL1A2 gene on
chromosome 7 code for alpha 1(I) and alpha 2 (I) preprocollagen chains, respectively. The
preprochains pass to the rough endoplasmic reticulum, where the leader sequence at the
amino-terminal end is cleaved off, and the proalpha chains are post-translationally modified
by several enzymes.59 Certain prolyl and lysyl residues are hydroxylated, some hydroxylysyl
residues are glycosylated to form galactosyl or glucosyl-galactosyl hydroxylysyl residues. Nlinked glycosylation occurs in the carboxy-terminal non-collagenous area and phosporylation
of serine residues in the N-terminal extension peptide area. These post-translational
modifications of type I collagen are tissue specific. The three chains are then assembled
together, starting from the carboxy-terminal end of the chains by interchain disulfide bond
formation. The modification of the procollagen chains goes on during the assembly of the
chains. Type I procollagen molecule, which is about 1.5 times the size of type I collagen, has
large extension peptides at both ends of the molecule. The carboxy-terminal propeptide
10
(PICP) is globular in structure; the amino-terminal propeptide (PINP) is partly helical, partly
globular. After the procollagen molecule has been assembled it is secreted into the
extracellular space via the Golgi complex.57 The propeptide moieties are then cleaved off
from the molecule by specific endoproteinases. The C-terminal propeptide (PICP) is removed
first by a specific endopeptidase (bone morphogenetic protein 1, BMP-1),60 after which the
molecule is layered onto the collagen fibrils as type I pN-collagen. The amino-terminal
propeptide is then cleaved off after some delay by another specific endopeptidase.61
The type I collagen molecules, approximately 360 kDa in molecular weight, 300 nm long and
1.5 nm in diameter, are layered on the surface of another fibril in a longitudinally staggered
quarterly array, forming collagen fibers. Collagen fibers are then stabilized by crosslink
formation.62, 63, 64 The first step is the deamination of the ε-amino groups of specific
telopeptide lysine or hydroxylysine residues to corresponding aldehyde derivatives, catalyzed
by the enzyme lysyl oxidase, a copper-dependent enzyme acting only upon certain telopeptide
lysine residues. After the first enzymatic step in crosslinking immature divalent ketoimine
crosslinks are formed by condensation of the aldehyde with a helical lysine or hydroxylysine.
Part of the divalent crosslink undergoes maturation to a trivalent pyridinium or pyrrole
crosslink, which occurs by further reaction of the ketoimine with a telopeptide aldehyde. The
nature of the crosslinks is determined by the post-translational modifications of the alpha
chains, especially the hydroxylation of specific lysine residues. In bone the level of lysyl
hydroxylation is relatively low as compared to that in non-mineralized tissues. Hydroxylysyl
pyridinoline is the most abundant crosslink in a variety of tissues, but not in bone, where lysyl
pyridinoline predominates, being almost specific for calcified tissue.65 There is also evidence
that some of the mature trivalent crosslinks are pyrrole cross-links, formed from the reaction
of a telopeptidyl lysine aldehyde and an immature crosslink hydroxylysino-5ketonorleucine.66, 67, 68 These pyrrole crosslinks probably form between three adjacent
collagen molecules whereas the pyridinium crosslinks connect two collagen molecules
together.63, 69
Collagen crosslinking is of utmost importance to the biomechanical properties of bone, to the
strength of bone,70 and to the mineralization of bone.71 The decrease in the number of both
mature trivalent and immature divalent crosslinks reduces bone strength and decreases its
mineral density.72 In addition to enzymatic crosslinking, formation of nonenzymatic linkages
by glycation and accumulation of glycation end products can take place with ageing of
collagen,73 affecting the properties of bone.
11
Figure 1: Type I procollagen / collagen molecule and formation of fibrils according to ref. 61, with
permission.
2.2.2 Non-collagen proteins
About ten percent of bone organic matrix is taken up by several non-collagenous proteins
such as proteoglycans, glycoproteins, arginine-glycine-aspartic acid-sequence (RGD)
containing attachment proteins and gammacarboxy glutamic acid (gla)-containing proteins.61
These proteins are produced by the osteoblasts at different stages of bone formation and
matrix mineralization, and they have specific functions during bone remodeling processes,
though some of these activities still remain to be elucidated.
2.2.2.1 Proteoglycans
Proteoglycans take up about ten percent of the non-collagen proteins in the bone matrix. At
sites where bone formation is about to take place a large chondroitin sulfate proteoglycan is
found. This protein with an apparent molecular weight of one million Da has a core protein of
approximately 390 kDa and chondroitin sulfate (CS) sidechains of 40 kDa attached to it. Its
proposed function is to capture space for bone formation. In a later phase of bone formation
this large proteoglycan degrades and is replaced by decorin and biglycan in the mineralized
matrix. Decorin with an apparent molecular weight of about 130 kDa has a core protein of 46
kDa and one CS sidechain attached to it, while biglycan (MW about 270 kDa) with a similar
size core protein has two glycosaminglycan side chains. Both of these proteins have 24 amino
acid leucine rich repeats (10 in decorin, 12 in biglycan), also found in other connective tissue
proteins, and believed to function in sequestering transforming growth factor beta (TGF-β)
and modulating its activity.74
These two proteoglycans are differently located in bone. Decorin location coincides that of
collagen, and its function is proposed to be in regulating fibril diameter.75 Both decorin and
biglycan are produced by osteoblasts during bone formation, but only biglycan expression has
been located in osteocytes.
12
Some cell surface-associated proteoglycans like heparan sulfate proteoglycan, syndecan, and
another proteoglycan, betaglycan, have been shown to have a role in mediating responses to
growth factors e.g. fibroblast growth factors (FGFs) and TGFs in the developing bone.76, 77
Bone cell cultures have been shown to synthesize hyaluronan, an unsulfated
glycosaminoglycan, which might have a role in bone morphogenesis.61
2.2.2.2 Bone glycoproteins
Alkaline phosphatase is a ubiquitously expressed enzyme, having at least four genes coding
for it in different tissues.38 The bone enzyme (Bone ALP) is one isoform of the
bone/liver/kidney or tissue non-specific enzyme, being differently glycosylated in different
tissues and even within one tissue. In bone two differently glycosylated isoforms have been
found.78 Alkaline phosphatase is a dimeric ectoenzyme with a molecular weight of
approximately 130 kDa, and it is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. For eighty years it has been known to be involved in bone
mineralization, and it is considered to be a hallmark of osteoblast lineage cells.79 Bone ALP is
expressed by osteoblasts, starting in early mineralization phase of bone development,80 and it
is known to be involved in matrix mineralization, though the exact mechanism is as yet
unresolved. It is found in high concentrations in extracellular matrix vesicles, where it is
proposed to provide sufficient amounts of phosphate for the mineralization to proceed by
hydrolyzing it from proteins or pyrophosphates.81 Some of the enzyme is released from the
membrane and liberated into the circulation, where, in addition to the two isoforms of Bone
ALP, also a minor form of the isoform containing the GPI anchor is found.78
Osteonectin (or SPARC or B-40) is a glycated and phosphorylated protein with a molecular
weight of approximately 45 kDa, expressed by osteoblasts somewhat later than Bone ALP.80
In addition to calcified tissues it has been located in other sites e.g. parietal endoderm, some
basement membranes and platelets. It is an acidic and cysteine rich protein, capable of
binding calcium.61 Its exact function is not known, but according to in vitro studies a role in
regulation of cell proliferation, cell-matrix interactions, angiogenesis and metalloproteinase
expression has been proposed.82
2.2.2.3 Bone RGD proteins
Bone matrix contains several cell attachment proteins, which have in common the RGD
(arginine-glysine-aspartic acid) amino acid sequence, responsible for mediating the
attachment of these proteins to cell surface integrins.61 The interaction of cells with the
extracellular matrix is essential for their proliferation, migration and differentiation.
Thrombospondins (TSPs) are complex proteins composed of three identical subunits with a
molecular weight of 150 kDa.83 In addition to the RGD sequence they have several domains
capable of binding matrix proteins, and the molecule has also a calcium binding sequence.
TSP is found primarily in basement membranes. In bone it is produced by osteoblasts and
located in the osteoid, where one of its functions is to activate TGF-β.84
Fibronectin is another large ubiquitous RGD protein (MW 250 kDa).61 Fibronectin is
composed of two unidentical subunits, both having nine domains for different functions – for
binding different matrix proteins, DNA and cell surfaces independently of RGD - in addition
to the RGD sequence. Fibronectin is either synthesized locally in bone during early phase of
osteogenesis or brought to bone by vasculature. Vitronectin or S-protein of serum is a
monomer of molecular weight of 70 kDa.61 It is synthesized primarily in the liver, and low
13
amounts are found in calcified tissues. Vitronectin, too, can bind several matrix and serum
proteins.
Osteopontin is a cell adhesion protein with a core protein of approximately 32 kDa.61 The
protein is posttranslationally modified to a differing extent, both phosphorylated at serine
residues and glycosylated by both N- and O-linkages. It was first isolated from bone, but has
since been found in other tissues, too. It can be produced by both osteoblasts and osteoclasts,
and it is synthesized during different phases of bone remodeling. Its RGD sequence is situated
in the mid region of the molecule, and it binds with great affinity the ανβ3 integrin, and it also
binds calcium. It has been shown to function as an inhibitor of mineralization and as a
mediator of cell-matrix and matrix-matrix/mineral adhesion.85 Osteopontin is typically found
in cell- and matrix-matrix interfacial structures.
Bone sialoprotein (BSP) is an RGD containing protein with a molecular weight of
approximately 75 kDa.61 It is highly glycosylated, about half of its molecular weight comes
from carbohydrate, and 12 percent of the carbohydrate is sialic acid. One third of the serine
residues of BSP are phosphorylated, and the protein can also be sulphated at tyrosine
residues. In addition to the RGD sequence the molecule has other sequences capable of
binding cells at the tyrosine-rich domains of the amino- and carboxy termini of the
molecule.86 BSP is specific to mineralized tissues, and it has been shown to be expressed in
advanced breast cancer, in atherosclerotic plaques and in trophoblasts. In bone it is mainly
produced by osteoblasts during the mineralization phase of bone formation, but also
osteoclasts have been shown to have mRNA for BSP. BSP makes about 15 % of noncollagenous organic matrix of bone.
Another sialic acid containing protein in bone is bone acid glycoprotein-75, an acidic
glycosylated and phosphorylated protein with 30 % sequence homology with osteopontin in
the amino-terminus of the molecule. It binds calcium, and there is some evidence that it
inhibits bone resorption.
2.2.2.4 Bone gla-proteins
Osteocalcin (OC) or bone gla-protein (gammacarboxy glutamic acid) is a 49 amino acid
polypeptide with a molecular weight of 5700 Da.8 It makes about 15 % of the noncollagenous proteins in bone matrix. The molecule, which is synthesized under the
stimulation of vitamin D as pro-osteocalcin, contains three glutamic acid residues, which are
posttranslationally carboxylated in an enzymatic vitamin K dependent reaction. The
propeptide is cleaved off simultaneously with secretion to the extra cellular matrix, where OC
binds via calcium to hydroxyapatite. This causes a conformational change in the molecule,
which becomes partly α-helical in nature, and enhances the accumulation of OC in the bone
matrix. OC is considered to be one of the two bone specific proteins,37 and it is produced
mainly by osteoblasts during the late mineralization phase, but some OC is also expressed in
osteocytes.87 OC function is still unknown, but roles in mineralization and regulation of bone
resorption have been suggested. Recent evidence indicates that osteoblasts express an
osteocalcin receptor, and OC may be a signaling molecule for osteoblasts to stop bone
formation.88
Matrix gla protein (MGP) is another gla-protein in bone matrix.61 It is larger than OC, its
molecular weight being approximately 15 kDa, and it contains five gla residues. It is
synthesized in an earlier phase of bone formation than OC, and its function has not been
established.
14
2.2.3 Growth factors and cytokines
Bone matrix contains a large variety of growth factors and cytokines, polypeptides with
molecular weights varying from 7.5 kDa to 30 kDa. They are produced mainly by the
osteoblasts, to some extent by osteoclasts and cells in the bone marrow, and they have
autocrine or paracrine effects on bone cells. These molecules are essential for the
morphogenesis and development of bone, as well as the regulation of bone (re)modeling. In
addition, arachidonic acid metabolites such as prostaglandins are produced by bone cells. All
these factors exert their effect by binding to their specific receptors, which starts a signaling
cascade by activating specific kinases to phosphorylate specific transcription factors.
2.2.3.1 Growth factors
Several members of the transforming growth factor-β (TGF-β) superfamily, such as the three
isoforms of TGF-β and many bone morphogenetic proteins (BMPs) or osteogenic proteins
(OPs), are expressed in bone at different stages of development.89, 90 All of these factors are
dimeric secreted proteins, synthesized as larger proproteins and cleaved at a consensus ArgX-X-Arg site to yield mature dimeric molecules.91 They all have a cluster of seven conserved
cysteine residues, a cysteine knot, at the C-terminal region of the molecule.92, 93 They exert
their biological function by binding to two types (I and II) of serine/threonine kinase
receptors, of which type II transphosphorylates the type I receptor, and thus initiates the
intracellular signaling by phosporylating transcription factors, Smads.93 According to current
knowledge several BMPs (BMP-1, -2, -6, -7/OP-1) can induce the differentiation of
mesenchymal cells to osteogenic cells, and also regulate osteoblasts in expression of bone
matrix proteins.94, 95, 96 BMPs can also regulate osteoclastogenesis and bone resorption, either
through osteoblasts or, according to recent evidence, directly via their receptors on
osteoclasts.97, 98 TGF-βs have multifunctional effects on osteoblasts: they both promote and
inhibit proliferation, and they stimulate growth factor and bone matrix component production
under certain conditions whereas in other conditions they inhibit it.99 The activity of TGF-βs
is regulated to a great part by TSP1.84
Insulin-like growth factors (IGF) I and II are both produced by bone cells and found in the
bone matrix,100 as are at least five of the seven known IGF binding proteins, IGFBPs,101, 102,
103
and the binding protein proteases.104 Both IGFs stimulate osteoblastic cell proliferation
and differentiation, and they are important mediators of the effects of several systemic
hormones such as glucocorticoids, estrogen and growth hormone, as well as of mechanical
load on bone remodeling, IGF-I mediating the effects of systemic hormones and IGF-II that
of mechanical load.105, 106, 107 IGFs act through binding to either of the two types of receptors,
IGF-1R or IGF-2R, and their activity is modulated by the binding proteins and the respective
proteases. Most of the binding proteins inhibit the binding of the IGFs to their receptors, but
IGFBP-3 and -5 enhance their activities.108 The regulation of bone remodeling by the IGF
system may also vary depending on the skeletal site.109
Fibroblast growth factors are a family of polypeptides that have a crucial role in the
regulation of cell proliferation, migration and differentiation in several tissues, including
bone. To date twenty members of the family are known.110, 111, 112 FGFs act through high
affinity cell surface receptors, which possess tyrosine kinase activity, FGFRs 1-4, and low
affinity receptors, heparan sulfate proteoglycans.76, 110, 113 In bone osteoblast lineage cells both
produce FGFs and respond to them. The effects of FGF-1 and 2 on osteoblasts have been
shown to depend on the stage of the cells: in early maturation stage FGFs increase
proliferation, in the later developmental stages they inhibit differentiation, as judged by
15
decreased expression of osteoblast markers such as type I collagen, alkaline phosphatase and
osteocalcin.114, 115, 116
Platelet derived growth factor (PDGF) is a dimeric polypeptide. PDGF isoform AA has been
shown to be expressed in bone, where it participates in the regulation of osteoblast
proliferation and, probably through osteoblasts, of bone resorption.117 Vascular endothelial
growth factor (VEGF) is a 44 kDa protein essential for angiogenesis. VEGF is expressed also
in osteoblasts, and its receptors VEGFR-1/Flt-1 and VEGFR-2/KDR/Flk-1, which belong to
the receptor tyrosine kinases of PDGFR family, are found in both osteoblasts and
osteoclasts.118, 119 In bone VEGF has its common effect on angiogenesis, but it has also been
shown to have a role in osteoclast recruitment and attachment as well as in osteoblast
differentiation.
2.2.3.2 Cytokines
Bone cells or cells in the bone marrow express several cytokines, which participate in the
regulation of bone remodeling mostly as response to the effects of other remodeling
regulating factors.
Interleukin-1α and 1β are 17.5 kDa proteins, both potent activators of bone resorption.120
They exert their effect by binding to their 80 kDa or 60 kDa receptors, IL-1Rs.121 IL-1
receptor antagonist (IL-1ra) binds to the same receptor, opposing the effects of IL-1.122 IL-1s
stimulate osteoclast formation from their precursors as well as their differentiation.
Interleukin-6 is a 26 kDa glycoprotein, which is produced by several cell types including
osteoblasts.123 In bone it has been shown to stimulate bone resorption by mediating the effects
of calciotropic hormones such as parathyroid hormone (PTH), of estrogen depletion or of
other resorption stimulating factors.124, 125 It acts through binding to its receptor (IL-6R) to
start a gp130 receptor subunit mediated signaling pathway.126 Both IL-1 and IL-6 have been
shown to stimulate bone resorption by activating several bone degrading
matrixmetalloproteinases.127 Interleukin-11 is also a multifunctional cytokine that stimulates
bone resorption with a gp130 mediated pathway.128 It is activated by different stimulus than
IL-1 and –6, and it has been shown to enhance cell adhesion via integrins.129 In addition to
interleukins that stimulate bone resorption also those having an inhibitory effect on bone
resorption such as interleukins - 4 and -18 are found in bone.130, 131
During recent years two members belonging to tumor necrosis factor alpha (TNFα) ligand
and receptor families have been found to be expressed by osteoblastic cells. These factors
have been shown to be crucial regulators of bone remodeling.
Osteoprotegerin (OPG), also called osteoclastogenesis inhibitory factor (OCIF) and TNF
receptor-like molecule 1 (TR1), was isolated and cloned by several groups, and has been
found to be a molecule capable of inhibiting osteoclastogenesis.132, 133, 134, 135 OPG is a soluble
secreted molecule with four cysteine rich domains, responsible for the molecule’s biological
effect, and with two so called death domains. It can act as a 60 kDa monomer or as a
homodimer formed by a disulfide linkage.136 It acts as a decoy receptor to a transmembrane
TNF ligand family member, which was identified independently by several groups secondary
to its receptor in T cells as well as in osteoblastic / stromal cells.137, 138, 139, 140 The molecule
was named TNF-related activation-induced cytokine (TRANCE), osteoclastogenesis
differentiation factor (ODF) or osteoprotegerin ligand (OPGL) and RANKL (receptor
activator of NF-κB ligand) by different groups, and was proven to be an important factor for
the development and function of T cells, dendritic cells and osteoclasts. It is a 316 amino acid
16
residue transmembrane protein with a TNF-family homologous domain in the extracellular
part of the molecule. In bone it acts by activating its signaling receptor RANK (receptor
activator of NF-kB), a member of the TNF receptor family, expressed on the membrane of
osteoclasts and their precursor cells, which uses the c-Jun N-terminal kinase (JNK) signaling
pathway.141 RANK is a 625 amino acid residue transmembrane receptor with four cysteine
rich domains, typical to TNF receptor family, in the extracellular part of the domain.
In addition to the cytokines mentioned above yet another secreted factor produced by the
osteoblasts, the macrophage colony-stimulating factor (M-CSF or CSF-1), is essential for the
proliferation of osteoclast progenitor cells, and for the differentiation of these cells into
osteoclasts.142, 143
2.2.3.3 Arachidonic acid metabolites
Prostaglandins, in bone especially those of E series (PGE2 and PGE1), are arachidonic acid
metabolites which locally exert diverse effects on bone metabolism. They modulate osteoblast
proliferation and function as response to systemic hormones and mechanical strain mainly by
regulating the activity of cytokines and growth factors.144, 145, 146, 147 They are also powerful
regulators of bone resorption, either stimulating or inhibiting osteoclast differentiation and
function, mostly through factors produced by osteoblasts, but also through direct effect on
osteoclastic receptors.148, 149, 150 Prostaglandins exert their effect in bone mainly through one
of their four receptors, EP4, which is an adenylate cyclase activating receptor. In addition to
prostaglandins, also several lipoxygenase metabolites of arachidonic acid, leukotrienes, have
been shown to activate osteoclasts to resorb bone.151, 152
2.2.4 Bone proteolytic enzymes
Bone matrix degradation is essential for normal bone remodeling. Two types of proteolytic
enzymes are responsible for the matrix degradation in bone, lysosomal cathepsins and matrix
metalloproteinases (MMPs), which seem to exert their effects in a manner depending on the
stage of the remodeling cycle as well as on the type of the bone being remodeled.153, 154
According to recent evidence, cathepsin K is the enzyme mainly responsible for type I
collagen degradation in acidic pH, an early event of the resorption process. Cathepsin K
belongs to cysteine proteases, enzymes synthesized as proenzymes and activated at acidic
environment.155 Active cathepsin K is a 27 kDa protein expressed in osteoclasts at high
concentrations. It is capable of cleaving type I collagen at multiple sites, including several
sites at the helical region.156 This leads to the unwinding of the triple helix which makes the
molecule susceptible to further action of proteolytic enzymes, including matrix
metalloproteinase action later in the resorption process at neutral pH.153 Other cathepsins e.g.
B, H, L and S are also expressed in bone, but to a much lesser extent than cathepsin K.157 In
addition to degrading type I collagen, cysteine proteases are capable of degrading noncollagenous proteins of bone extra cellular matrix.153
Some twenty members of the matrix metalloproteinase family are known to date, some of
them secreted proteins, and some membrane bound proteins, all capable of degrading extra
cellular matrices at neutral pH.158 They have structural similarities, including a zinc-binding
active site. They all are synthesized as proenzymes, which are activated by the cleavage of an
80 amino acid residue amino-terminal peptide. Their activity is regulated by their specific
tissue inhibitors, TIMPs (TIMP-1, -2 and –3) with which they form inactive complexes. They
have different substrate specificities, and therefore their expression differs depending on the
17
tissue and its developmental stage. In bone the most important MMPs expressed in osteoclasts
seem to be MMP-9, a 92 kDa protein which is also called gelatinase B because of its
gelatinolytic activity, and MMP-14, a membrane-type I MMP, MTI-MMP.159, 160, 161 They are
also expressed in osteoblasts and osteocytes, where in addition several other MMPs are
detected. These MMPs act in the remodeling process prior to osteoclast attachment, in the
early stage of the remodeling cycle, and among them are MMP-2 or gelatinase A, a 72 kDa
protein with gelatinolytic activity, MMP-13, also called collagenase-3, and MMP-1 or
interstitial collagenase, which are capable of degrading fibrillar collagens.162, 163 MMP-3 or
stromelysin-1, capable of degrading non-collagenous proteins of bone matrix, is also
expressed in osteoblasts early in the remodeling cycle.
In addition to being able to degrade bone, matrix MMPs have an important role in effecting
the recruitment of the osteoclast progenitor cells to the future resorption sites, and preparing
the environment for osteoclast attachment.164, 165, 166 They have also been suggested to have a
role in the coupling of resorption and formation together with bone lining cells.54, 167 The
expression of the matrix metalloproteinases and their inhibitors in bone, as well as their
activity, is regulated by factors that regulate bone remodeling.168, 169
2.3 Bone remodeling
Bone remodeling takes place by teams of cells called basic multicellular units (BMUs) or
bone remodeling units (BRUs) or osteons. The shapes of these units differ in the two different
types of bone. In cancellous or trabecular bone, which comprises about 15 % of the skeleton,
BRUs are saucer shaped so-called Howship’s lacunae. In compact or cortical bone, which
forms the major part of the skeleton, the remodeling takes place in cylindrical structures
called Haversian units. Although trabecular bone comprises the minor part of the skeleton, it
is metabolically the more active type of bone, and about 25 % of trabecular bone is remodeled
yearly, while about 3 % of cortical bone is renewed.1, 170
The cells are differently organized in the different types of bone, but the remodeling sequence
is the same in both systems. The remodeling cycle starts with an activation phase, during
which the lining cells are withdrawn from the bone surface, and the bone matrix is prepared
for the attachment of osteoclast precursor cells. The precursor cells migrate to the resorption
site where they are activated to form multinuclear bone resorbing cells. Activation phase is
followed by a resorption phase during which the mineral component of bone is dissolved, and
the organic matrix degraded. The resorption phase is followed by reversal phase during which
apoptosis of osteoclasts and recruitment of preosteoblasts take place. In the following
formation phase preosteoblasts differentiate to mature osteoblasts, which fill the resorption
cavity with new unmineralized organic matrix, osteoid. Eventually the osteoid is mineralized,
and the BMU goes to a quiescent phase. In trabecular bone the time from the activation to the
completion of the resorption is normally about one month, and the remodeling cycle is
completed in about three months. In cortical bone the remodeling cycle is somewhat longer,
taking about four months. Figure 2 shows the bone remodeling cycle in trabecular bone.
18
quisence
osteoclast recruitment
resorption
reversal
formation, early
formation, late
mineralization
quisence
Figure 2: Schematic presentation of bone remodelling cycle in trabecular bone.
2.3.1 Regulation of bone remodeling
Bone remodeling is regulated by both systemic hormones and local factors, which were
discussed above. The most important systemic hormones, which regulate the remodeling
process are calciotropic hormones, parathormone (PTH) and the hormonally active metabolite
of vitamin D, 1,25-dihydroxyvitamin D or calcitriol (1,25-(OH)2D), and calcitonin (CT).
These hormones are responsible for maintenance of the body calcium homeostasis, and they
are under the negative feedback control of extracellular fluid calcium concentrations. PTH is
the most important of them as a regulator of calcium homeostasis. Other systemic hormones
having a great impact on bone metabolism are estrogens, glucocorticoids, thyroid hormones
and growth hormone.1, 171 Most of these hormones affect bone metabolism through their
receptors on the osteoblasts, and their effects are often mediated by locally produced
cytokines, growth factors or prostaglandins.
2.3.1.1 PTH
Parathormone is an 84 amino acid residue peptide secreted from the parathyroid glands as a
response to lowered extracellular calcium concentration, sensed by calcium sensing receptors
on the parathyroid glands.172 PTH corrects calcium levels rapidly by increasing calcium
reabsorption in the renal tubules, and more slowly by increasing the renal hydroxylation of
25-OH-vitamin D to its active metabolite, which enhances the intestinal absorption of
calcium, and by enhancing bone resorption, leading to the release of calcium from bone.
PTH can have both catabolic and anabolic effects I bone, depending on the way of
administration. Continuous effect leads to increased bone resorption, intermittent effect to
increased bone formation. The increase in bone mass due to PTH action is seen in cancellous
bone, perhaps at the expense of the cortical bone mass. PTH acts by binding to one of its two
receptors, both transmembrane G-protein linked receptors (PTHR-1 and PTHR-2), and PTH
effects in bone are mediated through several different signal transduction mechanisms.173, 174
As the receptors are found only in the osteoblasts, PTH effects bone resorption through
osteoblasts, by upregulating the expression of several osteoblastic factors, crucial for bone
resorption. Among these factors are IL-6, IL-11, M-CSF and RANKL, known to be essential
for osteoclast proliferation, differentiation and activity.124, 128, 140, 175
19
Parathyroid hormone related peptide (PTH-rP) is a peptide first discovered in association of
humoral hypercalcemia of malignancy. Its amino-terminal end is 70 % homologous with
PTH’s amino-terminus (13 amino acids), and it mimics PTH effects on calcium homeostasis
and bone resorption, doing that through PTHR-1.171 It is essential for normal fetal skeletal
development, and in adults it is expressed in the lactating breast and in the skin, in addition to
being expressed by several malignant neoplasms.
2.3.1.2 1,25-(OH)2D
1,25-dihydroxyvitamin D (I,25-(OH)2D) or calcitriol is the hormonally active metabolite of
vitamin D. It is formed by 1-alphahydroxylation of 25-hydroxyvitamin D (25-OH-D) in the
proximal renal tubules as response to lowered circulating phosphate concentration under the
stimulation of PTH. 25-OH-D is formed in the liver by the hydroxylation of cholecalciferol
(D3), obtained from the diet or formed in the skin from 7-hydroxycholesterol by ultraviolet
irradiation, or of ergocalciferol (D2), found in small amounts in vegetables.
1,25-(OH)2D has effects on calcium homeostasis, on bone resorption and on cell
differentiation, also in other tissues than bone. One of its important effects in bone is its effect
on the mineralization of the newly formed bone by supplying calcium and phosphate for it
through stimulating their intestinal absorption. It also increases renal calcium reabsorption.
1,25-(OH)2D is a potent bone resorbing agent. The way it acts on bone resorption has not
been fully elucidated, but it acts through osteoblasts, on which it has two different kinds of
effects. The rapid effect, mediated by a membrane bound receptor, causes a calcium influx
into the osteoblast, and augments PTH action, leading to increased bone resorption and
calcium release from bone.176 1,25-(OH)2D has been shown to upregulate the synthesis of
RANKL and downregulate the expression of OPG, the decoy receptor of RANKL, leading to
increased osteoclastogenesis.177 The slow effect of 1,25-(OH)2D on osteoblasts is an effect on
osteoblast differentiation, mediated through binding to the nuclear vitamin D receptor (VDR).
This causes heterodimerization of the receptor with retinoic acid receptor (RXR), and leads to
initiation of gene transcription.178 One of its slow effects is to upregulate OPG mRNA
expression.179 In the late phase of osteoblast differentiation calcitriol diminishes type I
collagen synthesis, and enhances osteocalcin and osteopontin synthesis. It also upregulates
VEGF expression in the osteoblasts, which in turn might stimulate the endothelial cells near
by to produce osteoblasts differentiating growth factors.180, 181 A role in coupling bone
resorption and formation for 1,25-(OH)2D has also been suggested.176
2.3.1.3 Calcitonin
Calcitonin (CT) is a peptide of 32 amino acid residues, secreted from the perifollicular C-cells
of the thyroid gland as response to increased circulating calcium concentration. It has a shortterm lowering effect of the extracellular calcium concentration by decreasing bone resorption
through a direct action on the osteoclastic CT receptors.171 It has been shown that CT effects
osteoclast activity by causing loss of ruffled border, and changing the structure of the clear
zone F-actin.
2.3.1.4 Other than calciotrophic systemic hormones
Several systemic hormones have an important impact on the regulation of bone remodeling.
Among these hormones are sex steroids, especially estrogens, thyroid hormones and
glucocorticoids, the effects of which on bone will be briefly referred.
20
Estrogens are important regulators of both skeletal growth and bone remodeling in both sexes.
During skeletal growth estrogens mainly act by stimulating pituitary growth hormone
secretion, whereas regulation of bone remodeling takes place via osteoblastic or, as recently
shown, osteoclastic estrogen receptors, ERα or ERβ.182, 183, 184 In addition to the nuclear
receptors, several co-regulators, transcription factors and response elements are involved in
estrogen action, and they vary in different target tissues. Estrogens have been shown to have a
rapid effect on target cells, similar to that of 1,25-(OH)2D, mediated through membrane
bound receptor or binding protein and leading to calcium flux and activation of MAP
(mitogen activated protein) kinase pathways. In bone estrogen inhibits bone resorption, to a
great part through osteoblastic receptors by down-regulating the expression of bone resorbing
cytokines IL-1β, IL-6 and TNF.185 Estrogen has also been shown to promote osteoclast
apoptosis, mediated probably by TGF-β,186 and to suppress RANKL- and M-CSF-induced
osteoclast differentiation from progenitor cells through a nuclear ER-dependent mechanism
which does not require mediation by osteoblastic/stromal cells,184 or by inducing OPG
synthesis in osteoblasts.187
Thyroid hormones, mainly the most potent one of them, triiodothyronine T3, are required for
normal skeletal development. They effect cell proliferation and differentiation through
nuclear thyroid hormone receptors, TRα1 and TRβ1, which are expressed in osteoblastic
cells. Similarly to other nuclear receptors, they interact by heterodimerization with other
nuclear receptors – retinoic acid receptor, vitamin D receptor - to regulate gene expression in
these cells.188, 189 In proliferating preosteoblasts T3 has been shown to downregulate
expression of genes typical for proliferating cells like c-fos/c-jun. Later it stimulates the
expression of mRNA for markers reflecting osteoblast differentiation such as osteocalcin and
alkaline phosphatase.190 There is also evidence that T3 might be involved in the
mineralization process of osteoid by stimulating membrane bound alkaline phosphatase
release from osteoblasts.191
Glukocorticoids have diverse effects on bone remodeling. In physiological concentrations
they stimulate osteoblast progenitors to proliferate, they effect the differentiation of
osteoblasts by stimulating the synthesis of osteoblastic proteins, e.g. alkaline phosphatase,
osteopontin and bone sialoprotein. Depending on the developmental stage of the osteoblast
glucocorticoids either stimulate or inhibit type I collagen synthesis, and they inhibit 1,25(OH)2D induced osteocalcin synthesis. They have also been shown to have a role in the
mineralization of bone extra cellular matrix.192, 193, 194, 195 They exert their effects through
specific receptors (GR) that belong to the same nuclear receptor superfamily as other steroid
hormone receptors, vitamin D and thyroid hormone receptors, and the effector molecules
needed for nuclear receptor function. Some of their effects in bone are mediated by
modulating the activity of paracrine factors, growth factors and cytokines.196, 197
2.3.2 Proposed model for the regulation of bone remodeling
The factors initiating bone resorption are not fully elucidated, but it is known that lining cells,
some growth factors such as VEGF, and matrix metalloproteinases have important roles in
preparing the bone surface, about to be resorbed, for osteoclast attachment, and in the
recruitment of osteoclast progenitor cells to the future resorption site. According to the
current knowledge, crucial factors for osteoclast proliferation and differentiation are the
recently found membrane bound member of tumor necrosis factor (TNF) ligand family, the
receptor activator of NF-κB ligand (RANKL) produced by the osteoblasts, and the secreted
macrophage-colony stimulating factor (M-CSF), also produced by the osteoblasts.198 Cell-cell
21
contact between the osteoblast and the hematopoietic osteoclast precursor cell is needed for
RANKL to activate its receptor NF-κB (RANK) on the osteoclast precursor cell / osteoclast
membrane. The activation of RANK leads to several signaling pathways leading to
differentiation and maturation of multinuclear, TRACP and CT receptor positive
osteoclasts.42 Osteoclastogenesis is regulated by systemic hormones and local factors, which
to a great extent regulate the expression of RANKL, M-CSF and RANK.131, 199 RANKL is
also regulated by osteoprotegerin (OPG), its decoy receptor produced by osteoblasts, which
binds RANKL and thus inhibits its binding to RANK. RANKL is also needed for the
activation of osteoclasts and for maintaining their activity. Downregulation of RANKL and
upregulation of OPG leads to osteoclast apoptosis.
Figure 3: Formation of actively resorbing osteoclast according to the ref. 198, with permission.
In the reversal phase of bone remodeling osteoblast precursor cells are recruited to the
resorption cavity, and osteoblastogenesis proceeds under the control of systemic hormones
and local factors. Osteoblasts synthesize the components of bone organic matrix, and the
formed osteoid is then mineralized. The mechanism of coupling bone resorption to bone
formation has not been elucidated, and several possible factors have been proposed, including
growth factors TGF-β and BMPs, 1,25-(OH)2D, MMPs and bone lining cells.
After bone organic matrix has been laid down by the osteoblasts, mineralization of the osteoid
takes place. In the mineralization process collagen molecules probably form the structural
background for the deposition of mineral. Matrix vesicles, formed from chondroblasts and
osteoblasts, contain high amounts of alkaline phosphatase and other enzymes needed for
initiation of mineralization process, and they act as nucleation sites for crystal formation. The
process is regulated by extra cellular matrix macromolecules such as proteoglycans.
As discussed above, several systemic hormones and locally produced growth factors,
cytokines and arachidonic acid metabolites regulate bone remodeling in a phase dependent
manner; some factors have diverse, even opposite effects on bone cell differentiation
depending on the phase of the remodeling cycle. Most of the regulating factors, also those
stimulating resorption, act through their receptors on the osteoblasts, by regulating the
expression of RANKL and OPG. In addition to systemic hormones and local factors,
mechanical strain is an important regulator of bone remodeling. Mechanical strain is sensed
by the osteocytes, which signal its effect to the other cells of bone through cap junction net
work by producing mediator molecules such as PGE2 and nitric oxide, and which finally
leads to increased bone formation. Table 1 summarizes some important factors that regulate
bone remodeling, and the expression of RANKL and OPG.
22
Table 1: Some systemic and local factors, which regulate bone remodeling and the expression of RANKL
and OPG.
Effect on
resorption
Effect on
formation
Effect on
RANKL
Effect on OPG
PTH, continuous
K
L
K
L
PTH, intermittent
L
K
NK
NK
1,25-(OH)2D, rapid
K
L
K
L
1,25-(OH)2D, slow
L
K
L
K
E2
L
K
L
K
IL-1
K
L
K
L
IL-6
K
L
K
L
IL-11
K
L
K
L
TNFα
K
L
K
L
PGE2
K L
L K
K
L
TGF-β
L K
K L
L
K
BMPs
L
K
L
K
IGFs
L
K
NK
NK
Factor
Systemic
hormones
Local
factors
NK = not known
2.3.3 Disorders of bone remodeling
2.3.3.1 Osteoporosis and other benign bone disorders
Osteoporosis is the most common of metabolic bone disorders. It is a state, where bone
resorption eventually exceeds bone formation, leading to reduced bone mass and detoriated
bone microarchitecture, and making bone susceptible to fractures. Definition of osteoporosis
at present is based on bone mineral density (BMD), most often measured by dual-energy Xray absorptiometry (DXA) at the lumbar spine or proximal femur. By definition, osteoporosis
in a Caucasian female is a state were BMD is more than 2.5SD (standard deviations) below
the young adult average BMD value. Osteopenia (low bone mass) is defined as a state where
BMD is under 1SD but not more than 2.5SD below the young adult average BMD.200
Postmenopausal osteoporosis (type I osteoporosis) is one type of primary osteoporosis, and
by far the most common form of osteoporosis. It is caused by estrogen depletion at
menopause, which causes activation of osteoclasts and increased bone resorption, and leads to
a deep reduction of BMD during the first menopausal years, and which goes on but lessens
23
during later years. Postmenopausal osteoporosis effects mainly trabecular bone, and the most
common type of fracture is vertebral. Type I osteoporosis can present also in males, but is
much more rare than in females. In age-related or senile osteoporosis (type II osteoporosis)
bone turnover is decreased, and bone formation impaired, and the effect is seen both in
cortical and trabecular bone. Senile osteoporosis presents normally in population over 75
years of age, and presents only somewhat more often in females than in males. Juvenile and
idiopathic osteoporosis are rare types of primary osteoporosis.201
Secondary osteoporosis can be caused by several disease states as well as by medications, and
also by some life style factors, such as alcohol abuse, tobacco smoking and low physical
activity, which all are risk factors for osteoporosis. Hormones that in physiological
concentrations are essential for normal bone remodeling can in non-physiological amounts
lead to osteoporosis. Glucocorticoids in excess, as in Cushing’s syndrome or on
glucocorticoid treatment, accelerate osteoclastic bone resorption, at least partly through
decreasing intestinal calcium absorption and causing secondary hyperparathyroidism. They
also reduce osteoblast function and thus bone matrix formation, leading to imbalanced
remodeling with excessive bone resorption, and they can cause osteocytes apoptosis. Some of
their effects are probably mediated by rapid non-genomic interaction with bone cells.202
Thyroid hormone excess – hyperthyroidism or high dose thyroxine substitution - causes
greatly increased bone turnover, which eventually leads to imbalance between resorption and
formation and bone loss.203 In both primary and secondary hyperparathyroidism, excess of
parathyroid hormone, bone turnover is increased. In primary disease resorption depths are
reduced and bone architecture is therefore preserved, while in secondary disease large
resorption depths cause detoriated bone structure.203
Osteogenesis imperfecta is an inherited disorder, caused by one of several known point
mutations in either of the two genes coding for alpha 1 and alpha 2 peptide chains of type I
collagen. These mutations lead either to decreased collagen synthesis or impaired collagen
function and consequently poor bone quality.204
Chronic liver disease is often associated with osteopenia or osteoporosis, partly due to
reduced osteoblast function, partly because of impaired vitamin D metabolism.203 Other
conditions involving malabsorption are often associated with osteopenia or osteoporosis.
Rickets in children and osteomalacia in adults are caused by vitamin D deficiency or by
deficient vitamin D effect. These disorders are characterized by impaired mineralization of
bone organic matrix and reduced strength of bone.201
Increased number and greatly increased activity of osteoclasts, often with tens of nuclei, are
characteristic to Paget’s disease of bone. Not only bone resorption but also bone formation is
greatly enhanced, but the bone formation is irregular and results in a mixture of woven and
lamellar bone.205
Smoking is one of the lifestyle factors, known to be risk factors for osteoporosis and fracture
by influencing calcium and bone metabolism in several ways. Smoking may be antiestrogenic
decreasing calcium absorption from the gut, it reduces the hepatic metabolism of estrogens, it
might modulate the activity of cytokines and their receptors, and it can also directly effect the
osteoblasts.206, 207, 208
Table 2 summarizes benign disorders of bone remodeling.
24
Table 2: Different types of osteoporosis / benign disorders of bone remodeling.
Postmenopausal
Senile
Primary
osteoporosis
Idiopathic
Juvenile
Cushing’s syndrome
Hypogonadism
Endocrine
etiology
Hyperthyroidism
Diabetes mellitus
Acromegaly
Hyperprolactinemia
Genetic
diseases
Osteogenesis imperfecta
Hypophosphatemia
Glucocorticoid therapy
Heparin therapy
Iatrogenic
Secondary
osteoporosis
Cancer chemotherapy
Thyroxin therapy
Gonadotropin releasing hormone agonists
Chronic obstructive pulmonary disease
Rheumatoid arthritis
Chronic liver disease
Malabsorption syndromes
Miscellaneous
etiology
Pregnancy related
Lactation
Malnutrition
Immobilization
Smoking
Alcohol abuse
Paget’s disease of bone
Rickets / osteomalacia
2.3.3.2 Malignant bone disorders
Several cancers are associated with metastatic bone disease. Bone metastases from breast
cancer, the most common female malignancy in the western world, are mostly of lytic nature.
Cancer cells that metastasize to bone produce PTHrP and cytokines (IL-1, IL-6, TNFα, PGE2
25
etc.), which interact with osteoblasts to stimulate RANKL production, and decrease OPG
production, and thus induce osteoclastic bone resorption. Bone cells on the other hand
produce cytokines and growth factors that stimulate cancer cell growth. This vicious cycle
leads to greatly accelerated bone resorption, often with only some concomitant increase in
bone formation, and to lytic bone lesions, which in advanced disease can cause
hypercalcemia.209, 210
In prostate cancer, which is most common male malignancy in the western world, the
metastases are mainly of sclerotic nature. Cancer cells in bone secrete growth factors (FGFs,
BMPs, endothelin-1, TGF-β), which accelerate bone formation around the tumor cell
deposits, without prior bone resorption.209 The accelerated bone formation is accompanied
with increased bone resorption, but to a lesser extent.211, 212 Calcium is deposited in sclerotic
metastases, and hypocalcemia is frequently seen in this type of metastatic bone disease with
resulting secondary hyperthyroidism, which further causes increased bone resorption.
Multiple myeloma is a plasma cell malignancy, which is frequently associated with
destructive bone lesions, caused by diffuse osteopenia or localized osteolysis. Myeloma cells
produce several factors such as IL-1 and IL-6, which are known to stimulate osteoclastic bone
resorption by stimulating RANKL production, and a similar vicious cycle as seen in lytic
metastases of solid tumors, is involved also in multiple myeloma. In multiple myeloma there
is usually no concomitant increase in bone formation, and hypercalcemia is often a
complication in advanced disease.209
2.3.3.3 Treatment of bone remodeling disorders
The ultimate aim of the treatment of bone remodeling disorders is to prevent fractures and
other skeletal complications. Bone related disorders are currently treated with agents which
reduce bone resorption.213, 214 Most commonly used treatment of postmenopausal osteoporosis
is estrogen replacement therapy (ERT), combined with progestin (HRT) treatment to prevent
unwanted side-effects on endometrium, and treatment with bisphosphonates and calcitonin.
These compounds are also used for prevention of postmenopausal osteoporosis. More
recently selective estrogen receptor modulators (SERMs) have been introduced to be used
both in prevention and treatment of postmenopausal osteoporosis. Calcium supplementation,
together with vitamin D treatment, has also been used especially in the elderly. Only very
recently treatment with intermittent parathyroid hormone has been approved to be used in the
treatment of severe osteoporosis. To date this is the only treatment to stimulate bone
formation. HRT and bisphosphonates have been shown to increase BMD, and to reduce
vertebral fracture risk to about half of the risk in untreated population. Raloxifene, the only
SERM so far approved for the prevention and treatment of osteoporosis, increases BMD less
than do estrogens or bisphosphonates, but its influence on fracture risk is similar. With
calcitonin BMD increments are smaller. The increase in BMD as response to PTH therapy is
more pronounced than with antiresorptive therapies.
Bisphosphonates are the most commonly used agents in the treatment of secondary
osteoporosis, as well as bone related disorders due to malignancy.215 Both nonaminobisphosphonates such as clodronate, and aminobisphosphonates, e.g. pamidronate and
zoledronate, have been shown to be effective in reducing skeletal complications due to lytic
bone metastases from breast cancer or bone lesions due to multiple myeloma. In addition,
clodronate has been shown to prevent the development of bone metastases in primary breast
cancer.216 In sclerotic bone metastases from prostate cancer bisphosphonates have been used
26
mostly in advanced disease for bone pain palliation, believed to be due to increased bone
resorption.
2.4 Markers of bone turnover
2.4.1 Markers reflecting bone formation
Markers, which reflect bone formation are proteins produced by the osteoblast, and liberated
into the circulation where their concentrations can be measured. To date total activity of
alkaline phosphatase or, more specifically, the activity or concentration of the bone-specific
isoform of the enzyme, the concentration of different immunoreactive forms of osteocalcin,
and the circulating concentrations of type I procollagen amino- and carboxy-terminal
propeptides have been used as reflectors of bone formation rate.
Table 3 summarizes the markers reflecting bone formation rate and the methods available to
measure them. In the following chapters some methods are referred in more detail.
Table 3: Biochemical markers reflecting bone formation.
Marker
Method
Bone-specific alkaline
phosphatase (Bone ALP)
HPLC
electrophoresis, all isoforms semiquantitavely determined after pretreatment
lectin precipitation and calculation, activity measured
IRMA or ELISA, mass concentration measured
immunoextraction with a monoclonal antibody, activity measurement
Osteocalcin (OC)
several RIAs, bovine OC as immunogen, intact OC and several fragments
measured
several RIAs and IRMAs, or methods with other than radioactive label, with
human OC as immunogen, measuring intact OC, its N-Mid-fragment or both or in
addition smaller fragments
Procollagen I carboxy-terminal
propeptide (PICP)
Procollagen I amino-terminal
propeptide (PINP)
RIA or ELISA
RIA measuring intact PINP
ELISA and automated ECIA measuring intact PINP and Col 1 fragment of PINP
2.4.1.1 Bone alkaline phosphatase
Bone alkaline phosphatase is coded by the tissue non-specific ALP gene, which is expressed
in bone, liver and kidney.38 The products of the tissue non-specific gene are
posttranslationally differently glycosylated in different tissues, or even in one tissue, leading
to several tissue specific isoforms of ALP. In bone two differently glycosylated isoforms are
found, and the proportion of these isoforms might change in different pathological
conditions.78 Mainly bone and liver isoforms are found in the circulation, in some persons
also small amounts of intestinal isoenzyme circulate. In childhood the bone isoform
predominates whereas in adults about half of the ALP activity comes from the bone, the other
half from the liver. During pregnancy heat stabile placental isoenzyme contributes part of the
ALP activity in the circulation. Certain tumors produce placental-like isoenzyme of ALP.
27
Several methods for determining the different circulating forms of ALP have been described.
Electrophoresis has been the most widely used method in clinical chemistry. Irrespective of
the medium used, electrophoresis has to be run for native sample and for differently
pretreated sample in order to get the different isoforms and isoenzymes separated and
quantitated. Heat denaturation in 56 oC for ten minutes inactivates almost all bone isoforms,
neuraminidase handling of the sample modifies the mobility of the other isoenzymes but the
intestinal one.217, 218, 219 A wheat germ lectin precipitation method was introduced in mid
80’s.220 In this method almost all bone isoform is precipitated with lectin. The activity of
Bone ALP is calculated from the total and the residual activity using a formula, which takes
into account that not all Bone ALP is precipitated. Two immunoassays have also been
developed for measuring Bone ALP. The immunometric assay uses two monoclonal
antibodies directed against different epitopes of the isoform, measures the mass concentration
of Bone ALP, and is available in two formats, either as an immunoradiometric (IRMA) or an
immunoenzymometric assay (IEMA).17, 221 In the ELISA assay Bone ALP is
immunoextracted from the sample using a specific monoclonal antibody coated on a
microtiter well.18 After washing the well the activity of Bone ALP is measured. Because the
specificity of the antibodies used in the immunoassays is based on the differential
glycosylation of the enzyme protein, both methods have some cross reactivity with the liver
isoform, and they do not necessarily recognize all bone isoforms formed in pathological
conditions.222
2.4.1.2 Osteocalcin
Osteocalcin is one of the two proteins known to date as being specific for bone and dentin,37
and the only one of these two whose concentration in circulation has been used as a reflector
of bone formation. In the circulation osteocalcin exists in several immunoreactive forms.9
About 30 % of immunoreactive osteocalcin is intact molecule, one third is the so called Nmid fragment. The N-Mid fragment forms via proteolytic cleavage of the intact molecule
between two Arg residues at positions 43 and 44.223 The rest of the circulating osteocalcin
consists of several smaller breakdown products. Degradation of OC takes place both in vivo
and in vitro.
The early immunoassays for osteocalcin in the 80’s used bovine osteocalcin as the antigen for
raising the assay antibodies and to be used as labeled competing antibody and standards.8, 224,
225
Bovine osteocalcin shows great homology with human osteocalcin, was not so prone to
degradation during the purification process, and the antibodies against bovine OC had a 100
% cross reactivity with the human OC. These early assays recognized different circulating
immunoreactive forms of OC to varying extents, and different assays gave greatly differing
results. The knowledge of the different immunoreactive forms of OC, introduction of
monoclonal antibodies against human OC, and immunometric techniques have made it
possible to measure certain molecular forms of OC, and methods for undercarboxylated OC
have also been described.226, 227, 228, 229, 230
Some of the newly formed OC is liberated directly into the circulation, where it can be
measured. The immunoreactive OC has been shown to correlate with histomorphometrical
measures of bone formation rate.231 However, as OC is incorporated into the bone
extracellular matrix, some of the circulating OC might originate from degrading bone, and the
circulating immunoreactive OC thus rather reflects bone turnover, not just bone formation.232,
233
Even to date, no standard preparation of OC exists, and the results obtained with tens of
commercially available or in-house OC assays are not comparable.10, 11, 12, 234,
28
Currently it is recommended that when possible, methods recognizing the N-Mid fragment
are used. Intact OC degrades rapidly in vitro, and therefore the serum must be quickly
separated from the red blood cells. Even in serum the intact OC loses its immunoreactivity in
hours, if not stored in low temperature (minus 70 oC or lower).
2.4.1.3 Type I procollagen propeptides
Type I collagen is synthesized by the osteoblasts, starting at an early phase of osteoblast
differentiation, as a large procollagen molecule consisting of two procollagen I alpha 1 and
one procollagen alpha 2 chains.57 The chains are post-translationally modified and assembled
to a trimeric procollagen molecule, which is secreted into the extracellular space, where large
extension peptides from each end of the molecule are cleaved off by specific
endopeptidases.59 The carboxy-terminal extension peptide (PICP) is cleaved off first, the
amino-terminal peptide (PINP) after some delay. The propeptides are liberated into the
circulation in an equimolar ratio, and their concentrations can be measured by specific
immunoassays. Although type I collagen is not specific to bone it is believed that the
circulating type I procollagen propeptides are derived mainly from bone due to higher
osseous turnover as compared to other tissues.
2.4.1.3.1 Carboxy-terminal propeptide of type I procollagen PICP
The carboxy-terminal propeptide of type I procollagen is composed of three equal size
peptide chains (MW approximately 33 kDa each), held together by disulphide bridges. It is
globular in shape, and it has been post-translationally glycosylated containing high-mannose
type oligosaccharides. In the circulation only one form of PICP exists, and it is cleared from
the circulation via liver endothelial cell mannose receptors.235
PICP can be measured with a radioimmunoassay (RIA)13 or an enzyme immunoassay
(ELISA). Serum PICP concentrations measured with the commercially available RIA have
been shown to correlate with bone formation rate as determined by histomorphometry and
calcium kinetics.236, 237, 238
2.4.1.3.2 Amino-terminal propeptide of type I procollagen PINP
The amino-terminal propeptide of type I procollagen is a partly helical, partly globular protein
with two equal size alpha 1(I) chains and one shorter part of alpha 2(I) chain, and a molecular
weight of approximately 35 kDa. The molecule is phosphorylated, and it is cleared from the
circulation via liver endothelial cell scavenger receptors.239 In the circulation at least two
molecular forms of PINP are found, the intact trimeric form and a smaller monomeric peptide
from the Col 1 part of the proalpha 1(I) chain of the procollagen molecule.
Several immunoassays have been developed for measuring the circulating PINP
concentration. In these assays polyclonal antibodies have been raised against synthetic
sequences of proalpha 1(I) chain,14, 240 against a proalpha 1(I) chain purified from human
amniotic fluid,16 or an antigen purified from cultured human osteosarcoma cells.15 Of these
assays the RIA with a synthetic peptide containing amino acids 1-12 from the amino-terminus
of human proalpha 1(I) chain as the antigen showed good correlation with other indices of
bone formation (OC, Bone ALP, PICP), whereas no correlation was found with the synthetic
peptide-based ELISA, and the values in healthy adults were 100-fold higher than those of
PICP. The ELISA method using the antigen purified from human amniotic fluid recognized
two circulating forms of PINP, the intact PINP and the small Col 1 fragment. The latter is
29
cleared from the circulation via kidneys, and thus its concentration might be elevated in renal
insufficiency. The RIA using the antigen purified from human osteosarcoma cells measures
only the circulating intact trimeric PINP.
2.4.2 Markers reflecting bone resorption
Markers of bone resorption are almost solely type I collagen degradation products, amino
acids, free or peptide-bound crosslinks formed during collagen degradation and liberated into
the circulation, from where they are cleared via kidneys.19 - 26 In addition to type I collagenderived markers, methods for measuring tartrate-resistant acid phosphatase activity or
concentration, or specifically the osteoclast specific isoenzyme 5b, have been described and
suggested to be used as reflectors of osteoclast activity.27 Also methods for bone sialoprotein,
BSP, have been described.241 Although an osteoblast product, BSP is incorporated into the
bone matrix, from where it is liberated during extra cellular matrix degradation, and thus its
circulating concentration is believed to reflect bone resorption.
Table 4 summarizes the markers and methods that can be used for measuring bone resorption,
which are referred in more detail in the following chapters.
Table 4: Biochemical markers reflecting bone resorption.
Marker
Method
Type I collagen-derived markers
Urinary excretion Total Pyr, total DPD
of pyridinoline
Free Pyr, free DPD
and
deoxypyridinoline
Free Pyr and DPD
(crosslinks)
Free DPD
Peptide-bound
crosslink
excretion
Serum
crosslinked
telopeptides of
type I collagen
HPLC after hydrolysis
HPLC without preceding hydrolysis
ELISA
ELISA, automated methods (CIA)
Crosslinked amino-terminal
telopeptides, NTX
ELISA, automated CIA
Crosslinked carboxyterminal telopeptides, CTX
ELISA for CTX-beta, RIA for CTX-alpha
Crosslinked amino-terminal
telopeptides, NTX
ELISA
Crosslinked carboxyterminal telopeptides, CTX
ELISA or automated ECIA for the beta-isomer
Carboxy-terminal
crosslinked telopeptide ICTP RIA
or CTX-MMP
Tartrate-resistant acid phosphatase TRACP in
serum
Several assays, which measure either activity or concentration
Osteoclastic tartrate-resistant acid phosphatase
TRACP 5b in serum
Immunoextraction and measurement of activity
Bone sialoprotein BSP in serum
RIA (not commercially available)
30
2.4.2.1 Type I collagen degradation products
Urinary excretion of hydroxyproline (HOP), an iminoacid formed from proline and a
prerequisite for the triple-helical conformation of collagen, has been used as an estimate of
bone resorption rate.5, 6 However, HOP is partly metabolized in the liver, and the excreted
HOP can originate from various sources: from diet as well as from bone or from other
proteins like the C1q component of the complement,242 or from newly formed collagen. Thus
only about 10% of the excreted HOP originates from degrading bone, making it an unspecific
marker of bone resorption. Therefore, during the last decade HOP measurement has been
replaced by more specific assays, measuring either urinary excretion or serum concentrations
of the type I collagen breakdown products, free crosslinks deoxypyridinoline (DPD, lysyl
pyridinoline) or pyridinoline (Pyr, hydroxylysyl pyridinoline) or crosslink peptides from the
amino- or carboxy-terminal telopeptide areas of type I collagen (ICTP=CTX-MMP, CTX,
NTX).19-26 These degradation products can originate only from mature collagen, and they are
not further metabolized. Hydroxypyridinium crosslinks of collagen form during the
extracellular maturation of fibrillar collagens like type I collagen.62-72 Both deoxypyridinoline
(DPD) and pyridinoline (Pyr) are trivalent crosslinks that form between two adjacent collagen
molecules, and are crucial for the mechanical properties and strength of collagen. DPD is
almost exclusively found in bone and dentin whereas Pyr is found also in cartilage, ligaments
and vessels. In addition to mature cross-links, immature divalent crosslinks are found in bone,
and their proportion might increase with aging as well as in some pathological processes such
as malignancies.243, 244 Figure 4 shows the origin of the different breakdown products of type I
collagen, measurable currently with immmunoassays or with chromatography.245
Figure 4:The origin of type I collagen breakdown products, used as markers of bone resorption. From ref.
245 by courtesy of Dr Simon Robins.
Originally pyridinoline excretion was measured using reverse phase HPLC with fluorescent
detection, either after acid hydrolysis (total crosslinks) or without hydrolysis (free crosslink
fraction),19 but less cumbersome immunoassays have largely replaced the chromatographic
method, at least in clinical routine. The most commonly used sample is a two-hour sample
after voiding the first morning urine, and the crosslink excretion is proportioned to creatinine
excretion.246 In addition to measuring crosslink excretion, methods for assessing serum
concentrations of the crosslink peptides have been developed.
31
2.4.2.1.1 Methods for urinary free crosslink compounds
An enzyme immunoassay (ELISA) for measuring pyridinoline excretion to urine was
introduced and became commercially available in early 90’s.21 Both Pyr and DPD, either free
or peptide-bound with a molecular weight less than 1000 Daltons, were measured with the
assay. Somewhat later a monoclonal antibody-based assay measuring only free DPD became
available in an ELISA format,22 and somewhat later as an automated chemiluminescent
immunoassay (CIA).247 In healthy adults a constant proportion of 40 – 50 % of the pyridinium
crosslinks was shown to be excreted in free, non-peptide-bound form, and therefore
measuring only free crosslinks was considered an adequate reflector of bone resorption
rate.248 However, the ratio of free to peptide-bound crosslinks might change in states of
altered rate of bone turnover,249 the proportion of excreted peptide-bound crosslinks
increasing with increasing turnover.
2.4.2.1.2 Methods for urinary peptide-bound crosslink compounds
2.4.2.1.2.1 Type I collagen cross-linked amino-terminal telopeptides (NTX)
Type I collagen amino-terminal telopeptides (NTX) are collagen breakdown products
containing trivalent pyridinoline or deoxypyridinoline crosslink structure and two peptide
chains from the amino-terminal telopeptide region of one collagen molecule connected to the
helical region of an adjacent collagen molecule.23 The crosslink at the N-terminal telopeptide
region forms predominantly between alpha 1(I) and alpha 2(I) or alpha 2(I) and alpha 2(I)
chains of collagen, a feature that is found only in type I collagen in bone. The antigen was
originally isolated from adolescent human urine, and monoclonal antibodies were raised
against it. An ELISA method was developed using an antibody that recognizes the aminoterminal region of the alpha 2-chain of type I collagen.23 The antibody did not react with the
linear peptide sequence, but needed to have the conformation of the crosslink peptide, and the
binding was not dependent on the nature of the cross-link. Later an automated
chemiluminescent immunoassay (CIA) was introduced. The automated assay uses the same
antibody as the original ELISA, but the antigen used as standard and competing antigen is a
synthetic crosslink peptide. The structure of the NTX antigen is shown in Figure 5.
Figure 5. The structure of the NTX antigen.
32
2.4.2.1.2.2 Crosslink peptide from the carboxy-terminal telopeptide region of type I
collagen (CTX)
The CTX antigen is an octapeptide (Glu-Lys-Ala-His-Asp-Gly-Gly-Arg) from the
crosslinking site of the alpha 1(I) chain of the type I collagen carboxy-terminal telopeptide
region. The aspartyl residue of the peptide can exist in two isomeric forms, the beta-form
resulting from aging of type I collagen.250, 251 The non-isomerized alpha-form predominates in
states of high turnover. A polyclonal antibody base enzyme immunoassay (ELISA) was
developed for measuring the urinary excretion of the beta-isomerized form of CTX
(CrossLaps). Only the linear peptide sequence is required for the immunoreactivity in this
assay. A monoclonal antibody-based radioimmunoassay (RIA) was later developed for
measuring the urinary excretion of the non-isomerized alpha-isomer of the peptide.252 An
increased alpha-CTX to beta-CTX ratio was shown in untreated Paget’s disease of bone with
accelerated bone turnover.253 The origin and structure of the CTX antigen is shown in
Figure 6.
Figure 6. The origin and structure of the 8 amino acid sequence of the CTX antigen from ref 251, with
permission.
2.4.2.1.3 Methods for serum type I collagen crosslinked compounds
2.4.2.1.3.1 Assay for the crosslinked carboxy-terminal telopeptide of type I collagen
(ICTP, CTX-MMP)
The first immunoassay for measuring type I collagen degradation product in serum was a
polyclonal antibody-based radioimmunoassay measuring a trivalent crosslink-containing
carboxy-terminal telopeptide of type I collagen (ICTP).20 The antibody binding requires the
trivalent crosslink, either DPD or Pyr, and the phenylalanine-rich domains of telopeptide
region of the two alpha 1 chains to be present. The antibody does not recognize divalently or
non-crosslinked peptides. Recently it has been shown that the antigen is destroyed by
cathepsin K, the cysteine proteinase mainly responsible for collagen degradation in acidic pH
during normal conditions.254 Cathepsin K degrades the bond between the phenylalanine-rich
domain and the cross-link, resulting in the loss of immunoreactivity. The action of matrix
metalloproteinases on bone collagen leads to the formation of ICTP antigen, which therefore
has been proposed to be called CTX-MMP.255
33
Figure 7: The structure of the ICTP or CTX-MMP antigen, from ref 254, with permission.
2.4.2.1.3.2 Serum assays for NTX and CTX-β
β
Recently serum assays for the carboxy- and amino-terminal telopeptides of type I collagen
have become available. The assay for serum NTX utilizes the same antibody as the assay for
urinary NTX, and the same assay design as the manual ELISA assay for U-NTX.25 Serum
CTX-β concentration is measured with a sandwich method where the same monoclonal
antibody is used as capturing and as detecting antibody.26 The antibody recognizes the same
peptide sequence as the antibody in the urinary CTX-β-assay, but a crosslink and two alpha 1
chains are needed for antibody binding. The assay is available in manual ELISA format and
as an automated electrochemiluminescent immunoassay (ECIA).256 The latter one uses a
synthetic antigen as the standard.
2.4.2.2 Tartrate resistant acid phosphatase
Human serum contains two isoforms of tartrate-resistant acid phosphatase (TRACP), 5a and
5b, which are products of the same gene and differ from each other in glycosylation, 5a
containing sialic acid, 5b not.27, 257 The isoform 5b has been shown to originate from
osteoclasts, and isoform 5a from other sources, most probably from macrophages. Several
assays have been developed for measuring either TRACP activity or its mass concentration,
but thus far they have been disappointing as reflectors of bone resorption. A novel
immunoassay measuring specifically TRACP 5b activity has recently been developed.27 In
this assay TRACP isoforms 5a and 5b are both first captured to the solid phase by a TRACPspecific monoclonal antibody, after which the bound TRACP activity is measured at pH 6.1,
at which 5b is highly active and 5a almost completely inactive.
2.4.3 Markers of bone turnover in different clinical situations
The value of markers of bone turnover has been evaluated in a large number of clinical
studies. Serum concentrations of the markers of bone formation S-OC, S-Bone ALP and SPICP have been shown to vary with age, being at their highest during puberty. 258, 259, 260 They
also have been shown to correlate with growth velocity in healthy children and in children
treated for growth hormone deficiency or for short stature without hormone deficiency.259, 261
The marker values before treatment did not correlate with the response to treatment, but the
height gain and the increase in markers showed good correlations, and early marker change
34
predicted later height gain. Urinary and serum type I collagen-derived resorption markers
show a similar age dependent pattern as do the formation markers.262, 263 Serum ICTP was
shown to be very high in neonates, increasing further somewhat up to one month of age, and
then declining from values close to 100 µg/l to values around 30 µg/l at one year of age.264
Markers have been evaluated in several disease states and during treatments, which are
known to effect bone metabolism. Glucocorticoids (GC) are frequently used to treat
inflammatory diseases such as rheumatoid arthritis (RA) and asthma. They are known to
cause osteoporosis which is characterized by decreased bone formation and unchanged or
increased bone resorption.202, 265 In RA itself there is evidence of unbalanced bone formation
and resorption, in favor of resorption: the type I collagen-derived resorption markers are
elevated in active disease and their levels correlate with the disease activity,266, 267 whereas the
formation markers have been reported to be either normal or lowered.268, 269 In newly
diagnosed asthmatic patients type I collagen-derived marker values have been similar to
values in control subjects, but serum osteocalcin has been shown to be lowered in pediatric
patients with asthma.270 Oral use of GCs as well as inhaled GCs, depending on the dose,
further decrease bone formation, shown by decreasing levels of osteocalcin and PICP both in
adults and children.271, 272, 273, 274 On the markers of bone resorption the effect of GCs is less
clear, and they have been shown either to increase, decrease or remain unchanged. On the
other hand, some studies have shown no changes in any markers of bone turnover during
treatment with inhaled GCs.275, 276 In one study an age-dependent response in S-ICTP was
shown, increasing in young patients but decreasing in older patients.274 The changes in bone
turnover markers are seen while on treatment, and the values return to baseline levels after
withdrawal of GCs.
Hyperthyroidism is characterized by accelerated bone turnover, due to a direct effect of
triiodothyronine on hormone receptors in the osteoblasts.188, 189 The accelerated turnover is
reflected as increased levels of markers of bone formation osteocalcin and Bone ALP, and as
increased excretion of type I collagen-derived resorption markers.277 In some studies,
increased levels of markers of bone formation and resorption have also been measured during
thyroxine treatment, indicative of increased bone turnover.278, 279 Whether this increased bone
turnover leads to increased bone loss is still controversary. In some studies significantly
reduced BMDs have been reported, 280, 281, 282 in some studies the detrimental effect has been
seen only in postmenopausal women,280 whereas in some studies the effect has been the same,
irrespective of menopausal status.281, 282
Bone metastases are frequently seen in context of solid tumors such as breast cancer and
prostate cancer, and lytic bone lesions are characteristic to multiple myeloma.209 Several
markers of bone resorption have been shown to be elevated in patients with bone metastases,
whether lytic or sclerotic.283, 284 The diagnostic value of especially the urinary markers of
bone resorption is, however, lessened due to their poor clinical specificity: they are often
elevated also in patients without bone involvement. Nevertheless, some bone markers are of
value as prognostic tools as well as in the follow-up of disease outcome. The prognostic value
of serum ICTP has been shown in several different solid tumors and in multiple myeloma,32,
285, 286, 287
and its concentration correlates with both the tumor burden in bone and bone
pain.288, 289 The value of ICTP in the follow-up of disease outcome has also been shown.290, 291
The clinical sensitivity of elevated values of bone formation markers Bone ALP and PICP,
and thus their value in diagnosis of metastatic bone disease, increases from lytic to mixed to
sclerotic metastases, with low sensitivities when the bone disease is of lytic nature, but with
sensitivities approaching 100% in prostate cancer patients with bone involvement.32, 283, 288, 289,
292
Osteocalcin is only seldom elevated, even in patients with blastic bone disease, and
35
lowered results have also been reported, irrespective of the type of metastases.288, 293 Some
studies have shown low osteocalcin values to correlate with worse outcome of patients with
multiple myeloma or with lytic lesions from solid tumors.286, 294, 295
Smoking is one of the lifestyle factors known to be a risk factor for osteoporosis. Its effect on
the markers of bone turnover have been studied in a few studies 296, 297, 298, 299 In these studies
osteocalcin or Bone ALP were measured as reflectors of bone formation, type I collagen
breakdown products were measured for bone resorption. These studies showed unbalanced
bone formation and resorption in smokers, either due to decreased bone formation297, 298or due
to increased bone resorption.296, 299 Osteocalcin was shown to be lowered in current smokers,
whereas Bone ALP was similar in smokers and non-smokers. Bone resorption was increased
or unchanged in smokers as compared with non-smokers. In two of the above mentioned
studies smoking was shown to significantly decrease circulating concentration of 25(OH)
metabolite of vitamin D.296, 297
The mean values of most markers of bone turnover in postmenopausal osteoporosis have been
shown to be higher in osteoporotic subjects in comparison to healthy subjects.29, 300 However,
they cannot be used in the diagnosis of osteoporosis due to the overlap of the values between
the two groups of subjects. Marker values before intervention have been correlated with bone
mineral density (BMD), and they have been evaluated as predictors of future bone loss and
fracture.301, 302, 303, 304 Several population based studies have also shown bone resorption
markers and BMD to be independent factors for prediction of bone loss and subsequently of
fractures, bone resorption being increased and BMD lowered in subjects who will sustain
fractures.305, 306 The predictive value of bone formation markers is less well established, and
contradictory results have been published.305, 307, 308 Of greatest value for the clinician, bone
markers serve to monitor the efficacy of antiresorptive therapy. Significant decreases in
resorption markers can be seen as soon as one month after the start of an efficient treatment
whereas formation markers decrease significantly after three months on treatment, with
significant increases in BMD being observed in 1 to 2 years.300, 309, 310, 311 Also, marker
changes after 3 months on antiresorptive treatment have been shown to predict later BMD
changes, at least at sites where trabecular bone predominates.300
3 Aim of the study
The aim of the present study is to evaluate commercially available immunoassays for type I
collagen-derived markers as to their performance. In addition, preanalytical factors like
sample handling and storage, effect of hemolysis on serum markers, and diurnal variation of
the markers are studied. The methods evaluated are radioimmunoassays for the amino- and
carboxy-terminal propeptides of type I procollagen (S-PINP, S-PICP) and for the cross-linked
carboxy-terminal telopeptide of type I collagen (S-ICTP), enzyme immunoassays for
measuring the urinary excretion of pyridinolines (Pyr), free deoxypyridinoline (DPD) and
amino-terminal telopeptides of type I collagen (NTX), and the automated chemiluminescent
immunoassays for DPD and NTX. In addition, in one study S-PINP and U-NTX are
compared with the serum activity of tartrate-resistant acid phosphatase 5b (TRACP 5b),
measured with the novel immunoextraction method. In two studies osteocalcin (OC) and in
one study bone specific alkaline phosphatase (Bone ALP) are measured. The clinical value of
the type I collagen-derived markers is assessed in different situations: in healthy persons, in
children with asthma and on corticosteroid treatment, in patients with prior differentiated
thyroid carcinoma on and off thyroxine treatment, in metastatic bone disease, and in
36
postmenopausal osteoporosis before and after treatment with estrogen or bisphosphonate.
Also the effect of smoking on two markers is evaluated.
4 Materials and methods
4.1 Subjects and study designs
4.1.1 Study I
In study I serum concentrations of amino- and carboxy-terminal propeptides of type I
procollagen (S-PINP, S-PICP) were measured in healthy volunteers, control persons from
several studies, three of which are included here as original publications (studies II, III and
IV). The age range of the healthy females (n = 158) was 3.8 – 80.5 years, and of the healthy
males (n = 95) 0.9 – 70.9 years. The same subjects have been used for establishing reference
ranges for other markers referred here. Study I was designed to assess the performance of
intact PINP radioimmunoassay, to measure S-PINP in healthy persons, and to compare SPINP values to S-PICP values in healthy population.
4.1.2 Study II
The subjects in study II were prepubertal children (6 boys and 8 girls) with median age of 9.1
years (range 7-11). They all had newly diagnosed perennial asthma, and they had not been on
previous anti-inflammatory treatment. The study was designed to measure bone turnover in
these children before treatment, after they had been one month on high dose (800 µg/m2/day)
inhaled budesonide (Turbuhaler, Draco AB, Lund, Sweden), after 5 months on lower dose of
budesonine (400 µg/m2/day), and after 6 months on inhaled nedocromil (Tilade, Fisons, UK).
As markers of bone formation serum concentrations of amino- and carboxy-terminal
propeptides of type I procollagen (S-PINP, S-PICP) and osteocalcin (S-OC) were measured.
Bone resorption was assessed by measuring serum concentration of type I collagen carboxyterminal cross-linked telopeptide (S-ICTP), and urinary excretion of pyridinolines (U-Pyr),
free deoxypyridinolines (U-DPD), and amino-terminal cross-linked telopeptides of type I
collagen (U-NTX). The baseline values were compared to values of age and gender matched
controls (n=21).
4.1.3 Study III
The subjects in study III were 29 patients (25 women, 4 men, median age 45 years) with prior
differentiated thyroid cancer. The patients had been treated with near total thyroidectomy
followed by radioiodine ablation, in most patients 9 to 11 years before the study, and since
then they had been on thyroxine therapy. The dose of levothyroxine was sufficient to render
serum thyroid stimulating hormone (S-TSH) level undetectable as assessed with a second
generation TSH method (detection limit 0.05 mU/l). Bone turnover was assessed with several
markers of bone turnover: serum concentrations of amino- and carboxy-terminal propeptides
of type I procollagen (S-PINP, S-PICP) and of osteocalcin (S-OC), and serum activity of bone
specific alkaline phosphatase (S-Bone ALP) were measured as reflectors of bone formation,
and serum concentration of type I collagen cross-linked carboxy-terminal telopeptide (SICTP) and urinary excretion of hydroxyproline (U-HOP) were used as reflectors of bone
resorption. The marker values were compared to the values of age and gender matched
controls (n=38). In a subgroup of 14 patients the T4 treatment was stopped after baseline
evaluation for 5 weeks, and the marker measurements were repeated after 5 weeks.
37
4.1.4 Study IV
Study IV included 25 patients with breast cancer and confirmed bone metastases (mean age
56 years, range 33-79 years), and 12 breast cancer patients with no bone metastases (mean
age 56 years, range 38-76 years), and their age matched healthy controls. The patients had
normal renal function, they had no metabolic bone disease, and they were normocalcemic.
The study was designed to assess the value of three type I collagen-derived markers, serum
concentrations of amino- and carboxy-terminal propeptides of type I procollagen (S-PINP, SPICP) and type I collagen cross-linked carboxy-terminal telopeptide (S-ICTP), in diagnosis of
skeletal disease due to breast cancer. In addition, their value in the follow-up of treatment
response, either chemotherapy or bisphosphonate treatment, was assessed for 13 patients with
skeletal disease.
4.1.5 Study V
Study V included 89 breast cancer patients, who were divided in two groups. A group of 49
patients were patients with stable disease (SD), and these patients had no or minor metastases
and stable clinical outcome of the disease. The other group of 40 patients consisted of patients
with progressive disease (PD), and these patients had bone, liver or other metastases, and
clinically evaluated aggressive type of disease. The study was designed to assess the value of
the serum concentrations of amino- and carboxy-terminal propeptides of type I procollagen
(S-PINP, S-PICP), and the ratio of the propeptides (PICP/PINP) as indicators of aggressivity
of breast cancer. The patients in study IV were included in the patient population of study V.
4.1.6 Study VI
The subjects in study VI were healthy smoking (n = 63) and non-smoking (n = 67) women, 6
months to 5 years postmenopausal. The median age of smokers was 51.8 years (range 46 - 55
years), and of non-smokers 52.9 years (range 47 – 59 years). The subjects could be
osteoporotic, but no other systemic illnesses were allowed, and they were not to be on
medication known to effect bone and calcium metabolism. The study was designed to assess
the effect of smoking on the efficacy of transdermal estrogen replacement therapy on bone.
The efficacy was assessed by measuring bone mineral density (BMD) at several skeletal sites,
and by measuring bone turnover with one marker of bone formation, serum concentration of
amino-terminal propeptide of type I procollagen (S-PINP), and one marker of bone
resorption, urinary excretion of amino-terminal cross-linked telopeptides of type I collagen
(U-NTX), before ERT and after 1 and 2 years on treatment. Vitamin D (25-OH-D) was also
measured at each time point. In addition, the marker values at baseline were compared to
those of healthy premenopausal non-smoking women with a median age of 36.8 years (range
20 – 51 years).
Smokers were randomized to receive either 1.0 mg (n=32, group 1) or 1.5 mg (n=31, group 2)
of transdermal estradiol daily, and non-smokers 1.0 mg (n=46, group 3) for 2 years. Estrogentreated women with intact uterus received medroxyprogesterone acetate or dydrogesterone 10
mg daily for 12 days of each cycle (=month). Non-smokers without estrogen treatment (n=22)
served as a control group.
4.1.7 Study VII
Nine healthy postmenopausal women, all 68 years of age, participated in study VII. The study
was designed to assess the effect of treatment with oral clodronate for one month on markers
38
of bone turnover and their diurnal variation. The subjects received a daily dose of 800 mg of
clodronate for the first two weeks on treatment and 1600 mg of clodronate for the last two
weeks. Serum concentration of carboxy-terminal propeptide of type I procollagen (S-PICP)
was measured to assess bone formation, serum concentration of type I collagen carboxyterminal cross-linked telopeptide (S-ICTP), and urinary excretion of pyridinolines (U-Pyr)
and amino-terminal cross-linked telopeptides of type I collagen (U-NTX) were measured for
assessing bone resorption. To assess the diurnal variation of the markers, serum samples were
drawn at 2-hour intervals for 24 hours. The diurnal variation of the urinary markers was
assessed from 4-hour urine samples. In addition, serum concentrations of calcium (S-Ca) and
parathormone (S-PTH) and urinary excretion of Ca (U-Ca) were assessed. Diurnal variation
of the amino-terminal propeptide of type I procollagen (S-PINP) was established later for
samples taken before clodronate treatment.
4.1.8 Study VIII
The subjects in study VIII were women of over 45 years of age, and 1 to 3 years
postmenopausal from an oral clodronate dose-ranging study (Probone study).312 They all had
vertebral osteopenia, a lumbar spine bone mineral density (BMD) at least one standard
deviation below the mean for premenopausal women (T-score <-1). Subjects with diseases or
medications known to affect bone or calcium metabolism were excluded from the study. The
study consisted of a primary 3-year dose-finding study of oral clodronate (BONEFOS£,
Leiras Oy, Finland) and a 2-year extension study. Study VIII was designed to compare a
novel method for measuring the activity of tartrate resistant acid phosphatase from osteoclasts
(S-TRACP 5b) to two type I collagen-derived markers, serum concentration of aminoterminal propeptide of type I procollagen (S-PINP) and urinary excretion of amino-terminal
cross-linked telopeptides of type I collagen (U-NTX) as reflectors of the efficacy of oral
clodronate treatment. The study population consisted of 59 subjects who had been on placebo
in the primary study and were then switched to 800 mg clodronate for 2 years in the extension
study. The marker values in the placebo group of the primary study (n=28) were used to
calculate the intraindividual variation (CVi) of the markers from samples taken at entry and at 3,
12 and 24 months. With respect to each marker, the percentage of responders was determined
according to the least significant change (LSC) in the marker, calculated from these CVis. In
the extension study, samples were taken yearly (baseline, 1 year and 2 years). BMD of the
lumbar spine and at the upper femur was measured at baseline and yearly thereafter.
Studies I and II were approved by the Ethical Committee of the Department of Allergic
Diseases, Helsinki University Central Hospital. Studies III, VI, VII and VIII were approved
by the Ethical Committee of the Third Department of Medicine, Helsinki University Central
Hospital, and study VIII additionally by local Ethical Committees of five Medical Centers in
Helsinki, Turku, Tampere, Hämeenlinna, Seinäjoki and Joensuu. Study IV was approved by
the Ethical Committee of Vaasa Central Hospital, and study V by the Ethical Committees of
the Medical faculty of the University of Oulu and Vaasa Central Hospital.
Table 5 summarizes the study populations and the markers and methods used in each original
study.
39
Table 5: The study populations and markers used in each original study.
Original publication
I
II
III
IV
V
VI
VII
VIII
Subjects
Healthy volunteers, n=253
Asthmatic children, n=14, and
control children, n=21
Marker
Method
S-PINP
RIA
S-PICP
RIA
S-PINP
RIA
S-PICP
RIA
S-OC
RIA
S-ICTP
RIA
U-Pyr
ELISA
U-DPD
ELISA
U-NTX
ELISA
S-PINP
RIA
S-PICP
RIA
S-OC
RIA
S-Bone ALP
Lectin precipitation
S-ICTP
RIA
U-HOP
Prockop and Udenfriend
Patients with breast cancer with,
n=25, and without, n=12, bone
metastases, and healthy controls,
n=37
S-PINP
RIA
S-PICP
RIA
S-ICTP
RIA
Patients with breast cancer,
either stable (n=49) or recurrent
disease (n=40).
S-PINP
RIA
S-PICP
RIA
Postmenopausal smoking (n=63)
and non-smoking (n=67) women,
and premenopausal non-smoking
women (n=38)
S-PINP
RIA
U-NTX
CIA, ELISA
Patients with prior differentiated
thyroid carcinoma on, n=29, and
off, n=14, thyroxine treatment,
and healthy controls, n=38
Healthy postmenopausal women
(n=9)
Postmenopausal women with
vertebral osteopenia, n=59 (and
n=29)
S-PICP
RIA
S-ICTP
RIA
U-Pyr
ELISA
U-NTX
ELISA
S-PINP
RIA
S-PINP
RIA
S-TRACP 5b
ELISA
U-NTX
ELISA
40
4.2 Methods
4.2.1 Assay for carboxy-terminal propeptide of type I procollagen (PICP)
A competitive radioimmunoassay with PICP RIA kit from Orion Diagnostica, Oulunsalo,
Finland, was used. In this assay PICP was produced by collegenase digestion of type I
procollagen from culture medium of human skin fibroblasts, and it was purified by lectinaffinity chromatography. This PICP was used as immunogen to produce PICP specific
polyclonal antibody in rabbits, as competing antigen labeled with 125-iodine (125I), and as
assay standards. A known amount of labeled PICP and PICP in the sample competed for the
restricted amount of binding sites of the antibody. The labeled antigen-antibody complex was
separated from free labeled antigen by second antibody conjugated to solid phase, kaolin.
The assay standard range was 0 – 500 µg/l, and the kit contained 5 standards (0, 50, 100, 200
and 500 µg). The assay was performed according to kit instructions with some minor
modifications: The sample was incubated simultaneously with the competing antigen and the
PICP-specific antibody in 37 oC for two hours. The reaction mixture was then cooled to room
temperature (5 minutes in a waterbath), and a second antibody-kaolin was then added. After a
30 minutes' incubation time the solid phase was separated by centrifuging the tubes (+5 oC,
30 minutes at speed 1500g). The liquid was removed with suction, and the radioactivity of the
pellet was counted with a 10-channel gammacounter (Gammamaster™, Wallac, Turku,
Finland). A standard curve was run simultaneously with the samples, and the curve was fitted
and the results calculated with a program connected to the counter (MultiCalc™, Wallac,
Turku, Finland). The assay required 100 µl of sample for each determination, and samples
were assayed in duplicates. Childrens’ samples were analyzed using a sample volume of 50
µl, and the needed reagent volumes were also halved. One reagent kit was designed for 100
determinations. Samples with high PICP concentrations were diluted with physiological
saline. Standards, samples, competing antigen and antibody solutions were pipeted either
manually or by using an automated pipeting device (Tecan, USA).
4.2.2 Assay for amino-terminal propeptide of type I procollagen (PINP)
A competitive radioimmunoassay with intact PINP RIA kit from Orion Diagnostica,
Oulunsalo, Finland, was used. The assay principle was similar to that of PICP assay. Intact
PINP used as immunogen, competing antigen and standards, was isolated from pleural fluids
of cancer patients using ion-exchange, gel-filtration and reversed-phase chromatography. The
polyclonal PINP specific antibody recognized the circulating intact PINP, the cross-reaction
with the small monomeric Col1 domain being only 1%.
The assay standard range was 0 – 250 µg/l, and the kit contained 5 standards (0, 25, 50, 100
and 250 µg). In studies I, II and III a protokit was used. The assay design was similar to that
of PICP assay except for the sample volume requirement, which was 50 µl in PINP assay.
One reagent kit was designed for 100 determinations.
4.2.3 Assay for carboxy-terminal telopeptide of type I collagen
A competitive radioimmunoassay with ICTP RIA kit from Orion Diagnostica, Oulunsalo,
Finland, was used. The assay principle was similar to that of the propeptide assays. The ICTP
antigen was produced by digesting type I collagen from human femoral bone with trypsin or
bacterial collagenase. The digestion resulted in formation of a degradation product with
41
molecular weight of about 10000 Da, corresponding the circulating C-terminal telopeptide of
type I collagen. In the ICTP assay the free and antibody-bound labeled antigen were separated
by a second antibody and polyethylene glycol.
The assay standard range was 0 – 50 µg/l, and the kit contained 5 standards (0, 5, 10, 25 and
50 µg). The assay design was similar to that of propeptide assays with same incubation times.
Sample volume needed for one determination was 100 µl, and the samples were run as
duplicates. Similarly to PICP assay, hhildrens’ samples were analyzed using a sample volume
of 50 µl, and the needed reagent volumes were also halved. One reagent kit was designed for
100 determinations.
4.2.4 Assays for urinary pyridinolines
4.2.4.1 Pyridinoline (Pyr) enzyme immunoassay
Urinary excretion of free and small peptide-bound cross-links, either pyridinoline or
deoxypyridinoline, was determined using Pyriliks® kit from Metra Biosystems, Palo Alto,
CA, USA. The assay standard range was 0 – 3000 nmol/l, and the kit included 6 standards.
The assay was a competitive enzyme immunoassay using microtiter well as the solid phase
(ELISA). The competing antigen, pyridinoline, purified from human urine, was labeled with
biotin and bound to avidin coated wells. The polyclonal antibody produced in rabbits was
recognized by both pyridinoline and deoxypyridinoline, and the Pyr in the solid phase and in
the sample competed for the restricted amount of binding sites of the antibody. The amount of
Pyr-specific antibody bound to the competing antigen was assessed with a second antibody
(goat antibody against rabbit IgG) conjugated to the assay label enzyme, alkaline phosphatase
(ALP). The amount of ALP in the solid phase was quantitated by using para-nitrophenyl
phosphate as the substrate.
The samples (100 µl) for this assay were pretreated by centrifuging them through a filter
(Spinfuge filter), which retained peptides larger than 1000 Da. Pretreated sample (10 µl) was
incubated in the coated microtiter well with the antibody in an overnight (18-24 h) incubation
at +5 +3 oC. After the incubation the wells were washed, and a second antibody-alkaline
phosphatase conjugate was added. In an one-hour incubation at room temperature the enzyme
conjugate was bound to the Pyr-specific antibody on the solid phase. The unbound conjugate
was washed away, and the amount of label enzyme bound to the well was then measured by
adding the substrate. The enzyme reaction was stopped after 60 minutes with a base (sodium
hydroxide), and the intensity of color formed was measured within 30 minutes in a plate
reader at wavelength 405 nm (Multiscan, Labsystems, Helsinki, Finland). Standards were
assayed at each run. MultiCalc program was used for curve fitting and calculation of the
results, which were then corrected for creatinine excretion. Creatinine was measured with a
modified Jaffe reaction with reagents included in the reagent kit. The samples were analyzed
as duplicates, and the samples and reagents were manually pipeted. The reagent kit was
designed for 96 determinations.
4.2.4.2 Free deoxypyridinoline (DPD) enzyme immunoassay
Urinary excretion of DPD was determined using Pyriliks-D® kit from Metra Biosystems, Palo
Alto, CA, USA. A monoclonal antibody specific for free DPD crosslink was used in this
assay. The assay solid phase, microtiter well, was coated with the monoclonal antibody, and
the competing antigen, deoxypyridinoline, was conjugated to the label enzyme, alkaline
42
phosphatase (ALP). The amount of enzyme bound to the solid phase was assessed by using
para-nitrophenyl phosphate as the substrate.
Turbid samples were centrifuged for 5 minutes at 1500g, and the supernatant was used for the
assay. Samples were prediluted 1:10 with the assay buffer. The assay standard range was 0300 nmol/l, and the kit included 6 standards. Diluted sample (50 µl) was incubated in the
coated well with the competing antigen for 2 hours at +5+3 oC in the dark. The amount of
enzyme label bound to the well was detected as in Pyrilinks® assay. The pipeting of the
sample and the liquid reagent was done either manually or by using an automated pipeting
device (Tecan). MultiCalc program was used for curve fitting and calculation of the results,
which were corrected for creatinine excretion. Creatinine was measured either with the
modified Jaffe method in a microtiter well format or with a kinetic Jaffe method on a clinical
chemistry analyzer (Hitachi 911, Boehringer-Mannheim, Germany).
4.2.4.3 Free deoxypyridinoline (DPD) automated chemiluminescent
immunoassay
The assay was an automated assay on ACS:180 immunoanalyzer (Chiron, USA). In this assay
the same monoclonal antibody as in the enzyme immunoassay was used. The competing
antigen was covalently coupled to the assay solid phase, paramagnetic particles. The
monoclonal DPD-specific antibody was coupled to a polyclonal anti-mouse antibody, which
was labeled with acridinium ester. The amount of label in the solid phase was detected as
light intensity released after the acridinium ester was triggered to a higher energy state by
adding NaOH and nitrite. The manufacturer calibrated the assay with a full standard curve.
For each reagent lot the standard curve was fed to the analyzer, and the standard curve was
fitted for the analyzer by assaying two calibrators at weekly intervals. The overall incubation
time for all methods on the analyzer was 7.5 minutes, and the time from pipeting the sample
to getting the result was approximately 15 minutes. The analyzer was used as a patch analyzer
for DPD measurements, and 60 samples or controls could be assayed in one run. The samples
were assayed as single determinations, and the results were proportioned to creatinine
excretion, measured with a kinetic Jaffe method.
4.2.5 Assays for urinary amino-terminal telopeptides of type I collagen (NTX)
4.2.5.1 NTX enzyme immunoassay
Urinary excretion of NTX was determined by using a kit (Osteomark™) from Ostex
International, Seattle, USA. In this assay the solid phase, microtiter well, was coated with the
competing antigen, crosslinked N-terminal telopeptide from human urine. The monoclonal
antibody, specific for a peptide sequence in the alpha 2 chain telopeptide region when
attached to a crosslink, was conjugated to the assay label enzyme, horseradish peroxidase
(HRP). The amount of HRP in the solid phase was quantitated by using tetramethylbenzidine
in dimethylsulfoxide as the substrate.
The assay standard range was 1-3000 nmol bone collagen equivalents (BCE)/l, and the kit
included 6 standards. Sample (25 µl) was incubated for 90 minutes at room temperature.
After washing the wells the amount of label in the solid phase was determined with a color
reaction and the color intensity was measured at 630 nm and using a reference wavelength of
430 nm using a plate reader. The pipeting of the sample and the liquid reagent was done
either manually or by using an automated pipeting device (Tecan). MultiCalc program was
43
used for curve fitting and calculation of the results, which were proportioned to creatinine
excretion, measured with a kinetic Jaffe method.
4.2.5.2 NTX automated chemiluminescent immunoassay
The assay was an automated assay on Vitros ECi immunoanalyzer (Ortho Clinical
Diagnostics, USA). The analyzer used an individual streptavidin coated well as the solid
phase, and HRP as the label. The amount of label enzyme in the solid phase was detected by
using a chemiluminescent substrate. The automated NTX assay utilizes the same monoclonal
antibody as in the manual assay, but the competing antigen is a synthetic cross-linked
telopeptide. The antibody is labeled with HRP, and the competing antigen with biotin. For
each reagent lot a standard curve made by the manufacturer was read to the analyzer from a
magnetic card. The standard curve place was fitted for each analyzer by assaying two
calibrators at weekly intervals. The overall incubation time of the assay was 30 minutes, and
the analyzer was a random access analyzer. The samples were assayed as single
determinations, and the results were proportioned to creatinine excretion, measured with a
kinetic Jaffe method.
4.2.6 Other bone marker measurements
Osteocalcin (OC) was measured with a competitive radioimmunoassay (RIA) from Nichols
Institute, San Juan Capistrano, USA. The method utilized a polyclonal antibody against
human OC, and the competing antigen was human OC labeled with 125-iodine. The
immunocomplex was precipitated with a second antibody conjugated to cellulose particles.
The assay standard range was 0-50 µg/l, the assay sensitivity was 0.3 µg/l, and the intra- and
interassay coefficients of variation (CV) were 4.5-6.2% and 4.1-8.9%. The antibody
recognized mainly the intact OC and the N-Mid-fragment (1-43), but with a lesser avidity
also smaller fragments of OC.
Bone-specific alkaline phosphatase (Bone ALP) was measured with lectin-precipitation
method using a kit from Boehringer-Mannheim, Mannheim, Germany.220 In this method bone
isoform of ALP was precipitated with lectin, and the activity of Bone ALP was calculated
from the total and residual activity of the supernatant using a formula, which takes into
account the fact that not all Bone ALP is precipitated. The activities were measured according
to the Scandinavian recommendation on a clinical chemistry analyzer (para-nitrophenyl
phosphate as the substrate, pH 10.1, reaction temperature 37 oC).313 The intra- and interassay
coefficients of variation were 4% and 5%, respectively.
Urinary hydroxyproline (HOP) was determined by the method of Prockop and Udenfriend.5
Tartrate resistant acid phospatase isoform 5b (TRACP 5b) was measured with BONE
TRAP™ kit from Suomen Bioanalytiikka Oy, Oulu, Finland. This method was a novel
immuno extraction method, where both TRACP 5b and 5a were first extracted to the assay
solid phase with a monoclonal TRACP specific antibody. After that the enzyme activity in the
solid phase was measured in conditions (pH 6.1), where 5b isoform was fully active and
isoform 5a almost completely inactive. The intra- and interassay coefficients of variation
were <6%.
44
4.2.7 Other laboratory methods
Vitamin D metabolites 25-OH-D and 1,25-(OH)2-D in study III were extracted with
acetonitrile, and the metabolites were purified using SepPak C18 cartridges and acetonitrile as
the eluant. The metabolites were separated in SepPak Silica cartridges with hexaneisopropanol elution. Both metabolites were quantitated using saturation analysis with reagents
from Amersham International plc, Amersham, UK. The intra- and interassay imprecision for
25-OH-D were < 12% (CV), and for 1,25-(OH)2-D <15% (CV).
25-OH-D in study VI was measured by a competitive radioimmunoassay (RIA) after
acetonitrile extraction of the sample using reagents from DiaSorin, Stillwater, MN, US. The
assay sensitivity was 5 nmol/l, and the intra- and interassay precision as CVs was 5.9 – 8.9%
and 6.0 – 9.0%, respectively.
Intact parathyroid hormone PTH in studies III and VI was measured by an
immunoradiometric method (IRMA) with reagents from Nichols Institute, San Juan
Capistrano, US. The method uses two affinity-purified polyclonal antibodies against two
different epitopes of iPTH. The analytical sensitivity was 1 ng/l, and the intra- and interassay
CVs were <6% and <8%, respectively.
Serum and urinary calcium Ca (studies III, IV, V, VI) were measured with a colorimetric
method (ortho-cresolftalein complex) on a clinical chemistry analyzer. The intra- and
interassay CVs were <4%. Serum calcium was corrected for albumin concentration using the
equation: S-Cacor = S-Catot – 0.2 x (S-Alb g/l – 40). Albumin was measured with a
colorimetric method (bromcresolgreen).
Total activity of alkaline phosphatase ALP (studies III, V) was measured according to the
Scandinavian recommendation using para-nitrophenyl phosphate in diethylamine buffer, pH
10.1, as substrate and a reaction temperature of 37 oC. The intra- and interassay CVs were
<5%.
Serum magnesium Mg and inorganic phosphate Pi (study III) were measured by routine
methods on a clinical chemistry analyzer; the CVs of the assays were <5%.
Serum and urinary creatinine (study III) were measured by a kinetic Jaffe method on a
clinical chemistry analyzer. The CVs of the assay were <4%.
Serum free thyroid hormones FT4 and FT3 in study III were measured by
radioimmunoassays after equilibrium dialysis.314
Serum thyrotropin TSH in study III was measured by a second generation
immunoradiometric method (IRMA) with analytical sensitivity of 0.05 mU/l using reagents
from Behringswerke, Marburg, Germany.
Serum estradiol E2 in study VI was measured by a direct radioimmunoassay (RIA) with
reagents from Sorin Biomedica Diagnostics, Italy. The analytical sensitivity of the assay was
0.04 nmol/l, and the intra- and interassay CVs ranged from 3 to 12%.
4.2.8 BMD measurements
In study VI BMD was measured with Hologic densitometer (Hologic QDR-1000, Waltham,
MA). In study VIII three different brands of bone densitometers were used; Lunar (Lunar
45
DPX, Madison, WI), Hologic and Norland (Norland XR 26, Medical System Inc., Fort
Atkins, WI).
4.2.9 Assessment of assay performance
The method performance was tested by assessing its analytical sensitivity, intra- and
interassay precisions (repeatability, reproducibility), accuracy and linearity:
9 The analytical sensitivity was calculated from the response of at least ten zero standard
measurements. The analytical sensitivity of a competitive assay was the concentration
corresponding the mean response minus three standard deviations of the measurements.
9 The functional sensitivity of the automated NTX assay was assessed during 3 months by
serial analyses of a pooled sample with concentration close to the analytical sensitivity of
the assay.
9 The intra-assay imprecision was calculated by assaying pooled samples with varying
concentrations as ten replicates in one run. PINP assay precision was also tested by using
a sample volume of 10 µl instead of 50 µl (1 to 5 dilution). The 40 µls were not
compensated for.
9 The inter-assay imprecision was calculated from the values of control samples or pooled
samples measured in several runs (8 – 10) using one reagent lot and several lots.
9 The accuracy was tested by adding known amounts of the marker to samples of varying
marker concentrations; the accuracy was expressed as spiking recovery. Also deviations
of target values of commercial controls were used for assessing accuracy.
9 The linearity was assessed from serial dilutions of samples with marker concentrations
that fell within the standard curve range of the assay, and was expressed as dilution
recovery.
9 The dilution point for the samples was determined for each assay based on the linearity
and accuracy of the assay. The patient samples exceeding this concentration were diluted
with physiological saline.
9 Correlations of the two formation markers and several resorption marker assays were
established; also correlation between S-PINP and U-NTX was assessed.
4.2.10 Assessment of factors effecting results
A number of samples were stored at room temperature up to 4 days, at +5 +3 oC up to one
week, and at –20 oC for longer periods to test the effect of different ways of storing the
sample on the results. The effect of 3-5 freeze-thaw cycles on the results was also tested.
Adding grossly hemolyzed serum to unhemolyzed sera from five healthy volunteers tested the
effect of hemolysis on the results. The marker concentrations were measured for the
unhemolyzed samples and samples with increasing degree of hemolysis (0.25 – 5.0 g Hb/l).
Diurnal variation of several markers was assessed in postmenopausal healthy women to
assess the effect of timing the sample.
46
The short-term intra individual variation (CVi) of U-NTX and U-DPD was established in five
premenopausal women from samples taken four times during one month. The long-term intra
individual variation of S-PINP, U-NTX and S-TRACP 5b was calculated from the samples of
the placebo group subjects in study VIII. The samples were taken at baseline, 3 months, 12
months and 24 months. The CVis were used to calculate the least significant changes (LSC)
of the markers with 95% confidence intervals. The LSC takes into account the CVi and the
inter assay precision of the method, and it was calculated using the formula LSC = 1.96 x
sqr(2 x ΣCVi2/n). Changes larger than the LSC are considered clinically significant.
4.3 Samples
Blood for serum marker measurements was drawn in the morning between the hours 7:30 and
9:30. In the study VII samples were drawn every two hours for 24 hours. Venous blood was
collected either into normal vacum tubes or into gel tubes, the blood was allowed to clot for
30 to 90 minutes, serum was separated by centrifugation for 10 minutes at 1500 x g, aliquoted
and stored at -20 oC or -70 oC until analysis.
Two hour morning urine after voiding the first urine was used for urinary marker
measurements in adults. In the study VII 4-hour samples were used for establishing diurnal
variation of the markers. In children the first morning urine was used. The difference of the
results between these two types of urinary samples was tested with samples of 5 healthy
premenopausal females, the difference being non-significant. The samples were aliquoted and
stored at –20 oC until analysis. As pyridinoline crosslinks in urine are known to degrade in
ultraviolet light, exposure to UV-light was avoided by using colored tubes.
Commercially available control samples were used when possible, and if necessary, pooled
and aliquoted patient samples were used to control the precision of the assays.
4.4
Statistics
StatView program (studies I, IV, partly VI), SAS System (studies VII, VIII), and SigmaStat
for Windows (study II, III, V, VI) were used for statistics.
StatView program was used when factors affecting the results - sample storage, freezingthawing, and hemolysis - were tested; the comparisons were made using paired t-test and the
correlations were calculated either as linear regression or using Spearman’s rank correlation
test.
In study I and II correlations were calculated using Spearman’s rank correlation test. Marker
values in different groups were expressed as means or medians, standard deviations (SD) and
ranges. Comparisons were made by using Mann-Whitney test (between groups), Wilcoxon
signed rank test (within group), or Student’s two-sided two-group t-test (between groups) and
paired t-test (within group) where the distribution was normal. In study II the marker values
were also expressed as standard deviation scores (SDS), calculated from the distribution of
values in healthy children.
In study III data with normal distribution was presented as means + SD, medians with ranges
were used for skewed distributions. Comparisons were made by Student’s two-sided twogroup t-test (between groups) and paired t-test (within group) when data was normally
distributed, otherwise Mann-Whitney rank sum test and Wilcoxon signed rank test were used.
47
In study IV nonparametric tests were used for comparisons and correlations (Mann-Whitney,
Spearman). The marker data were presented as medians and ranges.
In study V both linear and nonlinear regressions were used. Student’s t-test or Mann-Whitney
rank sum test was used for comparisons. Receiver operator characteristics curves (ROC) were
used to assess the accuracy of the markers and their ratio in discriminating between stable and
progressive disease.
In study VI linear regression with StatView program was used to establish the correlation of
the markers in different groups according to postmenopausal status and smoking habits. In the
comparisons between the study groups, normally distributed variables were studied using
one-way ANOVA or two group t-tests, and those non-normally distributed were tested with
Kruskal-Wallis one-way ANOVA or the Mann-Whitney rank sum test, as appropriate. Paired
data were compared using paired t-test (normally distributed data) or Wilcoxon signed-rank
test (nonnormal data).
In study VII areas under curve (AUC) for the 24-hour study periods were calculated for each
parameter, and the AUCs were compared with paired t-test or Wilcoxon matched-pair test.
The repeated measures analysis of variance was used to study the difference of diurnal
variation between the study days; the analysis included time-effect. Day and measurements
done within the day were included as factors in the analyses. Huynh-Feldt adjusted p-values
were used for within factors, and the tests were performed two-sided.
In study VIII the markers were analyzed with analysis of variance for repeated measures with
mixed modeling, including treatment group, time-point and their interaction as factors in the
model. The percentage changes from baseline were assessed with one-way analysis of
variance. Pearson correlation coefficients were calculated to measure the strength of the linear
relationship between different marker changes and also between the marker and BMD
changes.
In all studies a p-value of equal to or less than 0.05 was considered significant.
5 Results
5.1 Performance of the assays
5.1.1 Performance characteristics
The performance characteristics of each assay are tabulated in Table 6. The control samples
were chosen to cover the concentration range of the standard curve, the concentration of the
lowest control being close to the concentration of the lowest standard containing the analyte,
and the concentration of the highest control being slightly higher than the second highest
standard. In each assay 3 or 4 control samples were analyzed, and at least one of them was
always analyzed twice, before and after the patient samples. In the automated assay for UDPD two of the three available control samples were run before the patient samples and one
after them. In the automated NTX assay the three control samples from the manufacturer
were analyzed once daily. In addition a pooled sample with concentration close to the
analytical sensitivity of the assay was analyzed daily.
48
Table 6: Performance characteristics of the assays.
Method
Unit of
the
marker
Assay imprecision as coefficients
of variation, %
Analytical
sensitivity
Accuracy
Interassay CV
Intraassay CV
Within
one lot
Between
several lots
Linearity
Value from
target, %
Spiking
recovery,
%
Dilution
recovery,
%
Intact PINP
RIA
µg/l
2.3
2.4 –3.5*
2.5 - 5.2
2.7 – 6.7
96 – 105
96 – 100
79 – 103
PICP RIA
µg/l
1.2
2.1 – 3.7
3.5 – 6.6
2.8 – 6.9
96 – 103
ND
85 - 101
ICTP RIA
µg/l
0.4
3.8 – 7.3
4.7 – 6.9
2.5 – 8.0
98 - 100
ND
86 - 105
Pyr ELISA
nmol/l
25
2.7 – 7.0
5.7 – 8.5
ND
101- 110
ND
98 - 112
DPD ELISA
nmol/l
3
1.4 – 5.3
2.8 – 6.2
7.3 – 14.2
97 - 108
ND
96 - 106
DPD CIA
nmol/l
5
0.5-3.0
3.3 - 6.0
0.9 – 8.5
97 - 103
96 - 111
84 - 100
NTX ELISA
nmol/l
30
3.0 – 7.0
4.5 – 6.8
6.1 – 10.5
99 - 107
90 - 102
91 - 105
NTX CIA
nmol/l
5**
2.0 – 8.0
5.1 – 9.8
4.8 – 10.8
107 - 110
94 -108
85 - 113
ND = not done
*The intra-assay CV in intact PINP assay when 10 µl of sample was used was 3.8 %.
**The functional sensitivity of NTX CIA was 8 nmol/l.
An extra standard was used in the serum assays, all of them conventional competitive radio
immunoassays, in order to improve the precision in the low response area of the standard
curves (high concentrations). This standard was prepared from the highest and second highest
standard provided by the manufacturer, and its concentration in intact PINP assay was 175
µg/l, in PICP assay 350 µg/l, and in ICTP assay 37.5 µg/l. This standard enabled the dilution
point of the samples to be extended in these assays. In addition, the standard curve was
slightly modified in PINP assay; standards 0, 5, 17.5, 50, 100, 175, 250 µg/l were used. In
intact PINP assay samples with concentrations higher than 200 µg/l were diluted, in PICP
assay the dilution point was 400 µg/l, and in ICTP assay it was 38 µg/l.
In the urinary resorption marker assays the dilution points were following: U-Pyr 2000
nmol/l, U-DPD ELISA 220 nmol/l, U-DPD CIA 300 nmol/l, U-NTX ELISA 2000 nmol
BCE/l, and U-NTX CIA 2500 nmol BCE/l
The use of 10 µl of the sample for intact PINP assay without adding the 40 µl of diluent to the
reaction mixture did not have an effect on the results as tested with Student's t-test for 5 adult
samples (p=0.80). This decreased the needed sample volume for childrens’ samples, and also
the dilution step of the samples was omitted.
5.1.2 Correlation of the assays
The correlation of the two propeptide assays was good. When the correlation was assessed
with samples of concentration range from 10 to 1400 µg/l for S-PINP and from 50 to 1300
49
µg/l for S-PICP, the Spearman rank correlation coefficient r was 0.82 (p<0.0001), and the
linear regression equation was y(S-PINP) = 1.63(S-PICP) – 137 for the whole population (n =
253). When the correlation was studied in different age groups, it was better in young adults
and children (females under 20 years, males under 25 years) than in adults. In the original
publication I the correlation was studied by gender in different age groups, in figure A
(adults) and B (females <20 years, males < 25 years) the correlation is shown in the two age
groups with the genders combined.
r2 = 0.875, y = 1.40x + 6.88
n = 67
100,0
90,0
1600.0
80,0
70,0
1200.0
1400.0
60,0
S-PINP
S-PINP
r2 = 0.712, y = 0.29x + 6.71
n = 176
50,0
40,0
1000.0
800.0
600.0
30,0
20,0
400.0
200.0
10,0
0,0
0.0
0
50
100
150
200
250
0
300
A
200
400
600
800
1000
1200
1400
S-PICP
S-PICP
B
Figure 8: Correlation of S-PINP and S-PICP assays in adults (females >20 years, males > 25 years) in A
and in young adults and children in B.
Table 7 summarizes the correlations of different resorption marker assays; correlation
coefficient, and the slope and intercept of the linear regression equation are shown. Samples
from studies I, II, VI and VII as well as of healthy controls from other studies were used to
assess the correlations.
1600
50
Table 7: Correlation of type I collagen-derived resorption markers and different methods for U-DPD and
U-NTX.
x
y
n
r
p
Slope
Intercept
ICTP RIA
DPD ELISA
74
0.954
<0.0001
1.22
1.35
ICTP RIA
NTX ELISA
74
0.957
<0.0001
50.7
-88.7
Pyr ELISA
DPD ELISA
74
0.980
<0.0001
0.16
-0.52
DPD ELISA
NTX ELISA
152*
0.975
<0.0001
35.0
-107
DPD ELISA
NTX ELISA
93**
0.769
<0.0001
10.0
1.30
DPD ELISA
DPD CIA
161**
0.965
<0.0001
1.11
-1.13
NTX ELISA
NTX CIA
176**
0.992
<0.0001
1.13
20.0
* childrens’ samples included, ** childrens’ samples excluded
Although the correlation between the methods for the same analyte was good, individual
values could be quite different when different methods were used. The mean ratio of DPD
CIA value to DPD ELISA value was 1.056, SEM 0.023, and the mean ratio of NTX CIA
value to NTX ELISA value was 1.167, SEM 0.017, indicating that both automated methods
yielded somewhat higher results than the ELISA methods.
5.2 Factors effecting the assay results
5.2.1 Effect of hemolysis
Hemolysis did not have any effect on the results obtained in PICP and ICTP assays. In PINP
assay strong hemolysis, 2.5 and 5 gHb/l, lowered the measured PINP concentration by 12%
and 20%, shown in Figure 9. The effect was significant as assessed by t-test, p=0.01 for both.
110
S-PINP % left
100
*
90
*
80
70
60
Hb 0
Hb 0.25
Hb 0.5
Hb 1.0
S-Hb g/l
Figure 9: Effect of hemolysis on S-PINP values.
Hb 2.5
Hb 5.0
51
5.2.2 Effect of sample storage
All type I collagen-derived markers showed good stability both in serum and in urine.
Samples for serum markers PICP, PINP and ICTP could be kept in room temperature for four
days, in +5 +3 oC for one week and in –20 +3 oC for years. Three freeze-thaw cycles did not
have any effect on the results. Urinary markers showed similar stability, but the room
temperature, which was not tested. Ultraviolet light degrades pyridinoline ring structure, seen
as decreased U-Pyr and U-DPD values if kept in daylight. On the contrary, the peptide bound
marker U-NTX increased if kept in daylight. Therefore the urinary samples were stored in
colored tubes.
5.2.3 Timing of the sample
The timing of the sample was deducted from the diurnal variation of the markers, assessed
from samples of nine healthy postmenopausal volunteers. For S-PINP samples of two
volunteers were excluded due to several grossly hemolyzed samples. Figure 10 A shows the
diurnal variation of serum formation markers PINP and PICP and the resorption marker ICTP
as percentages from the lowest value, and the diurnal variation of urinary excretion of Pyr and
NTX is similarly displayed in Figure 10 B.
% above the lowest value
A
140
130
120
S-PINP
110
S-PICP
100
S-ICT P
90
4
8'
24
20
16
8
12
80
hour
160
150
140
130
120
110
100
90
80
U-Pyr
U-NTx
8:
00
-1
12 2
:0
01
16 6
:0
02
20 0
:0
024
24
:0
04
4:
00
-8
% above the lowest value
B
hour
Figure 10: Diurnal variation of serum (A) and urinary (B) type I collagen-derived markers; the values at
each time point are expressed as percentage from the lowest value.
52
Type I procollagen propeptides showed their peak values at midnight, approximately 20%
higher than the lowest values at 10 am. The serum resorption marker ICTP was at its highest
in early morning hours, and fairly stable in the afternoon. The urinary excretion of Pyr and
NTX were at their highest in samples collected from 4 to 8 and 8 to 12, the highest excretion
being 40 – 50 % higher than the excretion in the afternoon.
The reference ranges of the serum markers were established in samples taken in the morning
between the hours 7:30 and 9:30. Urinary markers were assessed from second voided
morning samples in adults and first morning samples in children.
5.2.4 The least significant change of the markers
The short-term (one month) intra individual variation (CVi) of urinary resorption markers
NTX and DPD in five healthy premenopausal volunteers varied from 6% to 11%. Depending
on the method and its precision, the LSC for DPD varied from 28% to 49% and for NTX
from 34% to 46%.
Long-term (2 years) intra individual variation was calculated from the samples of the placebo
group patients in study VIII for S-PINP, U-NTX (ELISA method) and S-TRACP 5b. The
calculated LSCs were 32% for S-PINP, 55% for U-NTX and 27% for S-TRACP 5b.
5.3 Type I collagen markers in healthy subjects, study I
5.3.1 S-PINP and S-PICP
The marker values were very high in young children up to 1 year of age; they declined
somewhat during the following years, and then increased again during the pubertal growth
spurt. After the puberty the marker values declined eventually to the adult levels, in girls
somewhat earlier than in boys. In females the adult marker levels were reached between the
ages 18 to 22, in males between the ages 22 to 27. Table 8 displays the marker values in
groups according to age and gender.
The ratio of PICP-to-PINP mass concentrations should be approximately 3, as the propeptides
are liberated into the circulation in equimolar concentration. This was the case in adults, but
in children and adolescents the ratio was lowered. In Table 8 the PICP-to-PINP ratio is shown
by age gender, Figure 11 shows the ratio in the whole population.
Table 8: Type I procollagen propeptides and their ratio in healthy population according to age group and
gender.
Gender
female
Age group
n
S-PICP, µg/l
S-PINP, µg/l
PICP/PINP ratio
Median
Range
Median
Range
Median
Range
< 20 yr
37
260
93 – 480
405
52.2 – 820
0.80
0.44 – 2.36
20-50 yr
93
110
60 – 239
37.1
18.5 – 80.6
3.32
1.53 – 4.83
> 50 yr
28
125
66 – 187
43.8
23.5 – 89.8
2.81
1.38 – 4.46
< 25 yr
30
317
117 – 1380
589
35.5 - 1404
0.64
0.38 – 3.30
> 25 yr
65
130
64 - 253
47.4
21.8 – 89.0
2.94
1.64 – 4.94
male
53
In the groups of young adults and children, a significant negative correlation between S-PINP
and age (r = -0.70, p < 0.001 for females, r = -0.52, p = 0.004 for males), and a significant
positive correlation between the PICP-to-PINP ratio and age (r = 0.83, p <0.001 for females, r
= 0.51, p = 0.005 for males) was found.
log ratio
10,00
1,00
0,10
0
10
20
30
40
50
60
70
80
age
Figure 11: The ratio of PICP-to-PINP in healthy population as the function of age.
Based on the results on healthy subjects, the reference interval for premenopausal women
above the age of 20 years was set from 15 to 80 µg/l for S-PINP and from 50 to 170 µg/l for
S-PICP. The reference intervals for adult men above the age of 25 years were from 20 to 100
µg/l for S-PINP and from 50 to 230 µg/l for S-PICP.
5.3.2 Type I collagen-derived resorption markers
Type I collagen resorption marker values were also established using a part of the same
healthy population as was used for formation markers. Table 9 shows the values of the
urinary excretion of Pyr, DPD and NTX values by age and gender. For DPD and NTX the
values obtained with the original ELISAs are shown. Total pyridinolines was the first urinary
resorption marker assay evaluated, and the number of female subjects under the age of 20 and
over the age of 50 was so small that the values are not shown in the Table 9.
54
Table 9: Urinary resorption markers in healthy subjects.
U-Pyr, nmol/mmol Crea
Gender
Age group
n
Median
female
Range
U-DPD, nmol/mmol
Crea
U-NTX, nmol/mmol
Crea
Median
Range
Median
Range
21.1
5.8 – 112
490
53 - 3410
5.2
2.6 – 9.3
34
14 – 122
5.8
3.9 – 11.1
43
17 - 86
< 20 yr
44
20-50 yr
40
> 50 yr
16
< 25 yr
25
101
26 - 181
18.6
2.4 – 81.1
643
32 – 3472
> 25 yr
20
23
17 – 35
3.0
2.3 – 4.9
38
17 - 89
34
17 – 49
male
The ratio of NTX value to DPD value (peptide-bound to free cross-links) varied with age, the
relative proportion of excreted peptide-bound crosslinks being higher in children than in
adults, shown in Figure 12. A significant negative correlation was also found between the
NTX-to-DPD ratio and age in females < 20 years of age (r = -0.74, p<0.001), and in males <
25 years of age (r = -0.60, p < 0.001). After menopause a positive correlation between the
ratio and age was found (r = 0.40, p<0.001).
70.0
60.0
NTx to DPD
50.0
40.0
30.0
20.0
10.0
0.0
0
10
20
30
40
50
60
70
80
age
Figure 12: The dependence of the ratio of NTX-to-DPD (peptide bound to free crosslinks) on age.
Table 10 displays S-ICTP values in healthy population. Also S-ICTP was high in children,
but no difference was found in the values of adult females and males. The reference ranges
for various resorption markers in adults are shown in Table 11.
55
Table 10: S-ICTP in healthy population.
Gender
female
Age group
n
S-ICTP, µg/l
Median
Range
< 20 yr
22
14.6
4.0 – 24.5
20-50 yr
77
2.6
1.6 – 4.9
> 50 yr
22
3.1
2.0 – 4.5
< 25 yr
18
12.5
2.9 – 20.0
> 25 yr
46
2.8
1.1 – 4.6
male
Table 11: Adult reference ranges of type I collagen-derived resorption markers
Premenopausal women > 20 years
Men > 25 years
S-ICTP, µg/l
1.5 – 4.5
1.5 – 4.5
U-Pyr, nmol/mmol Cr
15 – 50
15 – 45
U-DPD, nmol/mmol Cr*
1.0 – 8.0
2.0 – 9.0
U-NTX, nmol/mmol Cr, ELISA
5 – 65
5 – 85
U-NTX, nmol/mmol Cr, CIA
15 – 80
20 – 100
* A correction factor was used in the automated DPD assay to have the same the reference range in both DPD assays
5.4 Type I collagen markers during inhaled corticosteroid treatment in
children with asthma, study II
In children with newly diagnosed asthma (n=14, age 7-11 years), the type I collagen-derived
markers of bone formation and degradation did not deviate from marker values in age and
gender matched control children before the treatment with inhaled budesonide was started.
Serum osteocalcin was, however, significantly lower in asthmatic children than in controls
(p<0.001, -3.4 SDs from the control mean). All the markers of bone formation decreased
significantly after one-month treatment with high-dose budesonide (800 µg m-2 day-1), and
similar significant decrements were seen in all markers of bone resorption but U-NTX. After
5-month treatment with lower dose of budesonide (400 µg m-2 day-1) all markers of bone
turnover were lower than at baseline. However, only U-NTX significantly changed from 1month time point. After half-a-year treatment with nedocromil all the type I collagen-derived
markers significantly increased from the values after inhaled corticosteroid treatment,
reaching the baseline values at 12 months. S-OC also increased during the treatment with
nedocromil, and reached above baseline values at 12 months.
Marker values of patients and controls at baseline as medians and ranges are displayed in
Table 12, and marker values of the patients before and after the treatments are displayed in
Table 13. Figure 13 A shows the changes in formation markers as standard deviation scores
(SDS), and Figure 13 B shows similarly the changes in resorption markers.
56
Table 12: Marker values in control children and in children with newly diagnosed asthma.
Controls, n = 21
Patients, n = 14
Marker
Median
Range
Median
Range
S-PICP, µg/l
320
190 – 460
320
240 – 570
S-PINP, µg/l
522
392 – 798
483
343 – 963
S-OC, µg/l
31.0
22.0 – 55.0
11.5***
4.7 – 33.5
S-ICTP, µg/l
14.9
10.1 – 21.2
14.6
11.4 – 21.4
U-Pyr, nmol/mmol Crea
128
93 – 181
150
96 -216
U-DPD, nmol/mmol Crea
18.4
12.1 – 31.8
18.4
13.6 – 24.4
U-NTX, nmol/mmol Crea
659
353 – 894
532
85.9 – 894
*** significantly lower than in controls, p < 0.001
Table 13: Marker values in asthmatic children before treatment, after treatment with high-dose
budesonide (1 month), low-dose budesonide (1 to 6 months), and nedocromil (6 to 12 months).
Baseline
1 month, n= 14
6 months, n = 14
12 months, n = 12
Marker
Median
Range
Median
Range
Median
Range
Median
Range
S-PICP, µg/l
320
240 – 570
250***
180 – 300
270**
190 – 420
290
190 – 510
S-PINP, µg/l
483
343 – 963
350**
210 – 557
322**
207 – 567
462##
315 – 753
S-OC, µg/l
11.5
4.7 – 33.5
9.0*
1.8 – 42.2
7.0**
2.3 – 11.4
21**##
10.1 – 38.5
6.6 – 14.8
##
8.2 – 20.0
#
82 – 172
##
12-0 – 30.1
#
209 – 783
S-ICTP, µg/l
U-Pyr, nmol/mmol Cr
U-DPD, nmol/mmol Cr
U-NTX, nmol/mmol Cr
14.6
150
18.4
532
11.4 – 21.4
96 -216
13.6 – 24.4
86 – 894
10.2**
111**
14.2*
473
5.5 – 15.4
67 – 169
7.7 – 20.0
69 – 1056
10.4**
106**
14.3*
366**
60 – 161
7.8 – 19.4
74 – 756
* p < 0.05, ** p < 0.01, *** p < 0.001 as compared to baseline, # p < 0.05, ## p < 0.01 as compared to 6 months
15.1
132
19.3
578
57
1
1
0
0
-1
S-ICTP
-2
S-PINP
S-OC
SDS
SDS
S-PICP
U-Pyr
-1
U-DPD
-3
U-NTx
-2
-4
-5
-3
0
1
2
3
4
5
6
7
8
9 10 11 12
0
1
Month
A
2
3
4
5
6
7
8
9
10 11 12
Month
B
Figure 13: Markers of bone formation (A) and resorption (B) in asthmatic children before and after
treatment, expressed as standard deviation scores from the values of healthy control children. The
children were treated with budesonide up to six months and with nedocromil from 6 months to 1 year.
The changes in S-PINP and S-PICP during both treatments (budesonide, nedocromil)
correlated positively (r = 0.73 and 0.70, p <0.01 and < 0.05) as did S-PINP changes with
changes in S-ICTP (r = 0.63 and 0.68, p < 0.05). In addition, S-PICP correlated positively
with S-ICTP during the treatment with budesonide (r = 0.66, p < 0.05). Positive correlation
was also found between changes in U-Pyr and U-DPD during both treatments (r = 0.70 and
0.83, p < 0.05 and < 0.01). Other significant correlations were not found between the marker
changes.
58
5.5 Thyroid hormone effects on the type I collagen markers, study III
The patients (n = 29) and their age and gender matched controls (n = 38) were well balanced
as to the baseline characteristics age, weight, height and BMI. Thyroid hormones free
thyroxine (FT4) and free tri-iodothyronine (FT3) concentrations were higher and thyroid
stimulating hormone (TSH) activity lower in patients than in controls, as could be expected.
Patients and controls were also well balanced as to the markers reflecting bone and calcium
metabolism, except for urinary excretion of phosphate (Pi) and magnesium (Mg) which were
higher in patients (Table 14). Also creatinine excretion was higher in patients.
Table 14: Markers reflecting bone and calcium metabolism in patients and controls; mean + SD or
median with ranges are shown.
Marker, unit
Patients
Controls
S-Ca_cor, mmol/l
2.45 + 0.10
2.46 + 0.08
S-Pi, mmol/l
1.05 + 0.17
1.08 + 0.13
S-Mg, mmol/l
0.87 + 0.08
0.90 + 0.07
64 + 22
62 + 21
106 (55 – 218)
111 (72 – 204)
39 (16 –77)
30 (13 –56)
U-Ca, mmol/24 h
3.6 + 1.7
3.4 + 1.8
U-Pi, mmol/24 h
32 + 10*
26 + 8
U-Mg, mmol/24 h
5.0 + 1.8*
3.9 + 1.4
S-25-OH-D, nmol/l
S-1,25(OH)2-D, pmol/l
S-PTHi, ng/l
* p < 0.05 for the difference between patients and controls
Markers of bone turnover during LT4 treatment
In the total population markers of bone formation, total ALP, OC, PICP and PINP were
significantly higher in patients than in controls, total ALP being 21% higher, p < 0.05, OC
35% higher, p < 0.001, PICP 10% higher, p < 0.05, and PINP 46% higher, p < 0.001. Bone
ALP was also higher in patients than in controls, but the difference was not significant
(Figure 14 A). Markers of bone resorption were also significantly higher in patients than
controls, ICTP by 21%, p < 0.05, and U-HOP by 37%, p < 0.001 (Figure 14 B). Table 15
shows the markers of bone turnover in female patients and their controls by menopausal
status. In premenopausal patients S-OC, S-PINP and both resorption markers were
significantly higher than in controls, whereas only S-PINP and U-HOP were higher in
postmenopausal patients than in their controls. On the whole, markers of bone turnover
indicated increased bone turnover in patients with prior differentiated thyroid carcinoma and
on thyroxine treatment.
59
A
B
Figure 14: Markers of bone formation (A) and resorption (B) in patients and controls; medians and
interquartile ranges are shown (from the original publication with permission).
Table 15: The markers of bone turnover in female patients and their controls by menopausal status; mean
+ SD or median with range are shown.
Premenopausal
Postmenopausal
Marker, unit
Patients (n = 15)
Controls (n = 22)
Patients (n = 10)
Controls (n = 13)
56 + 36
46 + 23
81 + 18
74 + 28
7.7 + 3.3*
4.6 + 3.0
8.8 + 3.7
8.5 + 4.3
S-PICP, µg/l
159 (27 – 236)
103 (66 – 164)
127 + 37
115 + 35
S-PINP, µg/l
56 (13 – 105)*
34 (22 – 52)
62 + 16*
44 + 16
3.6 + 0.9*
2.8 + 0.8
3.5 + 1.2
3.1 + 0.7
0.088 (0.049-0.137)*
0.062 (0.042-0.101)
0.089 + 0.028*
0.067 + 0.017
S-Bone ALP, U/l
S-OC, µg/l
S-ICTP, µg/l
U-HOP, µmol/24 h/m
2
*p < 0.05 for the difference between patients and controls
Bone mineral density at each measured site did not differ between patients and controls.
Markers of bone turnover after 5 weeks of iatrogenic hypothyroidism
In 14 patients the treatment with LT4 was stopped for 5 weeks, and the markers of bone
turnover were measured after this period of hypothyroidism. The withdrawal of LT4 caused a
significant decrease in S-OC (54%, p < 0.001), S-PINP (7%, p < 0.05) and S-ICTP (54%, p <
0.001), whereas S-PICP increased by 24% (p < 0.001) and S-1,25(OH)2-D by 29% (p <
0.01). The changes in bone turnover markers are shown in Figure 15.
60
Figure 15: Markers of bone turnover before and after interruption of LT4 therapy for 5 weeks (from the
original publication with permission).
5.6 Type I collagen markers in metastatic bone disease
5.6.1 S-ICTP, S-PICP and S-PINP as markers of metastatic bone disease in
breast cancer, study IV
In patients with breast cancer, metastatic to bone (BM+), serum concentrations of ICTP and
PINP were significantly higher than in healthy controls (p < 0.0001 and p = 0.046) whereas
S-PICP did not significantly differ in patients as compared to controls. The ratio of PICP-toPINP was significantly lower in BM+ breast cancer patients than in controls (p = 0.002),
shown in Table 16. Also in patients with breast cancer but without bone metastases (BM-) SICTP was significantly higher than in healthy controls (p=0.001), but the propeptides or their
ratio did not significantly differ between BM- patients and controls. ICTP was marginally
significantly higher in BM+ patients as compared to BM- patients (p=0.06), and the ratio of
the propeptides was significantly lower in BM+ patients (p=0.047). PICP and PINP did not
differ in the two groups of patients. In the BM+ patient group a significant positive
correlation was found between ICTP and PICP, r = 0.58 and p = 0.005, and for ICTP and
PINP, r = 0.60 and p = 0.005, and a negative correlation between ICTP and the PICP-to-PINP
ratio, r = -0.61, p = 0.004. In patients without bone metastases no correlations were found
between the markers.
Neither of these markers nor the ratio of the propeptides was clinically sensitive enough to be
used in diagnosing bone metastases, the clinical sensitivities being 56%, 24%, 30% and 52%
for ICTP, PICP, PINP and the ratio PICP-to-PINP. Also some patients without bone
metastases had S-ICTP and S-PINP exceeding the upper reference limit of the markers, and
the ratio of the propeptides was lowered in some patients, and the clinical specificities were
thus 93% for ICTP, 98% for PINP and 91% for the PICP-to-PINP ratio.
61
Table 16: Serum concentrations of ICTP, PICP, PINP and the ratio of the propeptides in breast cancer
patients with (BM+) and without (BM-) bone metastases and healthy controls.
Marker
BM+ patients, n = 25
BM- patients, n = 12
Controls, n=37
Median
Range
Median
Range
Median
Range
S-ICTP, µg/l
5.1**
2.2 – 90.6
4.0*
3.1 – 5.2
2.8
1.9 – 4.5
S-PICP, µg/l
116
61 – 343
110
77 – 177
117
82 – 178
S-PINP, µg/l
49*
18 – 1006
45
19 – 86
36
25 – 61
1.31 – 3.99
2.98
2.33 – 4.30
PICP/PINP ratio
1.98*
0.31 – 3.93
#
2.93
** difference between patients and controls, p<0.0001, * difference between patients and controls, p<0.05, # difference between BM+ and
BM-, p<0.05
In 13 BM+ patients follow-up samples after chemotherapy or treatment with bisphosphonate
were also available. Due to small patient number, no statistics were calculated for changes,
but S-ICTP, S-PINP and the PICP-to-PINP ratio and to a lesser extent S-PICP changed in
parallel with the course of the disease.
5.6.2 PINP and PICP in aggressive breast cancer, study V
Table 17 shows the metastatic status of the patients who were divided into two groups
according to their clinical status, to groups with stable disease (SD) or with progressive
disease (PD).
Table 17: The percentage of patients with different types of metastases.
Bone only, % of n
Visceral and soft
tissue only, % of n
Bone, visceral and
soft tissue, % of n
None, % of n
SD group, n = 49
38
15
23
24
PD group, n = 40
60
5
35
0
The mean of S-PINP was 7.2 times higher in PD group patients than in SD group patients
whereas the mean of S-PICP was only 1.7 times higher in the PD group. Also, the ratio of
propeptides was significantly lower in patients with progressive disease, as shown in Table
18. Figure 16 displays the box-and -whiskers plots of the propeptides and their ratio in the
two patient groups. In the PD group 2 of the 3 patients with no bone metastases had elevated
S-PINP, and they all had a PICP-to-PINP ratio 1.4 or lower. In the SD group one patient
without bone metastases had a low PICP-to-PINP ratio (1.3), but her S-PINP was normal.
62
Table 18: S-PINP and S-PICP concentrations and the PICP-to-PINP ratio in breast cancer patients with
stable or progressive disease.
SD group
PD group
Mean + SD
Difference
95% Cls
p
S-PINP, µg/l
38.1 + 2.9
276.1 + 79.1
77.9, 398.3
0.005
S-PICP, µg/l
100.2 + 5.0
174.3 + 19.7
33.1, 115.1
0.001
PICP/PINP ratio
3.07 + 0.18
1.02 + 0.07
1.66, 2.44
< 0.001
Figure 16: Box plots of S-PINP, S-PICP and their ratio in breast cancer patients with stable or with
progressive disease. O = 5 and 95 percentiles, horizontal bars = 10 and 90 percentiles, lower and upper
limits of the boxes = 25 and 75 percentiles. The solid line inside the box marks the median and the dotted
line the mean (from the original publication with permission).
The receiver operator characteristic (ROC) curves, shown in Figure 17, were calculated for
the propeptides, their ratio, and also for total alkaline phosphatase activity (ALP), which is
routinely measured in these patients to monitor bone and liver status. The optimal sensitivity
(0.90) and specificity (0.90) of S-PINP would be attained by using a cut-off value of 70 µg/l,
and those of PICP-to-PINP ratio (0.98 and 0.90) by using a cut-off value of 1.74.
Figure 17: ROC curves over a range of cut-off points for S-PINP, S-PICP, PICP-to-PINP ratio and S-ALP
(from the original publication with permission).
63
5.7 Type I collagen markers in postmenopausal osteoporosis
5.7.1 Effect of smoking on transdermal estrogen replacement therapy in
postmenopausal women, study VI
At baseline the formation marker S-PINP was similar in both non-smoking and smoking
postmenopausal women, and it did not differ from S-PINP of premenopausal women. U-NTX
was significantly higher in postmenopausal women as compared to premenopausal women (p
< 0.001), but it was similar in smokers and non-smokers. The correlation of the two markers
was poorer in postmenopausal smokers as compared to postmenopausal or premenopausal
non-smokers. 25-OH-D was 20% lower in postmenopausal smokers than in postmenopausal
non-smokers (p = 0.05). The baseline results in smokers and non-smokers are shown in Table
19, the correlation of S-PINP and U-NTX in different populations is shown in Table 20.
Table 19: S-PINP and U-NTX in postmenopausal smokers and non-smokers before ERT, and in
premenopausal non-smokers, and S-25-OH-D in postmenopausal women at baseline.
S-PINP, µg/l
Median
Range
U-NTX, nmol/mmol Cr
Median
25-OH-D, nmol/l
Range
Median
Range
Postmenopausal smokers
43.2
14.3 – 134.9
60.5*
24.4 – 171.4
36**
15 - 86
Postmenopausal non-smokers
38.4
8.7 – 92.7
59.4*
18.0 – 178.1
52
17 - 74
Premenopausal non-smokers
38.3
20.3 – 80.6
42.8
20.7 – 90.3
-
-
* significantly different from premenopausal non-smokers, p < 0.001, ** significantly different from non-smokers, p < 0.05
Table 20: Correlation of S-PINP and U-NTX in postmenopausal smokers and non-smokers at baseline,
and in premenopausal non-smokers.
r, p
Slope
Intercept
Postmenopausal smokers
0.34, <0.001
0.50
44
Postmenopausal non-smokers
0.62, <0.001
0.92
28
Premenopausal women
0.71, <0.001
0.95
10
The two groups of non-smokers, those allocated to receive estrogen and those who did not
want to start ERT, had significantly different S-PINP at baseline, being higher in control
group (median 59 µg/l vs. 35 µg/l, p < 0.05). The marker values in different study groups are
shown in Table 21. Other baseline characteristics, including BMD at lumbar spine and three
femoral sites, were well balanced between the study groups, the two groups of smokers,
randomized to receive either 1 mg or 1.5 mg of E2, and the two groups of non-smokers.
Table 21: Marker values in the study groups at baseline.
Smokers, 1 mg
E2
Smokers, 1.5 mg
E2
Non-smokers on
E2
Non-smokers
without E2
S-PINP, µg/l
median (range)
43 (14-135)
43 (18-85)
35 (9-93)
59 (24-119)*
U-NTX,
nmol/mmol Cr
median (range)
58 (27-151)
67 (24-171)
59 (18-178)
74 (34-141)
* p<0.05 as compared to other groups
64
The two doses of E2 resulted in a similar marker and BMD response in smokers, and
therefore the groups were combined for further analyses.
At 1-year time point the marker of bone formation, S-PINP, decreased significantly more in
smokers on E2 (-36%) than in non-smokers on E2 (-24%, p = 0.038). Also a significant
difference between smokers on E2 and non-smokers without treatment was seen (p < 0.0001),
S-PINP decreasing in the latter group non-significantly (-7%). Non-smokers on E2 and
without treatment did not significantly differ in response. At 2-year time point S-PINP did not
further change in smokers on E2, but increased in non-smokers on E2, the difference between
the groups being significant (p = 0.006). In non-smokers without treatment S-PINP was stable
from 1 to 2 years. Percentage changes of S-PINP are shown in Figure 18A.
U-NTX changed to a similar extent in smokers (-35%) and non-smokers (-33%) on E2 at 1year time point, whereas in non-smokers without treatment U-NTX slightly increased (+6%).
From 1 to 2 years U-NTX increased somewhat in smokers on E2, and it further decreased in
non-smokers on E2, leading to a marginally significant difference between the treated groups
at 2-year time point (p = 0.07). In non-smokers without E2 U-NTX returned to baseline at 2
years. Percentage changes of U-NTX are shown in Figure 18B.
5
A
15
-5
smoke rs + E
non-smoke rs
+E
non-smoke rs
-25
-35
-45
0 yr
A
B5
smoke rs + E
-5
-15
nonsmoke rs + E
nonsmoke rs
-15
-25
-35
1 yr
2 yr
-45
0 yr
1 yr
2 yr
B
Figure 18: Percentage changes of S-PINP (A) and U-NTX (B) in postmenopausal smokers on E2, nonsmokers on E2 and non-smokers without ERT.
ERT for 2 years improved the correlation of the markers in smokers, whereas the correlation
worsened in non-smokers without ERT during the 2-year follow-up, shown in Table 22.
Estrogen treatment did not have any effect on the vitamin D status of the smokers (data not
shown).
65
Table 22: Correlation of S-PINP and U-NTX in postmenopausal smokers and non-smokers on E2 before
the treatment was started and during the ERT, and in postmenopausal non-smokers without ERT during
the 2-year follow-up.
Smokers on E2
Non-smokers on E2
Non-smokers without E2
r
slope
interc.
r
slope
interc.
r
slope
interc.
Baseline
0.34
0.50
44
0.63
1.35
18
0.66
0.65
28
1 year
0.63
1.14
12
0.58
1.01
14
0.39
0.49
33
2 years
0.64
0.87
20
0.62
0.86
13
0.17
0.21
54
The BMD changes were similar in all treated groups, ERT causing an increase of BMD at all
measured sites, whereas women without treatment lost bone during the 2-year follow-up.
BMD changes at the lumbar spine and femoral neck are shown in Table 23, at the trochanter
and Ward’s triangle the changes were similar.
Table 23: The percentage change of BMD at the lumbar spine and femoral neck in smoking and nonsmoking postmenopausal women on ERT, and in postmenopausal women without treatment.
Lumbar spine
Femoral neck
1 yr, % change
2 yr, % change
1 yr, % change
2 yr, % change
Smokers on E2
2.1
3.6
1.4
2.2
Non-smokers on E2
1.7
2.6
0.9
2.4
Non-smokers without E2
-2.1
-2.4
-0.7
-0.2
In the estrogen-users the changes in bone markers correlated better to changes in the lumbar
spine BMD than to those in the femoral neck BMD. As related to the changes in the lumbar
spine BMD, the r values for the simultaneous changes in urinary NTX were –0.44 (p<0.0001)
at 1 year and –0.52 (p<0.0001) at 2 years, and for serum PINP –0.46 (p<0.0001) and –0.41
(p<0.0001), respectively. As to the femoral neck BMD, the respective coefficients ranged
from –0.20 (p=0.016) to –0.25 (p=0.003) for urinary NTX and from –0.16 (p=0.0049) to
–0.25 (p=0.003) for serum PINP.
Estradiol concentrations of the whole study population were measured at baseline and at 1and 2-year time points. On the daily dose of 1mg estradiol serum concentrations were nonsignificantly 10-20% lower in smoking vs. nonsmoking women, ranging from 0.25 nmol/l to
0.30 nmol/l in smokers. In smokers using the daily dose of 1.5 mg estradiol the concentrations
were 16-20% higher than in those on the lower dose, but the difference did not reach
significance.
5.7.2 Effect of short-term clodronate treatment on bone turnover, study VII
As already shown in 5.2.3, all measured markers had a clear circardian variation with a
significant time effect (p<0.0001 for all measured markers). S-ICTP had its peak value at 4
66
am, whereas the urinary excretion of the resorption markers was as their highest somewhat
later, at early morning hours. The peak values of formation markers were reached earlier, for
PINP at midnight and for PICP at 2 am.
Treatment with clodronate tended to attenuate the diurnal variation of U-NTX (p=0.07 for day
x time), but it did not have a significant effect on the diurnal variation of U-Pyr (p=0.13), SICTP (p=0.65) or S-PICP (p=0.99).
The area under curve (AUC) for each marker and for variables reflecting calcium metabolism
before and after the treatment is shown in Table 24. The significance of the change in AUC is
expressed as p-values. Four-week treatment with oral clodronate significantly decreased
urinary excretion of type I collagen-derived resorption markers, pyridinolines and NTX, but it
had no effect on serum resorption marker ICTP, nor on the formation marker PICP.
Treatment also significantly decreased serum calcium and urinary excretion of calcium,
whereas serum concentration of intact PTH increased.
Table 24: AUCs for markers of bone turnover and calcium metabolism before and after 4-week treatment
with oral clodronate.
AUC, mean + SE
Change in AUC
Analyte
Before
After
p
S-ICTP
69.4 + 6.2
72.8 + 4.2
0.41
U-Pyr
803 + 28
765 + 25
0.016
U-NTX
872 + 74
513 + 53
0.0002
S-PICP
2988 + 100
2987 + 105
0.99
S-Ca
55.9 + 0.4
55.1 + 0.3
0.030
U-Ca
11.2 + 2.7
7.5 + 1.6
0.021
S-PTHi
1028 + 71
1223 + 115
0.004
The effect of clodronate on osteoclastic bone resorption not only decreased urinary excretion
of NTX, but also attenuated its circardian variation, shown in Figure 19 A. The treatment did
not change the circardian pattern of the other measured markers of bone turnover, although
the AUC of Pyr significantly decreased after treatment, shown in Figure 19 B. The effect of
clodronate on osteoclastic activity was also seen as significantly decreased serum calcium
concentration as well as decreased urinary excretion of calcium. These changes were
compensated for by hyperparathyroidism, seen as a significant increase in circulating intact
PTH concentrations, shown in Figure 20.
67
A
B
Figure 19: Diurnal variation of urinary excretion of NTX (A) and Pyr (B) before and after treatment with
oral clodronate; mean + SE at each completed urine collection time point is shown (from the original
publication with permission).
A
B
Figure 20: Serum intact PTH (A) and urinary calcium excretion (B) before and after 4-week treatment
with oral clodronate in nine healthy postmenopausal women; mean + SE are shown (from the original
publication with permission).
5.7.3 Comparison of S-TRACP 5b with S-PINP and U-NTX as the reflector of
the efficacy of oral clodronate treatment in postmenopausal women with
vertebral osteopenia, study VIII
Marker values over time and changes in markers
At baseline of the extension study, the median value for S-TRACP 5b and S-PINP fell within
reference ranges for premenopausal women whereas the median U-NTX was above the
reference range. S-TRACP 5b was above the reference range for premenopausal women for
42% (25) of the subjects, who had been on placebo for the preceding 3 years, U-NTX was
elevated in 70% (41) of the subjects, whereas S-PINP was elevated in only 6.9% (4) of these
subjects. All the marker values decreased significantly during the first year of clodronate,
stabilizing thereafter. At the end of the study 8 (14%), 7 (13%) and 0 subjects had values
exceeding the upper reference range for premenopausal women for S-TRACP 5b, U-NTX,
and S-PINP. Marker values at baseline and after one and two years clodronate treatment
together with the reference values for premenopausal women are shown in Table 25.
68
Table 25: Reference values of the markers for premenopausal women and over time marker values in
subjects on clodronate for two years.
Clodronate group, median (range)
Reference range
of premenopausal
women
baseline
1 year
2 years
S-TRACP 5b U/l
2.40 – 4.00
3.77 (2.32 – 5.55)
3.06 (0.90 – 5.12)
3.06 (1.00 – 4.81)
U-NTX/Crea
nmol/mmol
5.0 – 65.0
74.7 (32.5 – 149.0)
31.5 (11.7 – 128.0)
36.3 (13.5 – 96.9)
S-PINP µg/l
15.0 – 80.0
53.1 (16.7 – 91.6)
26.0 (9.8 – 75.9)
24.5 (10.7 – 75.2)
The percentage changes in all measured markers from baseline to 1- and 2-year time-points
were significant (p<0.0001). The mean decrease in S-TRACP 5b was 21% (95% CI -25.5 to
-15.5) at the 1-year time-point and 18% (95% CI -23.3 to -11.7) at the 2-year time-point, the
mean decrements in U-NTX being 51% (95% CI -60.8 to -36.5) and 47% (95% CI -65.5 to
-28.7), and in S-PINP 46% (95% CI -54.8 to -37.3) and 42% (95% CI -52.5 to -30.9) at the
two time-points (Figure 21).
0
% change
-10
-20
T RACP 5b
NT X
-30
PINP
-40
-50
-60
0
1
2
T ime, years
Figure 21: Mean percentage changes in the markers at 1- and 2-year time-points.
BMD changes
Clodronate treatment for two years resulted in a mean change in the lumbar spine BMD of
+2.1% (95% confidence interval +1.1 to +3.1, p=0.0002), and a mean change in the
trochanter BMD of +2.6% (95% confidence interval +1.3 to +3.8, p=0.0001).
69
Correlations
Between marker percentage changes
Significant correlations existed between all the marker changes from baseline to the two other
time points (1 and 2 years), with correlation coefficients ranging from 0.34 (p=0.009)
between S-TRACP 5b and S-PINP at 2 years to 0.48 (p=0.0002) between TRACP 5b and UNTX at 2 years.
Between marker and BMD percentage changes
The correlations between the percent changes in markers and in BMD are shown in Table 26.
The percentage change in S-TRACP 5b from baseline to 1 year correlated with the percentage
change in lumbar spine and trochanter BMD at the 1- and 2-year time-points. The percentage
change in U-NTX from baseline to 1 year correlated with the percentage change in trochanter
and lumbar spine BMD at the 2-year time-point. The percentage change in S-PINP from
baseline to 1 year correlated with the percentage changes in lumbar spine BMD at the 1- and
2-year time-points, and also with the percentage changes in trochanter BMD at the 2-year
time-point.
Table 26: Correlation between marker percentage changes 1 year after start of the treatment with
clodronate and percentage changes in BMD at measured sites at 1 and 2 years. Correlation coefficient (r)
and significance (p) are presented where the correlation was significant or marginally significant.
BMD % change at lumbar
spine
1 year
S-TRACP 5b
% change at
U-NTX
1 year
S-PINP
2 years
-0.35, 0.0066 -0.36, 0.0068
BMD % change at
femoral neck
BMD % change at
trochanter
1 year
2 years
1 year
2 years
NS
NS
-0.37, 0.0041
-0.45, 0.0006
NS
-0.33, 0.014
NS
NS
-0.23, 0.087
-0.37, 0.0058
-0.28, 0.037
-0.39, 0.0032
NS
NS
NS
-0.36, 0.0081
NS = non-significant.
Responders
The calculated least significant changes for the markers were 26.6% for S-TRACP 5b, 55.1%
for U-NTX, and 32.4% for S-PINP.
The S-TRACP 5b change showed 39.7% of the subjects (23/58) at the 1-year time-point and
34.4% of them (20/58) at the 2-year time-point to be responders. The respective percentages
for U-NTX were 55.2 (32/58) at 1 year and 40.0 (22/55) at 2 years. In terms of the S-PINP
change, the majority of the subjects were responders, 72.4% (42/58) at the 1-year time-point,
and 79.3% (46/58) at the 2-year time-point. The mean percentage change of the whole study
group exceeded the LSC only for S-PINP. The percentage of responders to clodronate
treatment at the two time points by each marker is shown in Figure 22.
70
100
90
% responders
80
70
60
1 yr
50
2 yr
40
30
20
10
0
TRACP 5b
NTX
PINP
Figure 22: The percentage of responders to clodronate treatment by S-TRACP 5b, U-NTX and S-PINP.
Responders were those exhibiting a change larger than the LSC of the marker (26.6%, 55.1% and
32.4%).
6 Discussion
6.1 Assay performance and factors effecting the results
Based on the evaluation of the assays they all performed adequately well to be used in
assessing bone turnover. However, the first Pyr assay was cumbersome with the pretreatment
of samples and overnight incubation. With more specific antibody the pretreatment step was
eased. The precision of the assays over the concentration range used was acceptable in all
assays even when performed manually, it improved when automated pipeting device was
used for sample and reagent pipeting, and was clearly best in the automated assays for DPD
and NTX. Our results on the assay performance were in agreement with the results reported
by the groups who developed the assays, and also by others.13, 15, 21, 23, 24, 247, 315, 316, 317 In
addition, in the NTX CIA assay the analytical sensitivity was greatly improved as compared
to the NTX ELISA. The better assay performance of the two chemiluminescent assays was
not only due to the automation of the reaction steps but also to the more sensitive assay label
in the automated DPD assay and to the more sensitive detection technique of the enzyme
lable in the automated NTX assay.318
In the conventional RIAs for PICP and PINP the standard curve range was limited. By using
the extra standard between the second highest and highest standards included in the reagent
kit, the measuring range was extended to cover the adult reference range, but childrens’
samples were prediluted in PICP assay, and smaller sample volume was used in PINP assay.
Also in S-ICTP assay an extra standard was used, although the reference range fell to the low
concentration area of the standard curve. This standard improved the precision in the
concentration range above 20 µg/l, and enabled most childrens’ and pathological samples to
be assayed without predilution.
The correlation of the propeptide assays as well as all resorption marker assays was highly
significant. The correlation coefficients, the slopes and the intercepts of the linear regression
lines were dependent on whether childrens’ samples were included or not. When the ELISA
and CIA methods for U-DPD and U-NTX were compared, a good correlation between the
methods was found. However, both automated methods produced somewhat higher results as
71
compared to ELISAs. Moreover, we observed that individual results could be quite different
with different methods. The two DPD methods as well as the two NTX methods utilized the
same antibody, but it would be reasonable to assume that slightly individually differing
antigens in the different reaction mixtures could cause different results with different
methods. Also other study groups have reported good correlations between several type I
derived resorption markers, and also between different methods for the same analyte.317 In
that study no differences in individual results by different methods for the same analyte were
reported, in contrast to our results.
We have shown that hemolysis decreases the osteocalcin results in a dose-dependent manner,
independent of the method used. Therefore we undertook to study the effect of hemolysis on
the results of S-PINP, S-PICP and S-ICTP. The results of PICP and ICTP were unaffected by
hemolysis, but gross hemolysis (> 2.5 gHb/l) caused a significant decrease in S-PINP results.
Therefore, S-PINP results from grossly hemolyzed samples were not included in the data
presented here.
Type I collagen-derived markers were shown to be stable in both serum and urine. Serum
markers could be stored in room temperature for 4 days, refrigerated for one week, and deep
frozen in –20 oC for years. The stability of urinary markers NTX and DPD was not tested in
room temperature, but they were stable in freezer for 4 days, and for years in –20 oC. Both
serum and urine samples could be thawed and frozen several times having no effect on the
results. The stability results are in line with the results by others.245, 317 The robustness of
these markers makes it possible to assay the samples after storage periods of years in -20 oC,
and in this respect make these markers superior to labile markers such as intact osteocalcin
and TRACP activity.245
Ultraviolet light degrades free (deoxy)pyridinoline ring structure and thus decreases U-DPD
results.319 On the contrary, we observed that N-telopeptide results increase when samples
were kept in daylight. The reason for this could be that part of the N-telopeptides reside in
large aggregates and are not within reach of the antibody; the aggregates are degraded by the
UV light, causing an increase in measured U-NTX result. For these reasons urine samples
were stored in colored tubes.
All the type I collagen-derived markers have short half lives in the circulation and therefore a
clear diurnal variation, shown in study II in postmenopausal healthy women, but also by
others in premenopausal women.320 The peak values of formation markers were seen at
midnight, those of resorption markers later, in early morning hours. Thus after midnight the
formation marker values declined whereas the resorption marker values increased.
Glucocorticoids are known to cause uncoupling of bone formation and resorption. Serum
cortisol levels show their nadir at midnight, and rising cortisol could explain the uncoupling
of bone formation and resorption after midnight, although results against this theory have
been published.321, 322 In children, significant circardian rhythm for S-PICP, S-ICTP, U-DPD
and U-NTX but not for S-PINP was shown.323 As regards to serum concentration of CTX-β,
the diurnal variation is aggravated by meals, whereas diurnal variation of osteocalcin was not
affected by eating.324, 325 Furthermore, fasting did decrease several marker values in morning
samples of healthy premenopausal women, the effect being more pronounced in resorption
markers than in formation markers.326 Molecules with longer half lives, such as Bone ALP
and TRACP, show only non-significant circardian variations.327, 328
The circardian variation is an important contributor to the intra individual variation of the
markers, and thus to the least significant change (LSC) of the marker.329 The intra individual
72
variation (CVi) is largest for the urinary excretion of type I collagen degradation products,
especially for the peptide-bound crosslink compounds. The timing of the sample is therefore
of utmost importance, and the sample should preferably be taken in a fasting state.246, 326 In
healthy premenopausal women we calculated CVis of 6 to 11% for U-DPD and U-NTX in
samples taken during one month, in agreement with short-term CVis obtained for
postmenopausal women with a mean menopausal age of 17 years.330 The long-term CVis
calculated in study VIII were up to 68% for U-NTX and up to 44% for serum markers,
reflecting the population in early menopause when bone loss is at its fastest, and changes in
bone turnover accordingly at their highest. The LSCs calculated in study VIII were, however,
in accordance with LSCs reported in other studies.329, 331, 332, 333 The LSC for S-TRACP 5b,
27%, was smaller than LSC for S-PINP (32%), and similar to the reported LSC for S-Bone
ALP.329
6.2 Markers in healthy subjects
In healthy adults the marker concentrations were slightly higher in men than in women,
reflecting the fact that men achieve higher peak bone mass than women do,334, 335 S-ICTP
making an exception. In children and young adults all markers were high, declining to adult
levels between the ages 18 to 22 in females and between the ages 23 to 27 in males.
The ratio of type I procollagen propeptide mass concentrations in circulation is expected to be
close to 3, on a molar basis close to one. This was the case in adults, the mean ratio of PICPto-PINP being 3.2 in women and 2.9 in men. In children, S-PINP was much higher than SPICP, and thus PICP-to-PINP ratio was low. Even in young adults between the ages of 20 and
30 the ratio of propeptides was often under 2.0. An explanation to proportionally high S-PINP
as compared to S-PICP could be their different clearance from the circulation. They both are
cleared via liver endothelial cell receptors, PICP via mannose and PINP via scavenger
receptors.235, 239 Mannose receptors have been shown to be regulated by some hormones and
growth factors,336, 337, 338 resulting perhaps in higher clearance of PICP from the circulation
during growth and even in early adulthood. We found significant correlations between SPINP and age, and PICP-to-PINP ratio and age, in the groups of children and young adults. In
pubertal girls significant negative correlation has been reported between several markers of
bone turnover and estrogen.339 The estrogen effect could be the reason for the significant age
dependence of S-PINP and PICP-to-PINP ratio in our study in females under 20 years of age.
In males a similar correlation between S-PINP and age and PICP-to-PINP ratio and age was
found when the age range was extended to 25 years. It is well known that pubertal growth
spurt starts later in boys than in girls, due to androgen effect,334 and men gain bone mass for a
longer period of time, leading to a higher peak bone mass in men than in women.335 Some
very high S-PINP values and low PICP-to-PINP ratios were seen in pubertal boys indicating
growth spurt, as does serum concentration of the amino-terminal propeptide of type III
collagen.340 The growth spurt is not seen as elevations in S-PICP,259 leading to low PICP-toPINP ratio. In pubertal boys during the growth spurt the PICP-to-PINP ratio varied from 0.38
to 0.50. S-PINP value as high as in pubertal boys was also seen in the youngest child of study
I (under 1 year of age); however, the PICP-to-PINP ratio in this child was close to1.0. Data on
S-PINP in healthy children is scanty, but in one recent study it was measured in healthy
pubertal boys (n=155) and girls (n=151), among other markers of bone formation.341 In that
study S-PINP increased from prepuberty until the pubertal stage B3 in girls and G4 in boys;
in both sexes S-PINP correlated with height velocity which peaked at same stage where SPINP was as its highest. Similarly to our study, the highest values were seen in boys.
73
The type I collagen-derived markers of bone resorption were also high in childhood, and like
S-PINP and S-PICP, declined to adult levels somewhat earlier in females than in males. In
several studies involving larger number of healthy children and adolescents it has been shown
that the markers of bone resorption increase up to 2 months of age, then decrease reaching a
stable level at about the age of three years.262, 342 Depending on the marker, the levels in
children are even more than ten times higher than in adults. During puberty the resorption
markers increase, earlier in girls than in boys, and similarly to our study decline to adult
levels earlier in girls than in boys.343 The results obtained in different studies are, however,
somewhat contradictory, in one study the highest values during puberty were seen in girls,343
in another study, consistent with our results, in boys.341 We found a relationship between the
ratio of peptide bound crosslink (NTX) to free crosslink (DPD) excretion and age, the relative
proportion of NTX declining with age. This probably reflects the fact that during high
turnover relatively more of peptide bound crosslinks are excreted.249 Probably due to the same
reason, in studies by others, urinary excretion of DPD increased less than other resorption
markers in pubertal girls.341, 343 Our results on the values of the serum marker of bone
resorption, ICTP, are consistent with the results of others, values declining gradually to adult
levels after puberty. In one study S-ICTP was shown to have the best correlation to bone mass
of the markers measured, and it also correlated well with the height velocity.341 We found the
highest ICTP-values in pubertal girls, contrasting the results of urinary resorption markers.
No difference according to gender was seen in adults.
6.3 Markers in different disease states
6.3.1 In children with asthma, on and off inhaled corticosteroid treatment
Several markers of bone formation, S-PINP, S-PICP and S-OC, and of bone resorption, SICTP, U-Pyr, U-fDPD and U-NTX, were measured in study II to assess the effect of inhaled
budesonide (BUD) treatment on bone metabolism of asthmatic children. Before the treatment
was started type I collagen-derived markers were similar in asthmatic and healthy children,
but S-OC was significantly lower in asthmatic children, consistent with results reported by
others.270 Lowered OC could reflect impaired mineralization, caused by changes in cytokine
production due to the asthmatic inflammation.344 One-month treatment with high-dose
budesonide caused a decrease in all markers. The type I collagen-derived markers did not
markedly change during the 5 months of lower dose budesonide treatment, and they returned
to baseline levels after 6-month treatment with nedocromil. Also OC was stable during the
lower dose of budesonide, and increased above the baseline level, almost reaching the level of
healthy children during nedocromil treatment. Moreover, the good correlation of type I
collagen-derived markers of bone formation (PINP, PICP) and resorption (ICTP) during
budesonide treatment indicated that the balance between the two processes was maintained,
though the turnover was lowered. Slightly different results have been obtained in one
comparative study of the effect of inhaled BUD, fluticasone propionate (FP) or cromone on
bone turnover in 75 school-aged asthmatic children.345 In that study high-dose medication
was given for two months after which the dose was reduced to half. Four months’ treatment
with BUD reduced formation markers (PICP and OC) whereas ICTP-level did not change,
suggesting imbalance between bone formation and resorption. Moreover, in children with
growth or adrenocortical suppression the marker changes were more pronounced. In another
study with prepubertal asthmatic children inhaled corticosteroids (BUD or beclomethasone)
for one year reduced PINP, OC and ICTP whereas PICP slightly increased and Bone ALP did
not change.346 In the former study, the imbalance between bone formation and resorption
could have been caused by the longer treatment period with high-dose BUD, whereas the
results of the latter study are in line with our results, suggesting that the coupling between
74
bone formation and resorption is maintained during the inhaled budesonide, but bone turnover
is lowered. The small increase in PICP could be due to its changed clearance by the liver.
Out of the formation markers, the largest change was seen in S-PINP. Of the resorption
markers, S-ICTP (CTX-MMP) showed the most marked decrease, whereas the decrease in UNTX during high-dose budesonide was non-significant. This difference in response of the two
markers is different from the response seen in postmenopausal osteoporosis.300 The reason for
this difference might be the different mechanisms of bone modeling in children and
remodeling in postmenopausal women. The good sensitivity of S-ICTP is supported by its
good correlation with growth velocity, shown also by others.346
The results obtained in some studies of adults on inhaled corticosteroid treatment have shown
no changes in markers of bone formation (S-Bone ALP, S-OC, S-PICP) or resorption (U-Ca,
U-DPD), neither did the treatment have any effect on BMD.275, 276 In our study, BMD of the
children was not measured, but their height gain during the study year was similar to the
height gain of healthy children, supporting the marker data and the fact that inhaled
corticosteroids do not have deleterious effect on bone or growth as do oral formulations.273, 274
6.3.2 Markers in patients with prior thyroid cancer, on and off thyroxine
treatment
In study III bone turnover and bone mineral density were assessed in patients with prior
thyroid carcinoma and on thyroxine suppressive therapy. All the measured type I collagenderived markers of bone formation and resorption and OC were elevated in the patient
population of 15 premenopausal, 10 postmenopausal women and four men, who had been on
thyroxine therapy for 9 to 11 years, in comparison to age and gender matched controls. Bone
ALP was also higher in patients, but the difference did not reach significance. In several other
studies high bone turnover has been shown in hyperthyroidism and during thyroxine
treatment.277, 278, 347, 348 In one study PICP in patients did not differ from that of controls,348
and indeed, also in our study the median PICP was only 10% higher in patients than in
controls whereas median PINP was 46% and OC 35% higher. Bone ALP in our study was not
significantly higher in patients as compared to controls, a result not in agreement with the
results of several other reported results. The reason for this discrepancy could be the method
used in our study, lectin precipitation of bone isoform combined with electrophoretic
separation of the isoforms in polyacrylamide gel. The method might underestimate the second
bone isoform, which normally is found in minor amounts in the circulation, but can be greatly
elevated in situations characterized by high bone turnover.4, 78, 245, 349 Of the formation
markers measured in our study, PINP seemed the most sensitive one to reflect increased bone
turnover, but unfortunately, it has not been measured in other studies. For bone resorption we
measured only the excretion of HOP, quite an unspecific maker, and serum ICTP, known to
be produced by matrix metalloproteinases. Nevertheless, both of these markers indicated
increased bone resorption to about same extent as markers of bone formation reflected
increased bone formation.
In our study bone formation and resorption were in balance as judged by the measured
markers, and also by the bone mineral densities, which were not reduced in patients as
compared to controls. In some studies significantly reduced BMDs have been reported,348 and
in some studies the detrimental effect has been seen only in postmenopausal women,280
whereas in some studies the effect has been the same, irrespective of menopausal status.281, 282
No changes in bone turnover or bone mass have been observed in some studies when the
smallest possible dose to keep serum TSH suppressed has been used.350, 351 In our study
75
population, no reduction in BMD could be shown, despite of increased bone turnover and
standard doses of LT4 used. The reason for this could be high intake of calcium in the Finnish
population,352 which might counteract the high bone turnover, and keep the formation and the
resorption in balance, resulting in no bone loss.
After the withdrawal of LT4 therapy, the markers decreased significantly reflecting lowered
bone turnover, in agreement with results by others.277 PICP, however, made an exception.
Iatrogenic hypothyroidism caused a significant increase in PICP whereas PINP significantly
decreased. The increase in PICP after stopping the treatment, as well as it being less, although
significantly, increased during therapy in comparison to PINP, results most probably from the
action of thyroxine on mannose receptors. As discussed above, mannose receptors are
sensitive to growth factors and hormones, and they are responsible for the clearance of PICP
whereas PINP is cleared via scavenger receptors, which are insensitive to hormones. The
same phenomenon was also seen in growing children, who had lower than expected PICP
concentrations and therefore a lowered PICP-to-PINP ratio.
Serum OC decreased by 42% after five weeks of hypothyroidism whereas PINP decreased
only by 7%. The decrease in OC was almost of same magnitude as the decrease in ICTP,
54%. The method with which OC was measured was a competitive radioimmunoassay
against human OC, and the antibody recognized intact OC, N-Mid-OC and also smaller
breakdown products of the protein. OC is imbedded to bone organic matrix, and therefore OC
and its breakdown products are freed to the circulation during bone resorption. Moreover, our
study showed that during hypothyroidism the active metabolite of vitamin D increased.
Vitamin D stimulates OC synthesis which would increase de novo liberated circulating OC.
Therefore, with the method in our study, OC reflected both bone resorption and formation.
Bone formation was reflected by PINP, which in five weeks decreased much less than OC,
consistent with the fact that changes in markers of bone formation are seen later than changes
in markers of bone resorption, due to the bone remodeling cycle which starts with resorption,
followed by reversal phase and finally formation. Even in these patients with high bone
turnover, five weeks was too short a time for PINP to decrease as much as marker of bone
resorption, ICTP, or turnover, OC, decreased.
6.3.3 Markers in bone disease due to malignancy
In the study of type I collagen-derived serum markers in patients with breast cancer and bone
metastases, serum ICTP was significantly higher in patients than in healthy, age-matched
controls, the median and mean value being above the upper reference range of the marker.
Also its specificity in diagnosis, 93%, was better than that of urinary excretion of several type
I collagen breakdown products, urinary deoxypyridinolines or N-telopeptides.283, 284 In our
study it was, however, elevated only in 56% of the patients, and therefore was not sensitive
enough to be used for diagnosis of the bone disease. In later studies ICTP has been shown to
be a sensitive marker of bone metastases, whether lytic or sclerotic, and higher clinical
sensitivities than we obtained have been reported.284, 353, 354, 355 Some studies have shown the
value of ICTP in the follow-up of disease outcome.290, 291 In our study 13 patients with bone
metastases were followed up, and indeed, ICTP changes reflected the disease outcome.
Significance of the changes was not calculated due to the small number of patients. Several
recent studies have, however, shown that ICTP does not respond to the treatment with
bisphosphonates, the most common therapy for bone metastases, as sensitively as do the
excretion of peptide-bound type I collagen breakdown products, especially NTX.284, 356 The
reason for this different response lies most probably in the mechanism of action of
bisphosphonates. It has been clearly demonstrated that all bisphosphonates decrease
76
osteoclastic bone resorption by several mechanisms, one of them being the inhibition of the
activity of bone resorbing enzymes, both cathepsins and matrix metalloproteinases.357, 358
Nevertheless, in an in vitro model of multiple myeloma bisphosphonates have been shown to
have an opposite effect on one of the MMPs, the MMP2: the aminobisphosphonates
pamidronate and zoledronate upregulated MMP2 expression, and the upregulation was more
prominent with the more potent bisphosphonate zoledronate.359 NTX and CTX are produced
by cathepsin K and MMP9, whereas MMP2 and MMP13 are responsible for the formation of
ICTP.360 MMP2 is frequently expressed in tumors, and its increased expression during
bisphosphonate therapy could explain the unchanged levels of ICTP.
The markers of bone formation, PICP and PINP were elevated in only 24% and 30% of the
patients, respectively, reflecting the fact that most patients had osteolytic bone lesions, some
having mixed lytic and sclerotic lesions. The results of several studies support our data,
formation markers being elevated only seldom when the bone disease is lytic.288, 292 Although
the sensitivity of PINP was low in detecting bone metastases from breast cancer, we were the
first to show that the ratio of the two propeptides changes in these patients: the PICP-to-PINP
ratio was lowered from about 3 to less than 2 in more than 50% of the patients with bone
involvement, and only a few patients without bone lesions had a lower than normal ratio, the
specificity of the ratio being 91% when the cut-off value of 2 was used. The propeptides and
their ratio were then analyzed in a larger population of breast cancer patients, divided into two
groups according to the aggressiveness of the disease, into patients with progressive or stable
disease. The optimal sensitivity and specificity of 90% of S-PINP for aggressive disease were
attained, when a cut-off value of 70 µg/l was used. Similarly the optimal sensitivity and
specificity of 98% and 90% of the PICP-to-PINP ratio was attained using a cut-off value of
1.74. As the PICP-to-PINP ratio is lowered during growth and when bone mass is gained, as
well as during thyroxine treatment and in active Paget’s disease of bone,361 a reasonable
explanation to the altered ratio in advanced neoplastic disease would be that the cytokines and
growth factors secreted by the tumor enhance the clearance of PICP by the liver endothelial
cell mannose receptors. Another explanation to the discrepant propeptide levels could be the
formation of α1-trimeric variant form of type I collagen in malignancy,362, 363 the variant
PINP being measured in the assay used whereas homotrimeric PICP would not be recognized
by the assay antibody.
The prognostic value of S-PINP has been later shown in a study involving 300 node-positive
breast cancer patients. 364 Postoperative intact PINP was shown to be higher in patients who
developed metastatic disease, either to bone, visceral or soft tissues, and patients with high
PINP value had poorer survival as compared to patients with low PINP. Similar results of the
value of S-PINP were obtained in a study where the ELISA method that recognizes the intact
PINP and the Col1 fragment was used to measure S-PINP in 100 patients with recurrent
breast cancer.16, 365 In that study S-PINP was measured after the first recurrence, before
systemic treatment was started. S-PINP was shown to correlate positively with the tumor
burden, and patients with high PINP had shorter time to disease progression, shorter overall
survival and their response to systemic treatment was poorer than patients with low PINP
level. In line with our study, the highest PINP concentrations were seen in patients with bone
involvement, but also in patients with only soft tissue metastases the median PINP was higher
than in healthy controls.
6.3.4 Effect of smoking on markers
Study VI was undertaken to investigate the effect of smoking on the marker of bone
formation, S-PINP, and resorption, U-NTX, and on the response of transdermal estrogen
77
replacement therapy in early postmenopausal women. Also vitamin D status of smoking and
non smoking postmenopausal women was assessed.
At baseline smokers did not differ from non-smokers as to their marker values, in line with
the similar BMD values in the two groups. The BMD finding is confirmed by a recent metaanalysis on on the risk factors for fractures.366 However, in smokers the correlation of the two
markers was worse than in postmenopausal and premenopausal non-smokers, perhaps
indicating worse balance between bone formation and resorption in smokers. Unbalance
between bone formation and resorption has been reported in a few recent studies, either due to
decreased bone formation or increased bone resorption or both.296 - 299 In these studies serum
OC was shown to be lowered in smokers whereas Bone ALP was similar in smokers and in
non-smokers. In two of the studies bone resorption was shown to be increased in smokers as
compared in non-smokers.296, 299 In one of the studies this was shown especially in heavy
smokers with over 20 years since menopause, in non-heavy smokers markers did not differ
from those of non-smokers.296 In the other two studies no difference in bone resorption,
measured as urinary NTX or hydroxyproline, was seen.297, 298
Our finding of lowered serum 25(OH)D levels in smoking women is in agreement with
previous studies,296, 297, 367 hypovitaminosis D perhaps being a major contributor to reduced
calcium absorption in smokers.367 Furthermore, hypovitamonis D could at least in part explain
lowered OC levels in smokers, reported by others, as vitamin D stimulates the synthesis of
OC.
6.4 Markers in the follow-up of antiresorptive treatment of
postmenopausal osteoporosis
6.4.1 Estrogen replacement therapy
Study VI showed no difference in response to estrogen treatment between smokers and nonsmokers, shown by both BMD changes and changes in markers. Also, increasing the E2 dose
in smokers from the standard dose of 1 mg daily to 1.5 mg daily did not change the response.
Some studies have exhibited a lessened response of BMD to oral estrogen treatment in
smokers vs. non-smokers, supporting the suggestion that smoking may negate the protective
effect of oral ERT on BMD and hip fracture. 368 In one study, it was shown that the effect of
smoking on BMD was dose dependent, reducing the BMD increase in women using 1 mg oral
estradiol daily but not at all in those using 2 mg.369 With both estradiol doses, the serum
estradiol levels in smokers were half of that in non-smokers, but the level of 0.15 nmol/l in
smokers on 2 mg daily estradiol was adequate to keep the response similar as in non-smokers.
The reason for lessened BMD response to oral treatment resides in an increased hepatic
metabolism of estrogens resulting in lower estrogen levels among postmenopausal smokers.
In our study serum estradiol levels were non-significantly lower in smokers than in nonsmokers, ranging from 0.25 nmol/l to 0.30 nmol/l in women receiving 1 mg estradiol daily,
and being well above the critical level of 0.15 nmol/l in the above mentioned study. Our study
demonstrated that smoking does not diminish the response of the lumbar spine and femoral
neck BMD to transdermal estrogen treatment even when used in standard doses, transdermal
route of administration circumventing smoking-induced increase in hepatic metabolism of
estrogens.
Bone marker measurements confirmed that the bone-sparing effects of treatment with
transdermal estrogen were not negated by smoking. Urinary excretion of NTX was reduced to
78
a similar extent in both smoking and non-smoking women during the treatment. The
reduction was somewhat less than a 52% decrease during one year of oral estrogen treatment
in a study in which increases in BMD corresponded those obtained in our study.370 The
formation marker serum PINP diminished to a similar extent as urinary NTX, but seemed to
do so more in smokers than in non-smokers. Also, S-PINP increased in non-smokers during
the second treatment year whereas in smokers it remained stable. In two studies of
postmenopausal women, the circulating concentration of PINP decreased to about half of the
pre-treatment level during 1 year of transdermal ERT, consistent with our results.371, 372 In these
studies the subjects were a combined group of smokers and non-smokers, and the follow-up
periods were 1 and 2 years. The study with a 2-year follow-up did not show any increase in SPINP during the second year in subjects less than 67 years of age. The difference in the results of
our study could perhaps be explained by somewhat, though not significantly, higher circulating
E2 concentrations in non-smokers, which could stimulate osteoblast activity as well as suppress
osteoclast activity, leading to more positive balance in non-smokers during extended
treatment.373 Another explanation could lie in the metabolism of PINP. The formation of PINP
and its release to circulation is decreased by estrogen treatment through slowing bone turnover,
which decrease can be anticipated to be similar in smoking and non-smoking women. PINP is
eliminated from the circulation mainly via scavenger receptors on liver endothelial cells.239 It can
be hypothesized that smoking increases the removal of serum PINP through hepatic induction.
Subsequently, in the face of a similar reduction in input, the ongoing increase in removal might
result in a more pronounced drop in S-PINP level in smoking vs. non-smoking women during
ERT.
Our marker data also showed that ERT reverses the poor balance between bone resorption
and formation in smokers, the correlation of the two markers, S-PINP and U-NTX, reaching
similar level as in non-smokers after one year on ERT. On the other hand, the marker data
also showed worsening of the balance in non-smokers with advancing menopausal age and
without estrogen replacement.
6.4.2 Short-term clodronate treatment
In the study of the effects of short-term treatment with oral clodronate on markers of bone
turn-over and their diurnal variation, the response was seen only in the urinary excretion of
NTX. The AUC for NTX decreased by 41% whereas that of free Pyr decreased only by 4.7%,
and no change was seen in the AUC of S-ICTP or in the AUC of the marker of bone
formation, S-PICP. Further, treatment with clodronate lessened diurnal variation of NTX
only. Several studies have shown the different response to treatment of various markers. Six
weeks’ treatment with estrogen decreased urinary excretion of NTX and total
deoxypyridinoline, measured by HPLC, by 38% and 40%, but free pyridinoline excretion,
measured by ELISA, and S-ICTP decreased only by 16%.374 In one study the response to
alendronate treatment for one year in late postmenopausal osteoporotic women was assessed
with several markers of bone turnover.300 Of the resorption markers, the best response was
seen in urinary NTX, in S-ICTP and urinary free pyridinolines the response was poorest. Yet
another study showed that free deoxypyridinoline excretion responded to estrogen treatment
but not at all to treatment with bisphosphonate.375 A doubt was thrown as to the bone
specificity and origin of U-Pyr and S-ICTP, due to their unresponsiveness to bisphosphonate
treatment. It is known that pyridinoline crosslinks originate from cartilage as well as from
bone, whereas deoxypyridinoline is more specific to bone. The ELISA for free pyridinoline
excretion was the first available immunoassay for assessing urinary excretion of type I
collagen degradation product, and it measured both pyridinolines and deoxypyridinolines,
free and bound to peptides less than 1000 Daltons. Pyr assay has been greatly replaced by
79
more bone specific markers since the introduction of the ELISA measuring specifically free
DPD excretion, and especially the assays measuring peptide-bound crosslink excretion NTX
and CTX. In part the better sensitivity of U-NTX in comparison to free Pyr excretion as
response to bisphosphonates treatment could be explained by slowing down bone turnover
which in itself decreases peptide bound crosslink excretion.249 As regards to S-ICTP, the
explanation to its poor responsiveness to treatment in benign conditions such as osteoporosis
lies in the action of enzymes degrading bone organic matrix, the action of cathepsin K
destroying the ICTP antigen and leading to the formation of CTX and NTX.254 Matrix
metalloproteinases are the enzymes responsible for the formation of immunoreactive ICTP
antigen, and though functioning also normally, contribute to a lesser extent to bone
degradation in benign conditions.153 - 156, 162 - 165
Several studies have also shown the poor responsiveness to treatment of S-PICP. In the
alendronate study mentioned above, S-PICP showed the poorest response of about 20%
among several bone formation markers studied.300 The upregulation of mannose receptors by
growth factors during high bone turnover could be the reason for the poorer response in SPICP as compared with other formation markers. In our study S-PICP showed no response at
all. This was probably due to the short follow-up period. In the above mentioned alendronate
study300 it was shown that a significant response in a resorption markers is seen after about
one month’s treatment, and the marker values reach their nadir in 3 to 6 months where after
they remain stable. In formation markers the response is seen later – after about 3 months’
treatment they decline significantly, and reach their nadir in 6 to 12 months, a finding that can
be explained by the sequence of the bone remodeling cycle.
In addition to significant decrease in urinary NTX, clodronate treatment decreased serum and
urinary calcium levels and caused compensatory hyperparathyroidism, seen as increase in
serum intact PTH levels. These findings indicated that clodronate treatment for 4 weeks
efficiently suppressed osteoclast activity.
6.4.3 S-TRACP 5b in comparison with U-NTX and S-PINP as a reflector of
treatment efficacy with clodronate
Study VIII was planned to compare changes in S-TRACP 5b to changes in urinary excretion
of NTX and in S-PINP in the follow-up of clodronate treatment of osteopenic
postmenopausal women. TRACP 5b is an osteoclast-specific enzyme with only nonsignificant circardian variation, and therefore it could be expected to reflect bone resorption
more accurately than do type I collagen-derived markers.
In comparison to the reference range for premenopausal women, serum TRACP 5b
concentration was elevated in 42% of the subjects, U-NTX was elevated in 70% of the
subjects, whereas the marker of bone formation, S-PINP, rose above the reference range in
only 7% of the subjects. In addition, the median value of U-NTX was above the upper
reference limit for premenopausal women, but the median of S-TRACP 5b fell within the
reference range. These findings may indicate uncoupled bone formation and resorption in this
group of postmenopausal women with vertebral osteopenia. Moreover, urinary excretion of
NTX seemed to detect osteopenic subjects more efficiently than did S-TRACP 5b. In several
studies the mean values of U-NTX and S-PINP in postmenopausal healthy women have been
shown to be somewhat higher than in premenopausal women, but in osteoporotic
postmenopausal women the marker means are significantly higher.300, 376, 377 In one study STRACP 5b was shown to be elevated in 48% of 29 osteopenic women, a result consistent
with the result of our study.378
80
The changes in the two markers of bone resorption correlated well, but the percentage change
in TRACP 5b was less than half the percentage change in U-NTX. This may be explained by
clodronate action. As discussed above, bisphosphonates affect bone resorption at several
levels:358 They inhibit the adhesion of osteoclasts to bone matrix, preventing the formation of
actively resorbing cells, and they inhibit osteoclastic proteolytic enzymes. They also cause
osteoclast apoptosis, the mechanism and kinetics depending on the bisphosphonates in
concern: the bisphosphonates which contain amino groups do so with slow kinetics by
preventing protein prenylation. The non-amino bisphosphonates such as clodronate cause
quick apoptosis by forming complexes with ATP, thus blocking the mitochondrial ADP/ATP
translocase.379, 380 Depending on which bisphosphonate, different mechanisms may
predominate in vivo.381 The finding that TRACP 5b was reduced to a lesser extent than UNTX could be explained as follows: First, clodronate acts mainly by causing osteoclast
apoptosis, but does not affect the formation of early osteoclasts, which would still secrete
TRACP 5b into the circulation. Second, it inhibits the activity of bone resorbing enzymes,
leading to decreased amounts of type I collagen breakdown products.
The TRACP 5b changes predicted BMD changes no better than did the type I collagenderived markers, and the markers' early changes predicted later changes in BMD at the
lumbar spine and trochanter only. These results are in agreement with those of several other
studies showing early marker changes in a response to HRT or bisphosphonates correlating
with later BMD changes only slightly.28, 311, 324, 331, 332 The markers used in those trials have
covered all the markers measurable with commercially available reagents. Urinary NTX,
CTX, DPD, and S-CTXβ changes from baseline to 3 to 6 months have been found to be
significant predictors of spinal BMD changes only, as have been the changes in serum
formation markers Bone ALP or OC. In some studies, marker changes have also been
predictive for BMD changes at other skeletal sites, especially in trials involving elderly
populations,310 whereas some studies have found no correlations at all between early marker
changes and later BMD changes.303, 304
Our LSC for S-TRACP 5b, 26.6%, was much smaller than that for U-NTX, 55.1%. However,
TRACP 5b changes identified somewhat fewer responders to treatment with clodronate than
did NTX changes, and markedly fewer than did PINP changes. These results are not in
agreement with results for TRACP 5b changes as a response to HRT in healthy early
postmenopausal women.27 In that small study a highly significant mean decrease of 48% in STRACP 5b was apparent after six months on HRT, and a decrease larger than an LSC of
26.2% occurred in 13 of 15 patients on HRT. The difference between the results of these two
studies may be in part explained by the differing mechanisms of the interventions. Estrogen
replacement directly affects the number of TRACP secreting osteoclasts by inhibiting
cytokines essential for osteoclast formation,184 - 186 whereas clodronate treatment influences at
a later stage of osteoclast development and bone resorption.381
The marker of the greatest value in finding the responders to clodronate treatment was SPINP. Although published data on S-PINP as a marker of the efficacy of antiresorptive
treatment are scanty, some studies support our finding. In a study of subjects with surgical
menopause,377 among several markers of bone formation (Bone ALP, PINP, PICP, OC), SPINP exhibited the highest response to HRT. Of the resorption markers measured (U-CTXβ,
U-CTXα, U-NTX, S- S-ICTP, S-CTXβ, S-TRACP) S-CTXβ was the most efficient, showing
as high a response as S-PINP. S-PINP was also shown to respond similarly to HRT use in
natural menopause.376 The reason for S-PINP being superior to U-NTX in detecting the
81
responders to clodronate treatment is mainly the larger intra-individual variability in U-NTX;
the mean percentage decreases in these markers were of the same magnitude.
The goal of the treatment or prevention of osteoporosis is to prevent fractures. Therefore, the
best bone marker would be the one which best predicts the appearance of fractures. For the
purpose of calculating the efficacy of a treatment in fracture prevention, long-term studies
with large populations are needed. In a few such studies changes in bone markers have been
shown to be of more significance than changes in BMD. As a response to raloxifene therapy,
reduced fracture risk was related to the change in bone turnover but not at all to the change in
BMD.382 In addition, the bone formation markers S-Bone ALP and S-OC were more
advantageous than urinary CTX. Also, in one study low carboxylated to total OC ratio was
shown to be a strong predictor of fracture among elderly population.383 In yet another study,
the decrease in bone resorption markers predicted up to 40% of the effects of risedronate on
vertebral fracture risk, whereas the change in BMD explained only 28% of the reduced
risk.384 In the FIT study, BMD changes predicted only 16% of the effects of alendronate on
vertebral fracture risk.385
82
7 Conclusion
To conclude, the commercially available reagents for measuring type I collagen-derived bone
markers behave adequately well to be used in routine. The markers tested were robust, and
the samples could be stored at -20 oC for years without any influence on the results. Diurnal
variation contributes to the significant intra-individual variation of the markers, and timed
samples are needed to obtain comparable results in the follow-up. The least significant change
of serum marker PINP (amino-terminal propeptide of type I procollagen) was much smaller
than that of urinary marker NTX (crosslinked amino-terminal telopeptide of type I collagen).
Serum PINP stands out as a sensitive marker reflecting bone formation in a variety of clinical
situations. In healthy children and young adults it correlated with age, the highest values
being seen in pubertal boys. During high-dose budesonide treatment of asthmatic children it
was the most sensitive formation marker measured to reflect the treatment effect on bone
turnover. In patients with prior thyroid carcinoma, and on thyroxine treatment, S-PINP
sensitively reflected increased bone formation, and the effect of 5 weeks’ iatrogenic
hypothyroidism on bone formation was seen only in S-PINP. S-PICP (carboxy-terminal
propeptide of type I procollagen) increased during hypothyroidism, possibly because of its
lowered clearance from the circulation due to thyroxine withdrawal. In patients with breast
cancer S-PINP was prognostic for aggressive disease.
The PICP-to-PINP ratio is lowered in children and young adults; the lowest ratios in healthy
population are seen in pubertal boys. Lowered ratios were also seen during thyroxine
treatment and in patients with aggressive breast cancer, for which the ratio less than 1.74 was
highly prognostic. The reason for the lowered ratio during growth and in states of high bone
turnover lies most probably in the clearance of the propeptides from the circulation, the
clearance of PICP via mannose receptors of liver endothelial cells being sensitive to
hormones, cytokines and growth factors whereas the clearance of PINP is not.
Urinary excretion of NTX seems to be the marker which most sensitively reflects bone
resorption in adults, as judged by the increased excretion after menopause and in osteopenic
subjects. In children, the most sensitive resorption marker is serum ICTP (crosslinked
carboxy-terminal telopeptide of type I collagen). S-ICTP showed the largest response to
inhaled glucocorticoid treatment in asthmatic children among several markers of bone
resorption; the good correlation between S-PINP and S-ICTP, and their similar magnitude of
decrease indicated balanced bone formation and resorption even during high-dose treatment.
S-ICTP reflected increased bone resorption in patients with prior differentiated thyroid
carcinoma and on thyroxine treatment, and it responded to iatrogenic hypothyroidism. In
breast cancer patients elevated S-ICTP was indicative of bone metastases.
In the follow-up of antiresorptive treatment of postmenopausal osteoporosis S-PINP is the
most efficient one of the markers measured. It decreased to a similar extent as U-NTX, but
due to its smaller LSC (least significant change), it detected more responders to treatment
with clodronate than did U-NTX. The serum concentration of TRACP 5b (tartrate resistant
acid phosphatase isoform 5b) decreased less than the type I collagen-derived markers, but it
detected about as many responders to treatment as did U-NTX, again due to its smaller LSC.
Serum ICTP did not change at all as a response to treatment with clodronate for one month,
whereas urinary NTX decreased significantly. The treatment response in various markers, as
83
well as the magnitude of the response, seems to depend on the therapy and its mechanism of
action.
84
8 Summary
This study was undertaken to evaluate the performance of several commercially available
immunoassays for measuring type I collagen-derived markers of bone formation and
resorption, to study factors which affect the interpretation of the results, and to evaluate the
utility of these markers in different clinical situations.
The commercially available reagents for measuring type I collagen-derived bone markers
performed adequately well to be used in routine, the most precise methods being those using
automated techniques. Most of the type I collagen-derived markers are robust, and the
samples can be stored at -20 oC for years without any influence on the results. Gross
hemolysis caused lowering of S-PINP (amino-terminal propeptide of type I procollagen)
results whereas S-PICP (carboxy-terminal propeptide of type I procollagen) and S-ICTP
(cross-linked carboxy-terminal telopeptide of type I collagen) were unaffected. Diurnal
variation is an important contributor to the significant intra-individual variation of the
markers, and therefore timed samples are needed to obtain comparable results in the followup. The least significant change (LSC) of serum marker PINP was much smaller than that of
the urinary marker NTX (cross-linked amino-terminal telopeptide of type I collagen).
In healthy population propeptides S-PINP and S-PICP were high in children, declined to adult
levels earlier in girls than in boys, and S-PINP somewhat increased in women after
menopause. In children and in young adults S-PINP correlated negatively with age, and it was
proportionally higher than S-PICP, leading to lowered PICP-to-PINP ratio during childhood
and early adulthood. In adults the propeptide concentrations were somewhat higher in men as
compared to premenopausal women. The resorption markers, urinary excretion of NTX, and
the excretion of free pyridinolines, U-Pyr, and deoxypyridinolines, U-DPD, changed with age
similarly to the formation markers. However, the ratio of peptide-bound to free crosslink
excretion (U-NTX-to-DPD) was age-dependent, the relative proportion of NTX being higher
in children and young adults. Serum concentration of ICTP, was also high in children, and
declined to adult levels as did the other markers. In adults, no difference between genders in
S-ICTP was found.
In children with newly diagnosed asthma, before treatment the type I collagen-derived
markers S-PINP, S-PICP, S-ICTP, U-NTX, U-Pyr and U-DPD did not differ from marker
values in healthy children, but S-OC (osteocalcin) was significantly lower. During treatment
with high-dose inhaled corticosteroid for one month all markers decreased, S-PINP and SICTP showing the largest responses. The good correlation between formation and resorption
markers and the similar magnitude of the decrement in S-PINP and S-ICTP indicated
unchanged balance between bone formation and resorption, despite of decreased bone
turnover. During low-dose budesonide treatment for five months the marker levels did not
further change. Type I collagen-derived markers increased to baseline levels after six months
from withdrawal of the glucocorticoid, and S-OC increased above the baseline level, being
similar to S-OC in healthy children. As judged by the marker data, inhaled budesonide
treatment did not have a deleterious effect on bone metabolism and growth in children.
Markers of bone turnover, S-PINP, S-PICP, S-OC, S-ICTP and U-HOP, indicated increased
bone turnover in patients with prior differentiated thyroid carcinoma on high-dose thyroxine
treatment. In spite of increased bone turnover bone mineral densities were similar in patients
85
and in controls. After 5 weeks of iatrogenic hypothyroidism, due to stopping thyroxine
treatment, bone turnover decreased, as judged by decreased levels of S-PINP, S-OC, S-ICTP
and U-HOP. On the contrary, S-PICP increased significantly possibly because its altered
clearance by thyroxine withdrawal via liver endothelial cell mannose receptors, which are
known to be influenced by hormones and growth factors. S-PINP was the marker which
reflected altered bone formation whereas S-OC reflected both formation and resorption, as
judged by the magnitude of changes in the two markers.
S-ICTP and S-PINP were higher and the PICP-to-PINP ratio was lower in patients with bone
metastases from breast cancer in comparison to healthy controls and to patients with breast
cancer without bone involvement. The clinical sensitivities of 50 to 60% of S-ICTP and
PICP-to-PINP ratio are not high enough for these markers to be used in diagnosing metastatic
bone disease, despite their good clinical specificities of more than 90%. Increased S-PINP
and decreased PICP-to-PINP ratio were highly prognostic for aggressive breast cancer.
S-PINP was similar in postmenopausal smokers and non-smokers, and it was not significantly
different from S-PINP in premenopausal non-smokers. On the contrary, U-NTX was higher in
postmenopausal women as compared to premenopausal women, but smoking did not cause
any difference in U-NTX. However, the correlation of the two markers was poorer in
postmenopausal smokers in comparison to non-smokers, indicating perhaps unbalance
between bone formation and resorption, in the favour of resorption, in smokers. In a group of
postmenopausal osteopenic women, both smokers and non-smokers, U-NTX was elevated
above the premenopausal reference range in 70% of the subjects whereas the other measured
resorption marker S-TRACP 5b was elevated only in 40% of the subjects. In this group, SPINP was above the premenopausal reference range in 7% of the subjects, indicating
unbalance between bone formation and resorption in osteopenic women.
As judged by the BMD and marker results, smoking did not lessen the effect of transdermal
estrogen replacement therapy (ERT), and the standard dose of estrogen was adequate also in
smokers for the bone sparing effect of ERT. However, during the second treatment year bone
formation increased in non-smokers with concomitant stable bone resorption, seen as
increased S-PINP and stable U-NTX, whereas in smokers both markers were stable, perhaps
indicating more positive bone balance in non-smokers during extended treatment.
The response to short-term treatment with oral bisphosphonates clodronate was seen as
decreased levels of U-NTX and decreased amplitude of diurnal variation of U-NTX.
Clodronate did not influence the diurnal variation of other measured markers, U-Pyr, S-ICTP
and S-PICP, nor their levels. The most sensitive marker to find responders to clodronate
treatment for one and two years in early postmenopausal osteopenic women was S-PINP. The
change in S-PINP was larger than the LSC of the marker in 79% of the subjects after two
years on clodronate whereas the changes in markers of bone resorption, U-NTX and STRACP 5b, found only 40% and 34% of the subjects to be responders. Early marker changes
as response to treatment with transdermal estrogen or oral clodronate predicted later changes
in bone mineral density at sites where trabecular bone predominates. As judged by the results
of this study and studies by others, the response of individual markers of bone turnover to
different treatments varies depending on the mechanism of the intervention.
86
9 Acknowledgments
I am indebted to my supervisor Docent Matti Välimäki, MD, in many ways. He was the one
to arouse my interest in bone markers and bone metabolism, and it was his idea to compile
some of my work to a thesis. His ideas, energy, encouragement and advice have brought this
work to completion.
I am grateful to Docent Sirkka-Liisa Karonen, my other supervisor, for teaching me the
secrets of immunoassays, and for her many valuable advice. I also thank her for her
encouragement, continuous support and friendship during all these years.
I owe my deepest thanks to Professor Juha Risteli, MD, for his collaboration, valuable advice,
and for many ideas our long discussions arouse in me. I also wish to thank Professor Leila
Risteli, MD, for her collaboration.
I am grateful to my collaborator Docent Kalevi Laitinen, MD, for helping me in statistical
matters, for his valuable advice and comments, and for his support.
I am indebted to Docent Ritva Sorva, MD, for making it possible for me to cowork with the
research group at the Department of Allergic Diseases, and I also want to thank Dr Antti
Sorva, MD, PhD, for his collaboration.
I am grateful to all my collaborators, and special thanks I owe to Johanna Seppänen, MSc,
skillful statistician, who patiently explained statistical matters to me.
I owe my sincere thanks to the referees of my thesis, Professor Kalervo Väänänen, MD, and
Docent Jorma Salmi, MD, for their expert advice and constructive criticism.
Large part of the practical work was performed at the premises of United Laboratories Ltd,
and I owe my sincerest and deepest thanks to Dr Juhani Aho, MD, for letting me have the
facilities at my disposal. Without Dr Aho’s input this work would never have been possible.
I am grateful to Ms Leena Lehikoinen who not only collected samples but also kept them in
excellent order.
I owe my thanks to Seija Niemi, MSc, for answering my many questions on Orion
Diagnostica assays, and I also thank her for her friendship, and for many joyous moments
during congress trips. I am indebted to Mika Sorsa, MSc, who has been such a great help with
the computers, rescueing my manuscript at several stages. I am grateful to my friends and
colleagues at former Leiras Clinical Research for their support. It was enjoyable to work in
the enthusiastic and inspiring bone and oncology team, and the work had a great impact on
my thesis. I am also grateful to all my friends who have supported me in many ways during
these years, and especially I want to thank my friend Ms Kaija Linturi, who was always there
when needed.
I thank Miss Linda Mellberg for making my idea for the cover page to reality by drawing the
picture of our dog Titus, who shares a common interest with me, the bone.
87
I owe my deepest gratitude to my husband and collaborator Dr Eric Thölix, MD. He has been
a great proof reader of my manuscripts, he has supported and encouraged me to go on, and in
spite of some moments of despair and misery, these past years have been just wonderful.
I want to devote my warmest thanks to my daughters Johanna and Henna for their
understanding and patience. During these years they grew up to two lovely and independent
young ladies, who have supported and encouraged me in many ways. I am also indebted to
my mother, a philologist, who was dedicated to Icelandic sagas as I am to bone metabolism.
She would have loved to see my thesis completed.
This study was carried out at Helsinki University Central Hospital at the Division of
Endocrinology, Department of Medicine and at the Department of Allergic Diseases, at Vaasa
Central Hospital, at Oulu University Hospital, and at the Department of Clinical Chemistry of
Helsinki University. The studies were financed by the Finnish Cancer Research Foundation,
the Medical Research Council of the Academy of Finland, the Juha Vainio Foundation, the
Allergy Research Foundation, the Emil Aaltonen Foundation, the Finnish Anti-Tuberculosis
association foundation, the Technology Development Center, Leiras Oy, Orion Corporation
Orion Pharma and the Foundation of Clinical Chemistry. Reagents for some of the studies
were provided by Orion Diagnostica, Oulunsalo, Finland, and in part for one study by
Suomen Biotekniikka Oy, Oulu, Finland.
88
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