Calcif Tissue Int (1991) [Suppl] 49:$26-$30
Calcified Tissue
Use of Non-Collagen Markers in Osteoporosis Studies
Markku T. Parviainen, 1'2 Asta Pirskanen, 2 Anitta Mahonen, 2 Esko M. Alhava, 3 and Pekka H. M~ienp/iti 2
Departments of ~Clinical Chemistry, ~Biochemistry and Biotechnology, and 3Surgery, University of Kuopio, P.O.B. 6, SF-70211 Kuopio
21, Finland
Summary. Clinical and research laboratories routinely mea-
sure various hormonal and nonhormonal parameters of cal-
cium and phosphorus metabolism, and markers of bone turn-
over. Such measurements may help clinical decision-making
relating to metabolic bone disease and osteoporosis. Molec-
ular biological and cell-culture techniques are being used in
basic biochemical research on bone-cell metabolism. Results
may aid understanding of normal and abnormal regulation of
the bone-cell metabolism, and thus provide further insights
relating to the diagnosis and prevention of osteoporosis.
Key words: Biochemical markers - Osteoporosis - Osteo-
blasts - Osteosarcoma.
Osteoporosis is a feature of normal physiological aging as
well as of a number of disease states. Osteoporosis of aging
involves nonhormonal and hormonal factors. Estrogen,
1,25(OH)2D (calcitriol), and parathyroid hormone (PTH) are
critically concerned hormones. Osteoporosis arises from an
imbalance in bone remodeling: more bone is resorbed than
formed. Age-related osteoporosis shows itself in altered
urine biochemistry [1]. Excretion of hydroxyproline (HOP)
is increased, and excretion of calcium may be decreased. In
spinal osteoporosis, however, calcium excretion may be in-
creased with HOP excretion normal. Low plasma calcitriol
levels have been found in age-related osteoporosis. In spinal
osteoporosis, plasma calcitriol may be high. This could ex-
plain the increased bone remodeling found histomorphomet-
rically in spinal fracture patients [1].
Since bone matrix synthesis is performed by cells of os-
teoblastic lineage, matrix proteins synthesized by osteo-
blasts reflect the osteoblastic activity and bone formation.
They may be used as biochemical markers in osteoporosis
studies. The major macromolecular matrix components syn-
thesized by osteoblasts are type I collagen and bone
~-carboxyglutamic acid (Gla)-containing protein, osteocal-
cin. Osteoblasts also synthesize osteoclast-activating fac-
tors, prostaglandins, growth factors, and several enzymes,
such as procollagenase and alkaline phosphatase (Table 1).
PTH and calcitriol are major controllers of osteoblast activ-
ity, but other factors, including cytokines and growth fac-
tors, play important roles [2] (Table 2).
Assays of the Osteoporosis Markers in Serum and Urine
Alkaline Phosphatase
Serum total alkaline phosphatase determinations are of lim-
ited value for clinical diagnosis or therapeutic control of os-
International
9 1991 Springer-Verlag New York Inc.
teoporosis, up to now partly because of technical difficulties
in reliably interpreting alkaline phosphatase isoenzyme and
isoform analysis results. Modern separation methods allow
accurate and quantitative determination of the various alka-
line phosphatase isoenzymes and isoforms, including the os-
teoblast-derived isoforms [3-5]. Such methods are promising
diagnostic and investigative tools in clinical studies, includ-
ing osteoporosis investigations.
In a previous study [3], we found levels of the bone-
specific isoform of alkaline phosphatase to be age-related:
they were lower in patients 20-40 years old than in those
40--60 years old. Our findings were in agreement with those
of Duda et al. [6]. Kuwana et al. [4] found an age-related
decline in bone alkaline phosphatase in both sexes. In
women, however, bone alkaline phosphatase increased from
the sixth decade on. Duda et al. [6] found bone alkaline
phosphatase to be lower in young adults of both sexes than
in older people, with an increase from the sixth decade in
both sexes. The decline in bone alkaline phosphatase up to a
certain age obviously indicates decreased bone formation
and turnover.
Osteocalcin
Measurement of osteocalcin has revealed an increase in age-
related osteoporosis in many studies. This could reflect in-
creased resorption or synthesis as well. Some osteocalcin
antisera probably also measure osteocalcin fragments result-
ing from degradation as well as the freshly synthesized pro-
tein.
Serum osteocalcin levels decrease significantly in both
sexes as the age of 50 approaches (Fig. 1) [7]. After the age
of 50, there is an age-related increase, which is more marked
in women than in men. These findings are in agreement with
those of other studies [6-11]. Our results [7] differ from those
of Epstein et al. [10] in that we found osteocalcin in men to
be higher than in women. Our normal range for osteocalcin
[7] is, however, fairly low as compared with that in other
studies. We also found a close correlation between serum
osteocalcin and total alkaline phosphatase (P < 0.01). Levels
of serum osteocalcin were slightly high in long-stay geriatric
patients. There was a significant negative correlation (r =
-0.452, P < 0.05) between serum osteocalcin concentra-
tions and calcitriol levels in such subjects [7]. These findings
agree with those of Epstein et al. [10] but conflict with the
fact that calcitriol stimulates osteocalcin synthesis: adminis-
tration of calcitriol has been shown to increase serum osteo-
calcin in normal and osteoporotic postmenopausal women
[12, 13].
Recently, Vanderschueren et al. [8] have shown that age-
related changes in serum osteocalcin are closely reflected by
M. T. Parviainen et al.: Osteoblast Markers $27

Table 1. Major characteristics of the osteogenic cell lineage (modi- 4 ,

fied from [2]) [] Women
[] Men
1. Synthesis of macromolecular matrix components
Bone Gla-protein (BGP)
Type I collagen
2. Synthesis of autocrine and paracrine messages
Osteoclast activating factor (OB-OAF)
Prostaglandins
Growth factors
3. Synthesis of metalloproteins, their inhibitors, and their
T
activators
Procollagenase El
Collagenase proactivator
1
Tissue inhibitor of metalloproteinases
Plasminogen activator
4. Synthesis of alkaline phosphatase

0 ,
20-2s 30-39 40-49 50-59 60-69

Table 2. Major controls of the osteoblast phenotype (modified Age (years)

from [2])
Fig. 1. Mean circulating osteocalcin (BGP) concentrations (-+SE) in
1. Calciotropic hormones healthy women and men of different age groups. Numbers of sub-
PTH jects is as follows: age 20-29, women (4), men (3); age 30-39, women
Vitamin D metabolites (calcitriol) (17), men (16); age 40-49, women (5), men (12); age 50-59, women
2. Cytokines (15), men (8); age 60--69, women (3), men (31).
Interleukin-1
Tumor necrosis factors a and 13
",/-Interferon
Prostaglandins
3. Growth factors (GFs) (20)
Skeletal GF (lo)
Transforming GF-13
Insulin-like GF-1 (somatomedin C) 3
Epidermal GF
Acidic and basic fibroblast GFs (84)
Bone-derived GF (132-microglobulin)
:=L
v
2

(5
133

changes in osteocalcin in EDTA extracts of the cortical iliac 1

crest, suggesting that serum osteocalcin is a good indicator
of bone metabolism. They concluded that the overall de-
clines in bone osteocalcin in men and women and in serum
osteocalcin in men indicate decreases in bone formation and
turnover. Osteoblastic activity and bone turnover increase
for a time postmenopausally, as indicated by increases in
bone and serum osteocalcin levels. The increased turnover
results, however, from a negative bone balance. There are
obviously other reasons for age-related osteoporosis than
defective osteoblastic function [8].
Seasonal variation in serum osteocalcin has been re-
ported in recent studies [7, 14]. In ours [7], osteocalcin levels
were higher in summer and autumn than in winter. Serum
osteocalcin concentrations from October to November were
higher (by 37.1%, P < 0.01) than those between February
and March (Fig. 2). Thomsen et al. [14] found highest values
in February, lowest values in July. These conflicting results
can be at least partly explained by different geographical
locations of the populations studied.
Duda et al. [6] showed that serum osteocalcin and bone-
derived alkaline phosphatase are equally sensitive markers
for abnormal bone turnover in patients with many metabolic
diseases. F o r example, their determination gave concordant
results in postmenopausal osteoporosis. Determination of
bone-derived alkaline phosphatase allowed, however,
slightly better differentiation between normal and abnormal
subjects (see Fig. 4 in [6]).
0
I1-111 IV-V Vl-Vll VIII-IX X-XI
Month
Fig. 2. Mean circulating osteocalcin (BGP) concentrations (---SE) in
samples collected during different months. Numbers of subjects are
shown above the columns.
Urinary ~l-Carboxyglutamic Acid (Gla)
Urinary Gla levels can also be considered as markers of bone
metabolism. Gla is released from osteocalcin when the latter
is degraded. Since vitamin-K-dependent proteins other than
osteocalcin may release Gla, determinations of this sub-
stance in urine may be of low diagnostic value. In our stud-
ies, urinary Gla did not correlate well with age, or serum
calcium, total alkaline phosphatase, osteocalcin, or calcitriol
levels. In hyperparathyroid patients, urinary excretion of
Gla was, however, significantly lower than that in normal
subjects (28.3 -+ 13.6 p~mol/24 h vs 46.4 -+ 14.2 ~mol/24 h, P
< 0.05) [7]. Women exhibited somewhat lower excretion
$28 M. T. Parviainen et al.: Osteoblast Markers

rates than men (38.3 --- 12.3 ~mol/24 h vs 54.4 -+ 11.9 a)

ixmol/24 h). 8 0 84

60 AP
Other Markers -5 [] Cell Protein

E 40
Studies of the newly identified markers of collagen metabo- 0
0
lism, e.g., procollagen peptides in plasma (see elsewhere in
this volume), and urinary pyridinium cross-links [15], may t,"'- 20
make valuable contributions to osteoporosis studies.
E 0
0

Experimental Cell Studies

-20 |
The balance between bone formation and resorption is reg-
ulated by the action of hormones, growth factors, and other -40
100 10 1 0,01
substances in osteoblasts and osteoclasts. Human osteo-
blast-like cells and osteoblast-like osteosarcoma cells can
Calcitriol (nM)
serve as good models in in vitro studies of bone metabolism.
If the fundamental processes of bone metabolism are to
be understood, biochemical functions and modes of regula- b)
tion need to be precisely resolved. Study of cultures of cells
80 84
from normal human bone would facilitate understanding of
the in vivo situation. Primary culture of such cells is, how-
[] AP
ever, tedious and demanding. The cell numbers recovered 50
-6 [] Cell P r o t e i n
can be unsufficient for molecular biological studies. Contin- x_
uous cultures of clonal or transformed human or rat osteo- E 40
sarcoma cell lines have therefore been used as model sys- O
O
tems in many studies. However, such cells may exhibit drift
from the original phenotype with time, and may accordingly r- 20
exhibit changing patterns of hormonal responsiveness [16].
E
O 0

Human Osteoblast-Like Cells

~ -20
~7. ./

Cultures of human osteoblast-like cells have been estab-

lished without collagenase digestion from trabecular ex-
plants obtained from donors during surgery [17, 18]. This
method has been highly successful in isolating cells from
normal bone. These cells have been characterized in terms
of responsiveness to calcium-regulating hormones, and their
abilities to synthesize constituents such as osteocalcin [18]
and alkaline phosphatase (Fig. 3a). The calcitriol-induced
synthesis of osteocalcin can be antagonized by interleukin-1
and by the immunomodulator, cyclosporin A [18]. Sex ste-
roids and anabolic steroids have been shown to have partic-
ular effects on the proliferation of human-bone-derived cells
[17] (Fig. 3b).
Studies with Osteosarcoma Cells
For steroid hormones to have direct effects on bone cells,
high-affinity receptors must be present. Steroid-hor-
mone-receptor complexes interact with specific genes and
regulate their transcriptional activity [ 19, 20]. Homogeneous
populations of well-characterized human and rat osteosar-
coma cell lines, each phenotypically osteoblast-like, have
been used to reevaluate the existence of nuclear receptors
for calcitriol, estradiol, retinoic acid (an analogue of vitamin
A), and thyroid hormone in bone cells, for example [21-24].
The mechanism of biological response to a hormone is
ligand-dependent regulation of its receptor levels (homolo-
gous regulation). Calcitriol has been shown to up-regulate its
own receptor levels in human and rat osteosarcoma cells [25,
-40
1 0 1 0,01
Estradiol (nM)
Fig. 3. The effect of calcitriol (a) and estradiol (b) on the alkaline
phosphatase (AP) specific activity and cell proliferation (cell protein
was measured) in human bone derived cells in vitro. Each column
represents mean of three experiments and is expressed as an in-
crease or a decrease (%) from the control level.
26]. Examples of one hormone regulating the receptors of
another hormone (heterologous regulation) have also been
described. Regulation of calcitriol receptors is an important
mechanism of cellular responsiveness to calcitriol [27]. Sev-
eral hormones, e.g., retinoic acid, glucocorticoids and estro-
gen, have been shown to increase synthesis of calcitriol re-
ceptors [21, 28], increasing cell responsiveness to calcitriol
in specific tissues.
Steroid hormones regulate many functional responses in
bone cells. There are differences in hormone responsiveness
between different species and cell types. We have shown
[25] that calcitriol up-regulates its own receptor levels in
three different human osteosarcoma cell lines (MG-63, U2-
Os, SaOs-2). All of these cell lines exhibit alkaline phos-
phatase activity, which is characteristic of osteoblast-like
cells. Calcitriol is, however, capable of stimulating osteocal-
cin synthesis and alkaline phosphatase activity only in MG-
63 cells. U2-Os and SaOs-2 cells may therefore represent
different maturational stages of osteoblasts (Table 3). In rat
M. T. Parviainen et al.: Osteoblast Markers $29

Table 3. Effects of calcitriol on its binding, osteocalcin synthesis, Isopal System Analis/Belgium for the separation of AP isoen-
and alkaline phosphatase activity in different human osteosarcoma zymes. Clin Chem 33:958 (abstract)
cell lines. Calcitrioi treatment was for 24 h at 1 nM concentration in 6. Duda RJ, O'Brien JF, Katzmann JA, Peterson JM, Mann KG,
the binding and osteocalcin synthesis studies, and for 72 h at 10 nM Riggs BL (1988) Concurrent assays of circulating bone Gla-
concentration in the alkaline phosphatase study (modified from [25]) protein and bone alkaline phosphatase: effects of sex, age and
metabolic bone disease. J Clin Endocrinol Metab 66:951-957
Alkaline 7. Pirskanen A, Parviainen MT, Haukilahti M, M~ienp/i/i PH, AI-
[3H] 1,25(OH)2D3 Medium phosphatase hava EM (1988) Circulating osteocalcin and 1,25-dihy-
Cell bound osteocalcin (nmol pNP/ droxyvitamin D concentrations and urinary ~-carboxy-
line (fmol/106 cells) (ng/ml/107cells) min/mg) glutamic acid in healthy subjects and in patients with metabolic
MG-63 (C) 2.2 • 0.2 4.25 • 0.50 17 • 1 bone disease. In: Norman AW, Schaefer K, Grigoleit HG, von
MG-63 (T) 37.6 • 7.8 61.5 • 6.9 53 • 3 Herrath D (eds) Vitamin D. Molecular, cellular and clinical en-
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10. Epstein S, Poser J, McClintock R, Johnston CC Jr, Bryce G,
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30]. Calcitriol also increases levels of fibronectin, a cell- Gla protein concentrations in healthy adults. Dependence on
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