Bone Alkaline Phosphatase

36 Pages·2013·4.9 MB·English

TECOmedical Clinical and Technical Review
January 2013
Bone Alkaline
Phosphatase (BAP)
A biochemical marker
of bone turnover
Author:
Peter Haima, Ph.D.
Summary
The increasing number of drugs available for treatment of bone diseases requires the use of more rapid and
predictive methods to assess therapy efficacy. While detectable and significant changes in bone mineral den-
sity (BMD) take 18 to 24 months to develop, bone turnover marker have been shown to detect changes in bone
tissue within 3-6 months after starting anti-resorptive therapy. Therefore, measurement of bone turnover mar-
ker is increasingly recommended as a key component of therapy management: to rapidly identify therapy respon-
ders and non-responders, to assess therapy efficacy and to determine the optimal therapy and dose of treatment.
Moreover, since biochemical bone marker reflect the whole-body rates of bone turnover, the combined
measurement of bone marker and BMD provides more information on overall bone loss than BMD measurement at spe-
cific skeletal sites alone.
This paper presents an overview of all relevant clinical and technical data on Bone-Specific Alkaline Phospha-
tase (BAP), a biochemical marker of bone formation. Seven key criteria were formulated that need to be fulfilled by a
biochemical marker to be useful in assessing bone turnover and monitoring therapy. Using these criteria, BAP was com-
pared to other marker, whereby BAP demonstrated to be one of the most attractive bone turnover marker to date.
Important technical aspects as cross-reaction with liver alkaline phosphatase and BAP measurement in units of
activity (U/L) vs. units of mass (μg/L) are extensively discussed.
Summaries of the most important clinical studies with BAP as bone-turnover marker are presented. All clinical data have
been obtained with the Quidel® BAP Assay. Clinical study conclusions are:
Increased serum levels of BAP are seen in conditions characterized by excessive bone turnover including
postmenopausal women, osteoporosis, Paget’s disease, hyperparathyroidism, thyrotoxicosis, and metastatic
cancer, and are associated with rapid bone loss. BAP levels decrease following anti-resorptive therapy in a
dose-dependent manner. These short-term changes are inversely correlated with long-term changes in BMD.
BAP levels are correlated with bone growth in children and reflect pubertal growth stages.
In summary, it is demonstrated that BAP identifies rapid bone losers, and accurately monitors the efficacy of
hormone replacement-, bisphosphonate-, PTH analogue- and growth hormone-therapies.
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Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1 .1 Osteoporosis and bone remodeling ...................................................... 5
1 .2 Medical conditions affecting bone remodeling .............................................. 5
1 .3 Drug therapies for metabolic bone diseases ............................................... 6
2 Diagnostic methods for detecting metabolic bone diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2 .1 Diagnosis of metabolic bone disease, Bone Mineral Density (BMD) .............................. 7
2 .2 Bone marker for detecting bone disease and assessing therapy efficacy ......................... 7
3 BAP as a marker for detecting changes in bone turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3 .1 BAP background ..................................................................... 9
3 .2 Methods for measuring alkaline phosphatase .............................................. 9
3 .3 Measurement of BAP in protein mass (μg/L) and enzyme activity (U/L) ........................... 10
3 .4 BAP cross-reaction with liver alkaline phosphatase .......................................... 11
3 .5 Daily, dietary and age-related variation of BAP ............................................. 11
4 BAP clinical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4 .1 Summary ........................................................................... 12
4 .2 BAP responses to different clinical conditions and therapy .................................... 12
4 .2 .1 BAP indicates increased bone turnover ........................................... 12
4 .2 .2 Association between BAP levels and rapid bone loss ................................ 13
4 .2 .3 BAP reflects antiresorptive effect of HRT in postmenopausal women .................... 13
4 .2 .4 Change in BAP predicts BMD change in HRT- and placebo-treated women .............. 13
4 .2 .5 BAP rapidly identifies more responders to HRT than BMD ............................ 14
4 .2 .6 BAP reflects antiresorptive effect of alendronate in osteoporotic women ................. 14
4 .2 .7 Baseline bone turnover predicts turnover response to alendronate ...................... 15
4 .2 .8 Response to alendronate is more rapidly identified by BAP than by BMD ................. 15
4 .2 .9 BAP is more sensitive than Total AP to alendronate in osteoporosis ..................... 15
4 .2 .10 BAP responds to antiresorptive therapy as early as 8 weeks ........................... 16
4 .2 .11 BAP is more sensitive than TAP to bisphosphonates in Paget’s disease .................. 16
4 .2 .12 BAP and strontium ranelate therapy .............................................. 16
4 .2 .13 BAP and parathyroid hormone treatment .......................................... 17
4 .2 .14 Measurement of BAP in cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4 .2 .15 BAP and growth hormone therapy ............................................... 18
4 .2 .16 BAP in uremic patients ........................................................ 18
4 .2 .17 BAP is more sensitive than TAP in primary hyperparathyroidism ........................ 18
4 .2 .18 BAP is elevated in secondary hyperparathyroidism .................................. 19
4 .2 .19 BAP is elevated in Morbus Paget’s disease ........................................ 19
4 .2 .20 Determination of BAP in patients suffering from severe liver disease .................... 19
4 .3 BAP reference data ................................................................... 20
4 .3 .1 BAP reference data in premenopausal women ....................................... 21
4 .3 .2 BAP reference data in postmenopausal women ...................................... 21
4 .3 .3 BAP in postmenopausal osteoporotic women
4 .3 .3 on hormonal replacement or bisphosphonate therapy ................................. 22
4 .3 .4 BAP reference data in males ..................................................... 22
4 .3 .5 BAP reference data in children ................................................... 23
4 .4 BAP disease values ................................................................... 23
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Contents
5 Clinical validation studies of the Quidel® BAP Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
6 Special applications of the Quidel® BAP Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
6 .1 Use of BAP with cell culture supernatant .................................................. 28
6 .2 Use of BAP monoclonal antibody in western blotting,
FACS sorting and Immunocytochemical staining of osteoblasts ................................... 28
6 .3 Measurement of BAP in animal species ................................................... 29
7 TTechnical summary of the Quidel® BAP Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
8 Measurement of BAP in protein mass (μg/L) and enzyme activity (U/L) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
8 .1 Certificate of Analysis ................................................................. 32
9 Literature References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
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1 Introduction
1 .1 Osteoporosis and bone remodeling
Bone remodeling is an ongoing dynamic process consisting of bone resorption (due to osteoclasts digesting type I
collagen) and bone formation (due to osteoblasts). Normally, these processes are balanced, resulting in 10 %
replacement of the skeleton, each year. However, due to aging, disease or other conditions, bone turnover may
become imbalanced where bone resorption and formation occur at different rates. Osteoporosis is an age-related bone
disease characterized by low bone mass and micro architectural deterioration of bone tissue (see Figure 1). It is diagnosed
often after an already unacceptable loss of bone has occurred. In many cases a fracture leads to the initial diagnosis.
Fig . 1
Pictures provided by Pr. Daniel Chappard Université d’Angers, France
Normal & Osteoporotic Trabecular Bone
1 .2 Medical conditions affecting bone remodeling
Osteoporosis is the main clinical condition affecting bone remodeling, afflicting an estimated one – third of women aged
60 – 70, and two – thirds of women aged 80 or older. Osteoporosis can be prevented with proper diet, exercise, and
elimination of controllable risk factors; it can be treated with anti-resorptive therapies.
Metabolic bone disorders include:
• Hyperparathyroidism
• Hyperthyroidism
• Paget’s disease – a condition of abnormal bone formation
• Metastatic cancer to bone
• Nutritional rickets and osteomalacia
• Multiple myeloma
• Malabsorption syndrome
• Disorders caused by drug therapies:
· immunosuppressive drugs for treating cancer and organ transplants
· heparin, used in kidney dialysis
· phenytoin (Dilantin®) for epilepsy
· glucocorticoids (corticosteroids) for rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and asthma
· aluminium-containing antacids
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1 .3 Drug therapies for metabolic bone diseases
Physicians routinely treat patients with metabolic bone diseases. An overview of current and promising new therapies are
listed in Table 1.
Table 1 . Therapies for treatment of metabolic bone disease .
Therapy Application References
Anti-resorptive agents:
Estrogen Because of side effects,
1
(hormone replacement) for transitory time of peri-menopause only.
Phytoestrogen Natural source of estrogen as found in soy.
2
(hormone replacement) Used by women as a “safe” alternative to HRT.
Calcium Prevention of osteoporosis. 3, 4
Selective estrogen Prevent bone loss and the risk of vertebral fracture
receptor modulators (Raloxifene, (Evista))
Prevent bone loss and increase BMD
Bisphosphonates ((Alendronate (Fosamax); Risedronate (Actonel)). 5
Rigid administration is a disadvantage.
Treatment of osteoporosis and Paget‘s disease,
Calcitonin considered not as effective as bisphosphonates. 6, 7
Decreased tolerance with long-term use.
Active form of vitamin D given to post-menopausal
Vitamin D 8, 9
women who have osteoporosis in the spine.
Formation stimulating agents:
Increases BMD, however, clinical studies showed
Sodium fluoride
no decrease in vertebral fracture rates
PTH initially stimulates bone formation and later increases bone
Parathyroid hormone
remodeling; increases spinal BMD. Suggested for treatment of
(human recombinant 10
patients with persistent osteoporosis after prior alendronate
PTH (1-34))
treatment. (Teriparatide (Forteo))
Growth hormone therapy is used (and FDA approved) in the
Growth factors treatment of hypo-pituitarism and somatotropin deficiency of 11, 12
children and adults.
Agents inhibiting resorption and stimulating formation:
Strontium ranelate Inhibits bone resorption and stimulates bone formation. 13, 40
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2 Diagnostic methods for detecting metabolic bone diseases
2 .1 Diagnosis of metabolic bone disease, Bone Mineral Density (BMD)
Diagnosis of metabolic bone diseases can be established by measuring bone mass, i.e. bone mineral density
(BMD) at the hip, spine or other location. However, BMD is a static measure of bone composition, reflecting its hi-
story. A baseline BMD value does not offer any prediction of future bone loss or response to therapy. Moreover, since
biochemical bone marker reflect the whole-body rates of bone turnover, the combined measurement of bone
marker and BMD provides more information on the overall bone loss than BMD measurement at specific skeletal sites
alone. Finally, while BMD can indicate bone loss, it does not provide information on alteration or deterioration of bone
tissue structure.
The increasing number of drugs available for treatment of osteoporosis and other bone diseases requires the
use of more rapid and predictive methods to assess therapy efficacy. While detectable and significant changes
in BMD take 18 to 24 months to develop, bone turnover marker have been shown to detect changes in bone tissue
within 3-6 months after starting anti-resorptive therapy. Therefore, measurement of bone turnover marker is
increasingly recommended as a key component of therapy management: to rapidly identify therapy responders and non-
responders, to assess therapy efficacy and to determine the optimal therapy and dose of treatment.
2 .2 Bone marker for detecting bone disease and assessing therapy efficacy
To be useful in assessing the rate of bone turnover, and monitoring therapy, marker should:
A. show a difference in the rate of bone turnover pre-and post-menopause
B. demonstrate minimal analytical variation
C. significantly change in response to treatment
D. detect change in short time interval (months)
E. demonstrate minimal within person (biological) variation
F. preferably demonstrate little variation over the day
G. preferably demonstrate no influence to food intake
H. preferably demonstrate high stability in the biological specimen
An overview of current and promising new tests is shown in Table 2, together with the score on the criteria
mentioned above (adapted from Caulfield et al. [14]).
Based on these criteria, BAP (Bone Specific Alkaline Phosphatase) appears to be one of the most attractive bone
turnover marker .
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Table 2 . Bone marker and characteristics .
A B C D E F G H
(a)
Biomarker Method Main Pre- & Analytical Treat- Short Within Daily Food Sample
Sample post- variation ment time person variation intake stability
Type meno- response change variation
pause
Formation
5 days
BAP ELISA Serum + + + + + + + +
2–8 °C
Osteocalcin IRMA/ 4 hours
Serum + + + + ± + + -
Intact ELISA 2–8 °C
Osteocalcin 5 days
ELISA Serum + + + + ± + + +
N-mid 2–8 °C
5 days
PINP RIA Serum + + + + ± + + +
2–8 °C
5 days
PICP = CICP ELISA Serum NA + + + + ± + +
2–8 °C
Resorption
2 days
TRAP 5b ELISA Serum ± + ± ± NA + + ±
2–8 °C
7 days
DPD ELISA Urine + + + + + - + +
2–8 °C
1 day
Serum + + + + + - ± ±
2–8 °C
NTx ELISA
3 days
Urine + + + + ± - ± +
2–8 °C
1 day
Serum + + + + + - - +
2–8 °C
CTx ELISA
7 days
Urine + + + + ± - - +
2–8 °C
ICTP = 5 days
ELISA Serum - + - - NA ± + +
CTX-MMP 2–8 °C
Others
1 day
sRANKL ELISA Plasma NA + ±(b) - NA NA + ±
2–8 °C
Osteoprote- 1 day
ELISA Plasma NA + + - NA NA + ±
gerin (OPG) 2–8 °C
2 days
Cathepsin K ELISA Serum NA(c) + NA NA NA NA + ±
2–8 °C
Scores are: + (yes), - (no), ± (fair/indeterminate), NA (not available)
(a) Letters correspond to following, the marker: to bisphosphonate treatment of osteoporotic
A . shows a difference in the rate of bone turnover postmenopausal women [43]. However, PTH treat-
pre-and post-menopause ment
B . demonstrates minimal analytical variation of glucocorticoid induced osteoporotic women
C . significantly changes in response to treatment resulted in a fast (1 month) and sustained increase
D . detect changes in short time interval (months) of sRANKL levels [44].
E . demonstrates minimal within person (biological) variation (c) Cathepsin K is elevated in patients with established
F . preferably demonstrates little variation over the day rheumatoid arthritis.
G . preferably demonstrates no influence to food intake
H . p referably demonstrates high stability in the biological specimen
(b) In contrast to OPG, sRANKL showed no response
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3 BAP as a marker for detecting changes in bone turnover
3 .1 BAP background
Bone-specific alkaline phosphatase (BAP) is synthesized by the osteoblasts and is presumed to be involved in the
calcification of bone matrix, though its precise role in the formation process is unknown. BAP is one of a number of
different isoenzymes of alkaline phosphatase: bone, liver, kidney, intestine, and placenta. In the serum of most
healthy individuals, bone and liver isoenzymes of the tissue non-specific AP gene predominate in approximately equal
proportions.
The difference in glycosylation of the bone and liver isoenzymes (products of the same gene) has been exploited to ge-
nerate specific antibodies against BAP. BAP is considered to be a highly specific marker of the bone-forming activity of
osteoblasts.
3 .2 M ethods for measuring alkaline phosphatase
Diagnostic tests that are used for detecting alkaline phosphatase are:
• BAP ELISA – routine enzyme-linked immunosorbent assay, measuring BAP enzyme activity.
• BAP IRMA – routine immunoradiometric assay measuring BAP in protein mass units.
• Total alkaline Phosphatase (TAP) – routine automated laboratory test. Its high cross-reactivity to liver alkaline phos-
phatase makes this method non-specific for diagnosing bone disease.
• Electrophoresis – different in-house methods available. Non-standardized, results are inconsistent from lab-to-lab.
• Lectin Precipitation – different in-house methods, inconsistent from lab-to-lab.
Correlations between various methods are shown in Figure 2.
Fig . 2 . Correlation between BAP ELISA (Quidel®) and other methods for determining BAP .
Abb. 2a. Korrelation mit Elektrophorese. Abb. 2b. Korrelation mit Präzipitation.
(Data on file, Quidel Biosystems36) (Übernommen von Gomez et. al.28)
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Fig . 2 . Correlation between BAP ELISA (Quidel®) and other methods for determining BAP
Abb. 2c. Korrelation mit IRMA. (Data on file, Quidel Biosystems36) Abb. 2d. Korrelation mit TAP. (Übernommen von Gomez et. al.28)
3 .3 Measurement of BAP in protein mass (μg/L) and enzyme activity (U/L)
Bone Alkaline Phosphatase (BAP) is a marker for osteoblastic activity in vitro. BAP is typically measured by one of the two
methods, protein mass or enzyme activity.
Enzyme Activity
BAP is bound to a monoclonal antibody specific for the bone isoform. Other forms of Alkaline Phosphatase (e.g. liver, etc)
are washed away. Alkaline Phosphatase activity is measured via a chromogenic (color change), chemical reaction. In the
Quidel® BAP Assay, results are determined directly from this color change and expressed as units per liter (U/L). As the
enzyme activity is measured the preferred expression is U/L however, the enzyme activity can also be calculated in μg/L.
Protein Mass
In the IRMA method, the enzyme molecule BAP is directly measured by using two monoclonal antibodies
(Sandwich Assay) detecting two different epitopes; results are expressed in mass units (μg/L).
Correlation Protein Mass and Enzyme activity expressed in μg/L
BAP measured with the protein mass and the enzyme activity method, both expressed in μg/L, showed an excellent corre-
lation.
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