Clinical Chemistry of the Bone
(Download 
Clinical Chemistry of the Bone.pdf)
Bone is constantly being remodelled throughout life. There is coordination of bone resorption by osteoclasts and new bone matrix formation by osteoblasts, so that total bone mass remains approximately constant. The organic matrix of bone is approximately 90% type 1 collagen, which is synthesised by osteoblasts. Calcium salts are then precipitated on this matrix. Resorption occurs when enzymes are released by activated osteoblasts that degrade the bone matrix. Peak bone mass occurs in the third decade of life, with a slow decline thereafter, which is accelerated in women in the first few years after menopause [1]. Loss of bone is due to imbalance between resorption and formation, ie either excess resorption and/or deficient formation.
In many metabolic diseases of bone there is altered bone turnover, resulting in formation of abnormal bone (as in Paget's disease), or in bone loss (as in osteoporosis). Both of these situations can lead to fractures. These alterations can be detected and followed during treatment by measurement of biochemical markers of bone turnover. There are markers available for bone formation and for bone resorption. Complete investigation of bone disease may also involve measurement of calcium, parathyroid hormone, and vitamin D metabolites, which will not be discussed here. Calcium and parathyroid hormone were discussed in a previous newsletter (June 1993), which is available upon request to HAPS.
Markers of Bone Formation
These are products of osteoblasts (bone forming cells). The enzyme alkaline phosphatase has been used as an indicator of osteoblastic activity for many years. It is involved in making phosphate available for calcification of bone, and some enzyme leaks into the serum where it can be measured by routine biochemistry. However, it is not specific to bone. There are several isoenzymes of alkaline phosphatase which come from other tissue sources, particularly liver but also gut, placenta and some tumours. Methods for separating these isoenzymes are available so that bone specific alkaline phosphatase can be measured, but in the past these techniques have been time consuming and have only been available in reference laboratories. Recently a simple immunoassay has been developed, and if it lives up to expectations, this is likely to make measurement of bone specific alkaline phosphatase relatively routinely.
The other marker of bone formation that is used is osteocalcin. This is a bone matrix protein that is the second most abundant protein in bone after type 1 collagen. Its function is not clear but is involved in binding of calcium. When it is synthesised by osteoblasts a small proportion leaks into the circulation where it can be measured by immunoassay. There are several problems in measurement. Osteocalcin is renally excreted so levels are elevated in renal failure, but only significantly so in advanced renal failure (creatinine clearance less than 20-30ml/min) [2]. The main problem is the presence of degradation fragments in the serum, which probably come from bone resorption [3]. An immunoassay has to be chosen that only (or predominantly) measures intact osteocalcin as this is what reflects bone formation. Different immunoassays will give different results so when a patient is being followed over time it is important to keep using the same assay (ie the same lab).
Markers of Bone Resorption
These are mostly degradation products of collagen. Urinary hydroxyproline is the most extensively used marker of bone resorption. Hydroxyproline is a very common amino acid in collagen but is present in few other proteins. When collagen is degraded, hydroxyproline is released and is not reutilised. Most of it is metabolised by the liver, but a fairly constant proportion (10-20%) is excreted in the urine. Bone resorption is the major contributor to urinary hydroxyproline, but some also comes from dietary gelatin, other collagens (eg skin, cartilage), and other proteins [3]. There is also a contribution from procollagen peptides produced during bone formation, and collagen synthesised but not incorporated into bone. Dietary hydroxyproline can be controlled for by collecting a timed urine after an overnight fast and measuring the hydroxyproline to creatinine ratio, but no corrections can be made for the other factors mentioned.
Because of this nonspecificity, other markers have been looked for. Currently, the most specific markers appear to be the pyridinium cross links. These are tripeptides that join adjacent collagen fibrils to strengthen and stabilise collagen fibres. They are only present in significant amounts in bone and cartilage collagen. They are not present in procollagen and there is no dietary contribution. They occur in elastin but there is little turnover from this source. Two types of cross links occur, called pyridinoline (Pyr) and deoxypyridinoline (Dpd). Dpd only occurs in bone and dentine [3], so it is more specific than Pyr, which occurs in bone and cartilage, but Pyr is more abundent. Cross links are measured in urine (ratio to creatinine in a timed specimen), and either total cross links or just Dpd can be measured, depending on the assay used.
Clinical Use
Bone turnover markers have long been used in the diagnosis and monitoring of Paget's disease. Both bone formation and resorption are increased, and all markers are elevated. During treatment marker levels fall, but osteocalcin levels take longer to respond, so are less useful in this disease. Currently the most useful markers are bone specific alkaline phosphatase and pyridinium cross links.
Abnormalities of bone markers have been identified in many bone diseases, but attention is being focussed on osteoporosis. Osteoporosis is loss of bone mass with consequent risk of fracture of the weakened bone, which occurs in post menopausal women and in both sexes over the age of approximately 75 years. This is a major health problem (and expense) in the Western world with our aging population, but may be reduced in many post menopausal women with hormone replacement therapy. As only approximately 25% of post menopausal women will develop fractures, those at greater risk need to be identified and offered preventative therapy. The use of biochemical markers in osteoporosis is expanding but is still limited. They are not useful in diagnosis. Radiological procedures are necessary to visualise bone mass [1,4]. Biochemical markers can indicate bone activity but not absolute bone mass. Also bone mass varies from site to site, but biochemical markers reflect total activity.
Bone markers are more useful in directing and following therapy. Subgroups of osteoporotics can have excessive bone resorption or reduced bone formation. Those with excessive resorption will have elevated levels of bone turnover markers, and in this group antiresorptive therapy (such as oestrogens, bisphosphonates or calcitonin) may be of use [2,3]. Those with reduced bone formation will have low levels of bone turnover markers, and in this group bone formation stimulating agents (such as anabolic steroids) may be useful [2]. Once therapy has been instituted bone markers are very useful in monitoring response. A change in bone markers can usually be detected within a few months, but it can take 2 years for change to be detected on radiology [1]. Pyridinium cross links decline rapidly with oestrogen and other therapies. Osteocalcin and alkaline phosphatase also decline but less rapidly [4]. If the expected changes do not occur then higher doses or additional treatment may be indicated. New therapies can also be assessed by monitoring of bone markers [4].
What is Available at HAPS?
All the tests mentioned here are available through HAPS. Most are performed on site (hydroxyproline, total alkaline phosphatase, and pyridinium cross links). Bone specific alkaline phosphatase and osteocalcin are done at PaLMS by immunoassay.
References
1. M Caulfield. Biochemical markers of bone resorption. AACC Endo 1995; 13:47-55.
2. P Kelly, R Mason, J Eisman. The investigation of calcium and bone disorders. Recent Advances in Laboratory and Clinical Endocrinology No.4, Endocrine Society of Australia 1994.
3. C Price, P Thompson. The role of biochemical tests in the screening and monitoring of osteoporosis. Ann Clin Biochem 1995; 32:244-260.
4. L Demers. Biochemical markers of bone turnover. AACC Endo 1995; 13:163-166.
Written by: Carole Wilson, Bob Lojewski, Geoffrey Kellerman, John Dickeson
Clinical Chemistry, HAPS - May 1997
Reviewed by: Dr Huy Tran - January 2005
Back to Top of page...