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| Issue 11 2000 |  |
| LABORATORY MARKERS OF BONE METABOLISM |
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Dr Philip Clifton-Bligh Endocrinology, PaLMS tel: +61 2 9926 8388 e-mail: pclifton@doh.health.nsw.gov.au |
Dr Lisa Koe Biochemistry and Laboratory & Community Genetics, PaLMS tel: +61 2 9926 7453 e-mail: lkoe@doh.health.nsw.gov.au |
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Introduction Osteoporosis and its associated risk of bone fractures is a major health concern in Australia, with an estimated cost of over $700 million a year to the Australian community(1). The incidence of fracture is projected to double from 83 000 in 1996 by the year 2026 due to an increase of the elderly population(2). Hence, the prevention, diagnosis and treatment of osteoporosis is a worthwhile challenge for all health workers. Osteoporosis is currently defined by the WHO as a skeletal bone mineral density (BMD) > 2.5 SD below the young normal mean and the risk of fracture has been estimated to double for each standard deviation unit below the mean(1). Hence, radiological assessment of BMD is currently the main investigation to diagnose osteoporosis. Bone mass, however, is determined by the balance between bone formation and bone resorption with “uncoupling” of these two processes and increased bone turnover thought to underlie development of osteoporosis. Laboratory bone markers include those that reflect formation (osteoblast enzymes, by-products of collagen / bone matrix synthesis), or resorption (osteoclast enzymes, bone breakdown products). The clinical utility of each marker depends on its bone specificity, clearance mechanisms and ability to discriminate between formation and resorption activities. Recent evidence indicates that these bone markers have a role in predicting the future risk of minimal trauma fracture, as well as response to hormone replacement and other intervention therapy more rapidly than does BMD. Resorption markers have been demonstrated to respond 1-3 months after intervention, while formation markers responded after 6-9 months(3,4). These changes were reflected later in BMD at 12-24 months, suggesting that bone markers would be useful for monitoring early responses to therapy. The characteristics of bone markers are summarised in Table 1. Bone formation markers These markers reflect increased bone formation with or without increased bone resorption. Serum levels may increase with physiological growth, Paget’s disease, primary hyperparathyroidism, osteomalacia, renal osteodystrophy and conditions associated with high bone turnover. |
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Alkaline Phosphatase Several isoenzymes produced by bone, liver, intestine, placenta and kidney exhibit phosphatase activity at an alkaline pH. In bone, ALP activity reflects increased osteoblast or bone formation activity. Specific bone ALP can be investigated by measurement of the heat–labile fraction of total ALP, quantitation of ALP bands on electrophoresis and specific immunoassays. Osteocalcin Osteocalcin, or bone GLA protein, is a low MW, non-collagenous protein produced only by osteoblasts. Its metabolism is influenced by parathyroid hormone, 1,25 Vit D, and renal clearance. Vitamin K-dependent carboxylation of three glutamyl residues confers calcium binding ability. Osteocalcin may also be released during bone resorption and therefore levels may not strictly reflect bone formation activity. Carboxy and N-terminal pro-peptides of type I collagen (PICP and PINP) Type I collagen constitutes 90% of the organic component of the bone matrix and is synthesized as a procollagen precursor. Collagen is formed by the cleavage of the C– and N–terminals pro-peptides which can be measured in blood. The predominance of type I collagen in other tissues such as skin decreases their specificity as bone markers. Serum PICP is relatively stable at 4ºC and has been measured by radioimmunoassay with 3% and 5% intra- and interassay coefficients of variation, respectively(3). |
| Marker | Bone specificity |
Sample | Evidence based clinical utility |
| ALP |
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S | Increased bone formation activity |
| Osteocalcin | Bone-specific |
S |
Increased bone formation activity |
| PICP / PINP |
Present in skin type 1 collagen |
S | Higher PICP levels demonstrated in post-menopause osteoporosis c/w non-osteoporosis |
| Hydroxyproline | Poor specificity |
U | Not well demonstrated |
| trAcP | Poor specificity | P | Not well demonstrated |
| Pyr | Present in type ÏI collagen of tendon, ligaments, cartilage | U/S | Higher levels demonstrated in postmenopause osteoporosis c/w non-osteoporosis |
| Dpyr |
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U/S |
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| CTX | Present in skin type I collagen | U/S | Not well demonstrated |
| Present in skin type I collagen | U/S |
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| Abbreviations: Serum (S), Urine (U), hormone replacement therapy (HRT) | |||
| Bone resorption markers |
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Hydroxyproline Hydroxyproline is present mainly in collagen and is derived from proline by post-translational hydroxylation. It is not, however, a specific marker for bone function since it is also derived from muscle and skin collagen and from dietary gelatin. The value of urine hydroxyproline measurements has declined since more specific markers have been developed. Tartrate resistant acid phosphatase (trAcP) Several isoforms of this enzyme are present in osteoclasts, red blood cells and platelets, producing significant tissue non–specificity. The enzyme is also labile, with considerable loss of activity at temperatures >20ºC(5). Pyridinoline(Pyr) and Deoxypyridinoline(Dpyr) These crosslinks stabilise collagen chains and are formed during the maturation, not synthesis of collagen. Therefore, their release reflects bone resorption or collagen degradation. Dpyr is present only in bone and dentine and therefore is more specific than pyridinoline which is also present in type II collagen of tendon, ligaments and cartilage. The excretion of these markers into the urine, unchanged, provide convenient measurements for bone resorption. N-telopeptide and C-telopeptide of type I collagen (NTX, CTX) These markers are specific to type I collagen degradation. They reflect bone resorption, although their specificity are affected by the presence of type I collagen in skin tissue. They have nevertheless been shown in several studies to reflect responses to bisphosphonate therapy. Both NTX and CTX markers may be measured in serum or urine. |
| Specimen Information Serum Osteocalcin:
Dpyr and/or NTX
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| Recent studies on bone marker utility The evidence based clinical utility of bone markers has been demonstrated in two particular clinical circumstances; for identification of postmenopausal women with accelerated bone loss before significant effects on bone density are evident and to monitor early response to therapy, as the magnitude of response predicts the subsequent gain in bone density. |
| Predicting bone loss In early postmenopause, BMD may still be within normal limits, but accelerated bone loss may be present, as reflected by high bone fomation and even higher bone resorption marker levels. Eastell et al 6 reported that postmenopausal women with osteoporosis had 40% higher levels of urinary Dpyr, a bone resorption marker, than age matched postmenopausal women with normal bone mass, whereas serum osteocalcin, a bone formation marker, was only 11% higher. Kushida et al 7 reported that bone resorption markers Pyr, Dpyr and CTX were much higher in postmenopausal women with vertebral osteoporosis than in the cohort without osteoporosis, while formation markers, ALP and osteocalcin were raised to a lesser extent than resorption markers. Prospective studies have demonstrated that bone markers provide additional and complementary information to BMD assessments. The EPIDOS study concluded that a combination of urinary CTX and hip BMD had a higher specificity, for a given sensitivity threshold, for predicting hip fractures than BMD alone 8. Johansen et al 9 and Slemenda et al 10 independently showed osteocalcin to be a good predictor of changes in radial and lumbar BMD over 2-4 years in untreated postmenopausal women. More recently, Uebelhart et al 11 have shown that, using multiple regression analyses, a combination of single measurements of serum osteocalcin, fasting urinary Dpyr and fasting urinary hydroxyproline could predict the bone loss rate over 2 years with r=0.77 (p<0.0001). Predicting and monitoring therapy response Bone markers have also been shown to be useful in predicting likely response to therapy. Osteoporotic patients with highest bone turnover are considered to respond best to antiresorptive therapy, while it is suggested that osteoporotic patients with normal bone turnover may be more appropriately treated with calcium alone (12,13). In untreated osteoporosis patients, the relatively small decrease in BMD (1-3% per year) requires an interval of 18-24 months before significant effects of therapy can be detected 12. In contrast, reductions in urinary Dpyr by up to 50% may occur within 3 months as a successful response to antiresorptive therapy and provide much earlier indications of therapy response. The response of various markers to different therapies have been evaluated in several studies. A comparison of the response of many of the currently available bone markers to bisphosphonate therapy vs placebo was performed by Garnero et al 3. With the exception of free Pyr and CTX, the levels of all bone markers were reduced to normal premenopausal levels within 6-15 months with highly significant correlations between percentage changes in bone markers at 3 months and BMD at 24 months. |
| Which markers are best? The utility of several markers have been demonstrated, but it is as yet unclear which marker(s) are best and the choice often reflects clinical requirements for sensitivity and specificity, assay availability, complexity and costs as well as sample stability, handling and transport factors. Traditional markers such as ALP may be measured in many laboratories, while newer markers are now being compared to older markers in prospective clinical studies. Relevant issues which need to be addressed include inter-individual variability and the effects of circadian and other physiological variations on serial bone marker measurements. The Department of Endocrinology, PaLMS provides services for the measurement and interpretation of serum osteocalcin, urine NTX and urine Dpyr. Serum osteocalcin is measured by RIA, a method with imprecission of 11–12%. Method differences may result in variations between laboratories. Diurnal variations in urine Dpyr have been reported to be as high as 48%. Therefore, urine samples for Dpyr and NTX should be collected in the early morning, over 1 hour, following first void. A commercial ELISA kit measures urine NTX with a current imprecision of 5-10%, while Dpyr is measured by high performance liquid chromatography with an imprecision of 4-9%. The Medicare Benefits Schedule currently only rebates the cost of bone marker tests for patients with proven bone metabolism disorders or low mineral density. However, since bone markers are being shown to predict future risks, the costs vs benefits of bone marker measurement requires careful evaluation of current costs of investigation vs more long term savings by the prevention of fractures, in particular hip and spinal fractures which pose serious risks of morbidity and mortality. |
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References:
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