The Role of the Laboratory in Metabolic Bone Disease
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1 The Role of the Laboratory in Metabolic Bone Disease Howard Morris PhD, FAACB, FFSc(RCPA) President, IFCC Professor of Medical Sciences, University of South Australia, Clinical Scientist, SA Pathology Adelaide, Australia 5000
2 Overview: Metabolic bone disease includes osteoporosis with its consequent fractures and less common conditions such as rickets (in children)/osteomalacia (adults), hyperparathyroidism in its various forms, Paget s disease Musculoskeletal diseases impose a major burden on individuals and costs 37 Billion per year in the European Community alone
3 Overview: However for the body maintaining plasma ionized calcium homeostasis is critical for functioning of the neuromuscular system including the heart, lungs and skeletal muscle Thus homeostatic mechanisms act to prioritise plasma calcium levels over the status of other organs particularly the skeleton Calciotropic hormones, plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D are major players Endocrine activities maintain plasma calcium
4 Biomarkers of Osteomalacia Rickets in children/ osteomalacia in adults largely arises from hypocalcaemia or hypophosphataemia Biomarkers of bone metabolism (bone turnover markers) are raised Biomarkers to identify the cause focus on plasma calcium, phosphate and their regulatory hormones Vitamin D deficiency has historically been a major factor
5 Vitamin D Synthesis and Activity UV Skin 7-dehydrocholesterol Vitamin D 3 Liver CYP2R1 25D [INACTIVE] CYP24 Kidney CYP27B1 24,25(OH) 2 D 3 [INACTIVE] Plasma 1,25D [ACTIVE] Endocrine Activity Ca homeostasis 1,25D +1 VDRE
6 Critical serum 25D Levels for Osteomalacia amongst Indian Females New Delhi (Lat. 29ºN) Female patients Siblings Parents (90) Sister (51) Brother (74) Mother (49) Father (47) Age (years) 24 (9.5) 18 (7) 21(10) 43 (9) 50 (9) Serum 25D 14.4 (5.7) 18.3 (9.9) 30.4 (14.3) 24.5 (17.6) 49.8 (9.2) (nmol/l) (ng/ml) 5.8 (2.3) 7.3 (4.0) 12.2 (5.7) 9.8 (7.0) 19.9 (3.7) Serum PTH 26.1(16) 8.7 (8.6) 3.8 (3.4) 5.5 (3.8) 4.9 (3.7) (pmol/l) Biochemical 90/90 11/45 3/61 5/42 5/40 Osteomalacia Ray D et al. Clinical Endocrinology 2009; 71:
7 Vitamin D actions on bone through endocrine activities Plasma 1,25D regulates plasma calcium homeostasis through intestinal calcium absorption, renal tubular reabsorption of calcium through regulation of Calbindin 28k mrna, and bone resorption through osteoblast RANKL mrna Osteomalacia can be resolved by the endocrine action of vitamin D Critical level for plasma 25D 8 ng/ml (20 nmol/l )
8 Hip fracture patients arising from osteoporosis have provided new insights into requirements for vitamin D
9 Effects of vitamin D and calcium supplementation on risk of hip fracture: meta-analyses of data from clinical trials Risk of hip fracture is reduced on average 30% (P<0.001) with vitamin D and calcium supplementation in post-menopausal women Weaver CM et al., Osteoporos Int (2016) 27:
10 Vitamin D actions on bone Compelling data demonstrate that osteoporosis and fractures are prevented by maintaining serum 25D levels rather than plasma 1,25D, implicating autocrine/ paracrine actions of vitamin D acting through bone cells The critical level for plasma 25D 20 ng/ml (50 nmol/l)
11 Clinical and pre-clinical studies indicate different activities for the serum 25D and 1,25D metabolites on bone mineral status. All evidence suggests that the effect of serum 25D arises from withintissue synthesis of 1,25D
12 Vitamin D Synthesis and Activity UV Skin 7-dehydrocholesterol Vitamin D 3 Liver CYP2R1 25D [INACTIVE] CYP24 Kidney CYP27B1 24,25(OH) 2 D 3 [INACTIVE] Plasma 1,25D [ACTIVE] Endocrine Activity Ca homeostasis 1,25D CYP27B1 25D 1,25D +1 VDRE
13 Serum 25(OH)D, Bone CYP27B1 and Bone CYP24A1 are independent predictors of BS/BV Effect on BS/BV is independent of age, s1,25(oh) 2 D and PTH Independent variables Adj. R 2 P-value BS/BV = x 25(OH)D -8.3 x Bone CYP27B x Bone CYP24A x Gender Serum 25(OH)D Bone Cell Human Trabecular bone: Bone-derived 1,25D protects the plate-like structures 25(OH)D CYP27B1 1,25(OH) 2 D CYP24A1 Degradation Plate-like trabecular structure Acknowledgement: Ms Deepti Sharma, PhD student
14 Mean Wall Thickness (MWT) A measure of bone formation period Indicator of how long osteoblast work during each remodelling cycle 80X n=10 a n=7b n=9 c n=11 d Measured as the distance between the cement line and last completed bone packet High Vitamin D (78.5nmol/L) Preliminary data suggest that serum 25(OH)D levels are strongly related to MWT a vs b, p=0.8, NS a vs c, p=0.001 a vs d, p< b vs c, p=0.04 b vs d, p= c vs d, p=0.20
15 These data provide evidence for assessing serum 25-hydroxyvitamin D as a biomarker for bone quality The critical level for serum 25- hydroxyvitamin D is 20 to 24 ng/ml (50 60 nmol/l)
16 Biomarkers of Bone Metabolism Bone turnover markers assess the average activity of bone remodelling Mean Wall Thickness
17 Markers of Bone Resorption Degradation products of mature type 1 collagen or products of osteoclast cells
18 Type 1 Collagen Crosslinks in Urine or Serum measures of osteoclast activity PYD or DPD Assay available in serum and urine Assay available in serum and urine Assay available in urine and serum for free or total
19 Tartrate-Resistant Acid Phosphatase (TRAP5b) a measure of osteoclast number rather than activity Kim, Lee et al Bone 2012; 51:
20 Markers of Bone Formation Products of osteoblasts expressed during different phases of osteoblast development or type 1 pro-collagen products
21 Bone turnover markers assess the average activity of bone remodelling cycles
22 Alkaline Phosphatase (ALP) Total serum alkaline phosphate is largely a mixture of bone and liver isoenzymes plus some intestinal isoenzyme Bone-specific alkaline phosphatase assays are available as immunoassays; ALP is synthesised by osteoblasts and is essential for mineralisation
23 Serum Osteocalcin Understanding of its physiology has become more complicated recently with identification that the under-decarboxylated form is recognised as a hormone regulating whole body energy metabolism Difficult to measure because it is present in serum as different forms and fragments
24 Type I Pro-collagen comprises a triple helix of three collagen molecules, two of A1and one molecule of A2 Proteolytic cleavage sites PINP PICP Synthesized within the osteoblast endoplasmic reticulum and propeptides released as the complex is secreted from the osteoblast Aminoterminal propeptide (PINP) comprises 3 domains: NH 2 -terminal globular domain (most immunogenic domain) Helical domain (H) and COOH-terminal domain Current assays utilise various antibodies to this relatively large protein Adapted from Koivula M-K, et al, Clin Biochem (2012) doi: /j.clinbiochem
25 Limitations for current clinical usefulness Variation biological including diurnal, fasting or feeding, age & sex and analytical Interpretation of values for the individual patient critical values and reference intervals
26 Diurnal variation of serum CTX in male volunteers effects of time and food intake Wichers M, et al Diurnal rhythm of Crosslaps in human serum. Clin Chem 1999, 45,
27 Variability of BTMs with age, sexhormone status and amongst individuals U-NTX S-Bone ALP Garnero, P, Sornay-Rendu, E, Chapuy, M-C, Delmas, PD. Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J. Bone Miner Res. 1996, 11,
28 Limitations for current clinical usefulness Biological variation diurnal, fasting or feeding, age, sex and analytical Interpretation of values for the individual patient critical values and reference intervals
29 Can BTM measurements be used to assess risk of fracture? The level of bone turnover marker is not a strong predictor of fracture risk independently of or better than bone mineral density as indicated by current evidence Can changes in BTMs assess fracture risk following treatment? Several studies have shown in general that the larger the decrease in BTM, the larger the reduction in fracture risk following anti-resorptive therapy
30 Changes in s-ctx following treatment with oral and iv bisphosphonates Saag K et al, Bone 2007; 40: ca Figure 6 S-βCTX (ng/ml) Alendronate 70mg Zoledronate 5mg Time (weeks) alendronate given per oral weekly and zoledronic acid given as a single IV dose
31 Change in Bone Turnover and Hip Fracture in Alendronate-Treated Women: The Fracture Intervention Trial (FIT) One-year change in bone ALP and hip fracture risk among ALN-treated women. Percent change in bone ALP and predicted risk (log OR) of hip fracture (solid line) and 95% CI (dotted lines) from logistic regression model.
32 Summary Measurement of vitamin D status is a biomarker of a nutrient deficiency contributing to metabolic bone disease and is easily corrected Serum 25D levels > 8ng/mL (20 nmol/l) are sufficient to prevent osteomalacia/rickets in children Vitamin D and dietary calcium are essential nutrients: they are both necessary to reduce osteoporosis and decrease fracture risk in the elderly. Serum 25D levels 20 ng/ml (50 nmol/l) are required
33 Summary The clinical use of bone turnover markers is problematic as a result of pre-analytic and analytic issues The most robust evidence suggests that their clinical usefulness is for monitoring efficacy of treatments for osteoporosis With anti-resorptive therapies the measurement of a bone turnover marker provides the earliest evidence of efficacy and the lower the fall in bone turnover the higher the reduction in fracture risk Preliminary evidence suggest that with anabolic therapies the increase in bone formation marker predicts response to therapy
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