bone health

1. BONE HEALTH
PRESENTER: DR. JYOTI SHARMA
MODERATOR:DR. SOJIT TOMO

2. INTRODUCTION
• Bone – mineralized connective tissue
• Type I collagen
• Contains approx. 98 to 99% of Calcium
• Calcium and phosphorus helps in mineralization
• Dynamic tissue

3. BONE REMODELLING
• Approx 10% to 30% of the skeleton is remodelled each year, with individual
variation.
• Osteoclasts - resorb bone
• Osteoblasts - forms new bone at bone resorption site
• Osteocytes - nourish the skeleton and regulate bone cell activity
• Occurs in discrete packets known as bone remodeling units
• Remodelling cycle - activation, resorption, reversal, formation, and
termination/resting phases.

4. REMODELLING CYCLE

5. CALCIUM
• The skeleton contains approximately 99% of the body’s calcium
• Soft tissues and extracellular fluid contain about 1% of the body’s calcium.
• The free calcium fraction is the biologically active form
• About 80% of protein-bound calcium is associated with albumin

6. EQUILLIBRIUM OF CALCIUM IN SERUM
Calcium can be redistributed among the three plasma pools, acutely or chronically, by alterations in
the concentrations of protein and small anions, changes in pH, or changes in the quantities of free
calcium and total calcium in the plasma

7. FUNCTIONS
OF CALCIUM
FUNCTIONS:

8. CALCIUM ESTIMATION
• Total, albumin-adjusted, and free calcium
• In Serum and Urine
• Ion-selective electrode (ISE)
• Photometry - o-Cresolphthalein Complexone Method
Arsenazo III Method
• Atomic absorption spectrophotometry(Reference method)

9. PHOSPHORUS
• Inorganic and Organic phosphate
• Inorganic phosphate is measured.
• Inorganic phosphate exist as monovalent (H2PO4− ) and divalent (HPO4 2− ) phosphate
anions.
• The ratio of (H2PO4 − to HPO4 2−) is pH dependent
• 1 :1 in acidosis
• 1 :4 at pH 7.4
• 1 :9 in alkalosis
• Regulation of phosphate involves kidneys, intestine, and skeleton.

10. FUNCTIONS
• Major component of hydroxyapatite in bone
• Provides phosphate for extracellular and intracellular pool.
• Component of nucleic acids, phosphopholipids, ATP, creatine phosphate
• Muscle contractility, neurologic function, and electrolyte transport.
• Cofactors - nicotinamide-adenine dinucleotide phosphate (NADP).
• Activity of enzymes(adenylate cyclase, 25-hydroxyvitamin D-1α-hydroxylase)
• Buffer

11. PHOSPHORUS ESTIMATION
Reaction:
• Phosphate + ammonium molybdate phosphomolybdate complex (at 340
nm) acidic pH
or reduced molybdenum blue (600 to 700 nm)
• Fasting sample preferred because of diurnal variation

12. MAGNESIUM
• The total body magnesium content is about 25 g (~1 mol), of which about 55%
resides in the skeleton .
• One-third of skeletal magnesium is exchangeable and is thought to serve as a
reservoir for maintaining the extracellular magnesium concentration.
• Within cells, most of the magnesium is bound to proteins and negatively charged
molecules
• Extracellular magnesium accounts for about 1% of the total body magnesium
content

13. FUNCTIONS
• Cofactor for more than 300 enzymes in the body.
• MgATP as a substrate for numerous enzymes that require ATP
• Allosteric activator of many enzyme (adenylate cyclase, Na+-K+adenosine
triphosphatase (ATPase)
• Oxidative phosphorylation, glycolysis, cell replication, nucleotide metabolism,
and protein biosynthesis.
• Neuromuscular excitability

14. ESTIMATION
Total magnesium
• Photometry, fluorometry, flame emission spectroscopy, and atomic absorption
spectrometry[reference method]
• Calmagite +magnesium coloured complex ( 530 to 550 nm)
• Enzymatic methods: uses Mg2+-ATP as a substrate.
• The rate of the enzyme-catalyzed reaction α concentration of magnesium.
ALKALINE

15. • Free (Ionized) Magnesium
• ISEs with neutral carrier ionophores
• Current ionophores or electrodes have insufficient selectivity for magnesium over
calcium.
• chemometric correction required for calcium interference.
CONTD…

16. Normal values:
CALCIUM PHOSPHORUS MAGNESIUM
TOTAL(mg/dl) 8.8 – 10.3 2.5 to 4.5 1.7 to 2.4
IONIC(mg/dl) 4.6 – 5.3 _ 1.09 to 1.45

17. HORMONES REGULATING
BONE METABOLISM

18. PARATHYROID HORMONE
• PTH is synthesized and secreted by the parathyroid glands
• Regulates free calcium and phosphate.
• Acts directly on bone and the kidneys, and indirectly on intestine (via 1,25(OH)2d
• Circulating PTH demonstrates a circadian rhythm and an appropriate variation in
end-organ function throughout 24 hours
• Biological active form – Intact PTH
t1/2 of 4 minutes

19. INTRA-OPERATIVE PTH ESTIMATION
• Asses the completeness of parathyroidectomy and to facilitate minimally invasive
parathyroid surgery.
• PTH is measured just before the incision and again at 20 minutes after resection
of the hyperfunctioning parathyroid tissue.).
• A decline of 50% or more is usually considered indicative of the removal of all
hyperfunctioning tissue.

20. PTH ESTIMATION
• Noncompetitive (sandwich) immunoassays are commonly used for the
measurement of intact PTH
• PTH assays were developed to measure the intact molecule of PTH only and were
called intact assays.
• Reference intervals :
• Intact PTH 10pg/ml to 65 pg/ml or 1.1 pmol/l to 6.8 pmol/l
• PTH - 6pg/ml to 40 pg/ml or 0.6 pmol/l to 4.2pmol/l

21. PARATHYROID HORMONE–RELATED PROTEIN
(PTHrP)
• Causing the state known as humoral hypercalcemia of malignancy (HHM) in
various cancers
• Acts on parathyroid receptor, increases renal reabsorption calcium and activity of
osteoclasts
• Competitive immunoassays have been used to measure
• Non-competitive immunoassays can measure 1 to 2 pmol/L in normal subjects
• PTHrP concentrations in patients with HHM upto 26 pmol/L

22. VITAMIN D
• Active form 1,25(OH)2D
• Regulated, primarily by PTH, phosphate, calcium, FGF23, and 1,25(OH)2D
• Maintain calcium and phosphate concentrations via intestine, bone, kidney, and the
parathyroid glands
25(OH)D
• Is main circulating form of vitamin D
• longer half-life and is less affected by day-to-day variation, exposure to sunlight, or
food intake

23. Contd..
• Reference range
15 to 60 pg/mL (36–144 pmol/L)
• Dual mass spectrometry – reference method
• Commercially measured by radiochemiluminescence or chemiluminescence

24. INTEGRATED CONTROL OF BONE METABOLISM

25. Patterns of Calcium , Phosphorus and Vitamin D in various
disorders

26. CALCITONIN
• Synthesized from the parafollicular or C cells of the thyroid gland
• Stimulated by circulating calcium
• Inhibits activity of osteoclast
• Marker for medullary thyroid carcinoma (familial form) and helps in
monitoring of patient after treatment.
• Procalcitonin - as a potential infection marker

27. CALCITONIN ESTIMATION
• Noncompetitive immunoassays (IRMA, ELISA, EIA, ICMA)
• Basal calcitonin concentration in adults is about 10 pg/ml (2.9 pmol/L)
• The mean concentrations are lower in women (5.8 pg/ml[1.7 pmol/L]) and higher
in men (8.8 pg/ml[2.6 pmol/L])

28. FGF 23
• Increases fractional excretion of phosphate by the kidneys
• Secreted by bone cells
• Decreases active form of vitamin D [1,25(OH)2D)] by decreasing the activity 25-
hydroxyvitamin d 1-alpha-hydroxylase.
• Decrease plasma phosphate concentrations.
• In CKD, FGF23 increases to counter increasing plasma phosphate.
• Responsiveness to FGF23 declines reducing fgf23’s ability to lower plasma
phosphate in kidney failure.

29. BIOCHEMICAL MARKER FOR BONE TURNOVER
FOR BONE FORMATION
• Propeptides of type I procollagen-
PINP and PICP
• Bone alkaline phosphatase- BALP
• Osteocalcin (bone gla protein [BGP]) –
OC
FOR BONE RESORPTION
Type I Collagen Telopeptides
• N-telopeptide (NTX)
• C-telopeptide (CTX)
• ICTP
Pyridinium Cross-Link
• Free deoxypyridinoline (DPD) and
pyridinoline (PYD)
• Total deoxypyridinoline
(DPD)apyridinoline (PYD)
Tartrate-Resistant Acid Phosphatase 5b
(TRAP5b)

30. TYPE I PROCOLLAGEN

31. PINP AND PICP( PROPEPTIDES OF TYPE I PROCOLLAGEN )
• For each molecule of type I collagen deposited in the tissues, one molecule of
each propeptide is released
• Degraded by the endothelial cells of the liver and from kidney in minor quantity
• Clinical significance
• Response to treatment for osteoporosis therapies
• Bone formation markers in clinical practice.
• The ratio of picp/pinp in serum ~3 in adults
• In children it is less than1.387.
• A low picp/pinp ratio is associated with aggressive breast cancer.

32. ESTIMATION
• EIA is commercially available
• Intact PINP and total PINP.
• Only RIA for intact PINP has been approved by the US Food and Drug Administration (FDA).
• Reference Intervals.
• For PICP : 38 to 202 µg/L in men
50 to 170 µg/L in women
• For intact PINP:20 to 78 µg/L in men
19 to 84 µg/L in women
• PINP concentration is very high in neonates and young children

33. BONE ALKALINE PHOSPHATASE
• Isoforms: alkaline phosphatase of liver, bone, and kidney
• Hydrolyze inorganic pyrophosphate (PPi) to generate phosphate
• produced by osteoblasts during the matrix maturation phase 1
• Clinical Significance.
• In diagnosis and monitoring of Paget disease of bone.
• Increased in metabolic bone diseases - osteoporosis, osteomalacia and rickets,
hyperparatyroidism, renal osteodystrophy

34. OSTEOCALCIN
• Noncollagenous protein of the bone matrix
• Produced by osteoblasts, odontoblasts, and even chondrocytes.
• But it may serve as a site of deposition for hydroxyapatite crystals.
• Undercarboxylated osteocalcin - better predictor of fracture
• Metabolized mainly in the kidneys and in the liver
• T1/2- 5 minutes.
• Increased in hyperparathyroidism, acromegaly, and paget’s disease.
• Decreased in hypoparathyroidism , hypothyroidism and in patients on
glucocorticoid therapy.

35. BONE RESORPTION MARKERS
Type I Collagen Telopeptides [Amino-telopeptides (ntx) and
carboxytelopeptides (ctx)]
• They occur in the amino-terminal and carboxy-terminal regions of the collagen molecule.
•
• Released into the circulation as collagen is degraded.
•
• excreted in the urine
• Estimating relative risks of hip fracture in postmenopausal women and in predicting
complications of osteoporosis
• Reductions of urinary ctx and ntx in the range of 50% to 60% with 3 to 6 months of
antiresorptive therapy

36. Pyridinium Cross-Links (Pyridinoline and Deoxypyridinoline)
• Found in a unique ratio in the bone it is 3 to 3.5:1
• Excreted in urine in free form (40%) and in peptide-bound form (60%).
• Suggests active bone resorption.
• To study the effects of hormone replacement therapy
• Estimation - High-pressure liquid chromatography (HPLC) or Immunoassays.
• Marked diurnal variation that is seen with urinary pyridinolines, (peak late at
night and early in the morning)

37. METABOLIC BONE DISEASES
• Osteoporosis
• Osteomalacia and Rickets
• Disorders of Bone and Mineral in Chronic Kidney Disease (Renal Osteodystrophy)
• Paget Disease of Bone
• Osteogenesis Imperfecta

38. OSTEOPOROSIS
• Decreased bone mass
• More common in females
• Advanced age(>40), female gender, and
post-menopause
• Bone resorption exceeds bone formation
• Divided etiologically into primary and
secondary

39. CONTD…
• Both bone resorption and fomation markers increased and used in monitoring
the effects of antiresorptive therapy.
• The International Osteoporosis Foundation and IFCC recommend one bone
formation marker (serum PINP) and one bone resorption marker (plasma CTX)
to be used as reference markers

40. Osteomalacia and Rickets
• Vitamin D deficiency or phosphate
depletion
• Defective mineralization of bone
• Osteomalacia
• In adults
• bones gradually become soft
• the ratio of osteoid to mineralized bone
increases over time.
• Weakness, skeletal pain and
deformities, and fractures
• decrease in skeletal radiodensity.

41. Rickets: Defective mineralization of bones in children before the closure of
epiphyseal plate of long bones
• Vitamin D–dependent rickets - type 1 is an inherited defect in 25(oh)d-1α-
hydroxylase that causes impaired formation of 1,25(OH)2D.
• Vitamin D–dependent rickets type 2 is an inherited - very high plasma
concentrations of 1,25(OH)2d - resistance to 1,25(OH)2D - defects in the
1,25(OH)2D receptor
• Vitamin D–resistant rickets. - X-linked dominant inherited trait characterized by
renal phosphate wasting.

42. Renal osteodystrophy
• Bone abnormalities in end-stage renal disease (ESRD) patients
• Osteitis fibrosa cystica - increased bone turnover due to secondary
hyperparathyroidism, a consequence of decreased levels of 1,25(OH)2D3 and
ionized calcium
• Osteomalacia and adynamic bone disease - poor mineralization of bone resulting
in the accumulation of surface osteoid (unmineralized bone).
• Amyloidosis – α2 microglobulin deposition

43. Contd..
• Biochemical findings
• Hyperphosphatemia and Hypocalcemia
• Increased PTH
• 1,25(OH)2D is decreased. ALP is increased in patients with hyperparathyroidism
or osteomalacia.
• High magnesium concentration (2–4 mg/dL [0.08–0.16 mmol/L]) especially in
those taking magnesium-containing antacids

44. Paget Disease of Bone
• Osteoclastic bone resorption, followed by replacement of bone in a
chaotic fashion
• SQSTM1 mutation
• Has late onset, patchy bone involvement
• May involve one bone (monostotic) or several bones (polyostotic)
• Increased concentration of bone markers(PINP) and total ALP seen
• Diagnosis is made on basis of clinical and radiological findings and
total ALP

45. Osteogenesis Imperfecta
• Brittle bone disease - inherited connective tissue disorders similar low bone
mass, minimal trauma fractures, and subsequent bone deformity
• Lethal in fetal life and in neonates.
• Abnormal production, posttranslational modification, and metabolism of
collagen, particularly type I collagen
• Frontal bossing of the skull, bluish sclera, yellowish teeth, barrel chest/pectus
excavatum, joint laxity, vertebral compression, and growth retardation

46. • Acid phosphatase is increased in type VIII
• ALP increased in post fracture, and increased CTX and PINP in some types of OI
(type III)
• Abnormal urine pyridinoline cross-links ratios in different types of OI
• Bone formation and resorption markers can provide evidence of the therapeutic
effect of bisphosphonate treatment.

47. Thank you