Corso Integrato di Medicina di Laboratorio Insegnamento di Biochimica Clinica Metabolismo minerale e

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Corso Integrato di Medicina di
LaboratorioInsegnamento di Biochimica
ClinicaMetabolismo minerale e biomarcatori del
turnover osseo
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Main constituents of bone

• Cells
• - osteoblast (osteocyte)
• - osteoclast
• Organic matrix
• - Collagen (type I)
• - Other proteins (osteonectin 23, osteocalcin
  15, sialoprotein 9, others)
• Inorganic phase
• - Ca/P in the form of cristalline hydroxyapatite

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Calcium Physiology

• Calcium normally tightly regulated between
  approximately 2.20 and 2.60 mmol/L
• Several controlling factors
• PTH
• Vitamin D and metabolites
• Calcitonin
• Complex relations between
• GI uptake
• Renal Clearance
• Bone

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Calcium metabolism
Skeleton
25 mmol/day
25 Mol (99)
Gut
Kidney
13 mmol/d
300 mmol/day
2.20 mmol/L (30mmol)
3 mmol/d
290 mmol/day
Plasma/ICF
10 mmol/day
15 mmol/day
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Plasma Calcium

• Ionised Calcium
• Physiologically active fraction
• Cellular effects
• Control of PTH
• Bound Calcium
• Albumin main binding protein (50)
• Physiologically inactive
• Complexed Calcium
• Salts - calcium phosphate
• Total Calcium Ionised Bound Complexed

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Calcium Distribution in Plasma
Ionised Calcium 1.0 mmol/L
Total Calcium 2.0 mmol/L
Bound Calcium 0.95 mmol/L
Complexed Calcium 0.05 mmol/L
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Laboratory Measurement of Calcium

• Routine measurement is Total Calcium
• Cost
• Convenience
• Doesnt necessarily reflect ionised calcium
• Total Ca affected by albumin concentration
• pH influences ionised Ca

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Effect of Experimental Changes in Plasma Albumin
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Relation of Plasma Albumin to Calcium in Hospital
Patients
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Adjusted Calcium

• Calcium values can be corrected for changes in
  albumin
• Ca(adj) Ca (40 - Alb) x 0.025
• Reference interval is unchanged but patient
  samples are adjusted for better diagnostic
  performance
• Ca(adj) 2.15 - 2.65 mmol/L
• Ca(adj) 8.6 - 10.6 mg/dL

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Calcium homeostasis - PTH action
-ve feedback
PTH
125-DHCC
Decreased Ca Clearance
Increased Ca Absorption
Increased Ca Turnover with Net Resorption
Plasma/ICF Ca
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PTH Control
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Vitamin D Metabolism
VitD3
25-hydroxycalciferol (25-HCC)
VitD3
ACTIVE
INACTIVE
1,25-dihydroxycalciferol
24,25-dihydroxycalciferol
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Pathophysiology of Calcium

• Disorders of homeostatic regulators
• PTH
• vitamin D
• Disorders of the skeleton
• bone metastases
• Disorders of effector organs
• gut - malabsorption
• kidney
• Diet

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Causes of Hypercalcaemia

• Hyperparathyroidism
• Malignancy
• Lytic lesions
• Humoral (PTHrp)
• Drugs
• Thiazide diuretics
• Hyperthyroidism
• Excess absorption
• Vitamin D intoxication / Milk alkali syndrome
• Bone disease immobilisation
• Renal disease
• Artifact - venous stasis causing high albumin

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Hyperparathyroidism

• Commonest overall cause of hypercalcaemia
• Primary / Secondary forms seen
• Adenomas common / hyperplasia
• Associated with
• Increased bone turnover / resorption (biomarkers)
• Hypercalcaemia / Hypophosphataemia
• Long standing damage Osteitis Fibrosa Cystica

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Bone Metastases

• Common in several malignancies
• Lytic
• Breast / Lung
• Kidney / Myeloma
• Sclerotic
• Prostate
• Associated with
• Pain
• Pathological fractures
• Hypercalcaemia
• Raised bone markers

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Investigating hypercalcaemiaSorting out the
common causes

• Consider Adjusted Ca - look at the albumin
• Check drug history
• Exclude excess vitamin D intake
• Check for renal failure
• Simultaneous Ca PTH measurements
• Consider rarer causes more complex
  investigations

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Investigating hypercalcaemia
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Is it Hyperparathyroidism or Bone Metastases?
Hyperparathyroidism
Metastases
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Causes of Hypocalcaemia

• Hypoproteinaemia
• Vitamin D deficiency
• Dietary / Malabsorption
• Hepatic disease
• Renal Disease
• End organ Vitamin D resistance
• Hypoparathyroidism
• Inadequate intake of calcium
• Pseudohypoparathyroidism
• End organ (renal) PTH resistance

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Osteomalacia

• Due to vitamin D deficiency
• Adult form - widened osteoid seams with lack of
  mineralisation
• Classic childhood rickets - widened epiphyses
  poor skeletal growth
• Failure of vitamin D metabolism or effectiveness
  e.g. in renal / liver disease and some rare
  genetic forms
• Associated with
• Hypocalcaemia with secondary hyperparathyroidism
• Raised bone markers

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Is it Hypoparathyroidism or Vitamin D Deficiency?
Vitamin D Deficiency
Hypoparathyroidism
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Phosphate

• Major intracellular ion - small proportion in
  plasma
• Involved in high energy reactions e.g. those
  involving ATP - Na/K pumps
• Deficiency can cause weakness and dysfunction
• Severe depletion can be fatal

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Phosphate Deficiency - Causes

• Hyperparathyroidism
• Excess losses
• renal tubular damage
• GIT
• diabetes (diuresis)
• Poor intake
• malnutrtion

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RIMODELLAMENTO OSSEO
Precursori osteoblasti (OB)
Precursori osteoclasti (OC)
MIDOLLO OSSEO
Cellule di superficie
Cellule di superficie
OB
OC
Attivazione
Mineralizzazione
Osteoide
OSSO
Linea cemento
Sintesi osteoide
Superficie a riposo
Iniziale erosione
Osteone completo
Fase riassorbimento 2-4 settimane
Fase formazione 4-6 mesi
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Bone Disorders

• Metastatic disease
• Hyperparathyroidism
• Pagets Disease
• Osteomalacia / Rickets
• Osteoporosis

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Osteoporosis

• Common disease especially of elderly
• Generalised loss of bone with propensity to
  fractures - spine, hip
• Etiology is largely unknown
• No diagnostic abnormalities are seen in
  biochemical tests
• Diagnosis relies almost exclusively on DEXA/Xray

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Investigation of Bone Diseases

• Gross Stucture
• Xray
• Bone Mass (Calcium)
• DEXA
• Cellular Function / Turnover
• Biochemistry
• Microstructure / Cellular Function
• Biopsy

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Biomarkers of bone formation
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Alkaline Phosphatase

• Measured by the laboratory in Liver and Bone
  profiles
• Total ALP activity in serum derived from all
  isoenzyme (isoform) fractions. In healthy adults
  approximately
• 50 liver
• 50 bone
• Specific isoenzymes (isoforms) can be measured
  where there is diagnostic doubt

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Bone Alkaline Phosphatase (B-ALP)

• Phosphatase involved in mineralisation
• Released by osteoblasts
• Release stimulated by increased bone remodelling
• Childhood / Pubertal growth spurt
• Fractures
• Hyperparathyroidism
• Primary
• Secondary
• Pagets

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Biosynthesis of osteocalcin

• De novo by osteoblasts
• Intracellular precursor of 10 kDa
  (pro-osteocalcin)
• Dependent on vitamin K and CO2
• Modulated by vitamin D
• Expression of synthesis with hydroxyapatite
  deposition

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1
49
Osteocalcina intatta
1
49
44
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Frammento N-MID
C-terminale
1
19
43
20
Frammento MID
N-terminale
20
49
MID C-terminale
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Propeptidi del procollagene I
PINP
PICP
Collagene
Regione a tripla elica
Regione globulare
Regione non tripla elica
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Biomarkers of bone resorption
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Fosfatasi acida

• 4 isoenzimi (osso, prostata, lisosomale,
  eritrociti)
• Lisoenzima osseo è prodotto dagli osteoclasti
• Gli isoenzimi sono differenziabili in base alla
  inibizione con tartrato

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• Idrossiprolina
• Prodotto da modificazioni postsintetiche del
  collagene (idrossilazione di residui di prolina)
• Rilasciato in seguito alla degradazione del
  collagene, escreto con le urine, ma per il 90
  riassorbito
• Assorbito attraverso lintestino, necessita una
  dieta priva di collagene prima del dosaggio
• Non specifico per il collagene di tipo I

• Galattosilidrossilisina
• Prodotto da modificazioni postsintetiche del
  collagene (glicosilazione di residui di
  idrossilisina)
• Presente prevalentemente nel collagene di tipo I
• Rilasciato in seguito alla degradazione del
  collagene, escreto con le urine
• Determinato nelle urine solo con HPLC

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Crosslinks del piridinio
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Crosslinks del piridinio
Piridinolina (PYD)
Desossipiridinolina (DPD)
Cartilagine Osso Tendini/legamenti Connettivo dei
vasi
Osso Dentina (Aorta) (Legamenti)
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Telopeptidi del collagene I
NTX
CTX
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Bone markers vs. bone biopsy and bone mineral
density
Advantages
Disadvantages

• Non-invasive
• Reflect current levels of bone remodelling
• Predict rate of bone loss (markers of resorption)
• (Unlimited) number of sequential measurements

• Do not assess bone mass
• Do not distinguish bones involved
• Risk of non-specificity (metabolism)
• Sensitive to fractures

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BIOMARKERS AND BONE LOSS
P0.0005
Plt0.0001
P0.006
Garnero et al, JBMR 1999
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Effect of Alendronate Therapy on Serum CTX

100
Placebo
1 mg alendronate
5 mg alendronate
50
10 mg alendronate
20 mg alendronate
months
6 12 18 24
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Prediction of bone mass response to
antiresorption therapy by biomarkers
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BONE MARKERSBiological variability and critical
differences