CALCIUM AND ITS SIGNIFICANCE IN BONE CALCIUM AND ITS SIGNIFICANCE IN BONE

METABOLISMMETABOLISM

Jana Jana Jurcovicova

Department of Normal, Pathological and Clinical PhysiologyDepartment of Normal, Pathological and Clinical Physiology

CALCIUMCALCIUM

• 2% of body weight• 99% in bones and teeth in form of mineral• 1% in body fluids

• Plasma (Extracellular fluid)

2.25 – 2.75 mmol/L• Cell

cytosol 10-5 – 10-4 mmol/L

endoplasmic reticulum 0.3 – 0.8 mmol/L

PLASMA CALCIUMPLASMA CALCIUM

diffusible• 48% (50%) Ca2+ ionized • 6% (10%) combined with anions (citrate,

phosphate) – non-dissociated

nondiffusible• 46% (40%) combined with plasma proteins• combination with proteins depends on pH

Guyton and Hall, 1996

IONIC COMPOSITION OF ICF AND ECFIONIC COMPOSITION OF ICF AND ECF

Ion ECF

mmol/L

ICF

mmol/L

Na+ 136-146 20

K+ 3.8-5.4 150

Ca2+ 2.25-2.75 c. 10-4

ER 0.8

Cl- 97-109 3

HCO3-

A-

22-26

155

10

0

ROLE OF CALCIUMROLE OF CALCIUM

• release of transmitters from synapses

• “second messenger” (intracellular)

• neuromuscular transmission and muscle contraction

• protein folding in endoplasmic reticulum

• stimulation of secretory activity of exocrine and endocrine glands

• blood coagulation

• catherin activation

• bone mineralization

RELASE OF TRANSMITTERS FROM SYNAPSES Ligand-gated ion channelsLigand-gated ion channels

Voltage-gated ion channelsVoltage-gated ion channels

SYNAPSESSYNAPSES

Synapses Chemical – secreting neurotransmitters Electrical - direct opening of fluid channels that conduct electricity from one cell to another (gap junction)

1. action potential

2. voltage gatedCa++

channels

3. exocytosis ofAch vesicles

4. binding to Ach receptors

5. Influx of Na+ into muscle cellplate potential

6. more opening of Voltage gated Na+ channels

NEUROMUSCULAR TRANSMISSION AND MUSCLE CONTRACTION

REGULATION OF INTRACELLULAR CALCIUMREGULATION OF INTRACELLULAR CALCIUM

Calcium pump

Calcium pump

conc. 2.25-2.75 mM

10-4 mM

0.8mM

sensorproteins

store operated Ca2+ entry

“SECOND MESSENGER

Inositol phospholipid pathway: phospholipase C – Inositol 1,4,5, triphosphate IP3 - ER - Ca2+ – PROTEIN KINASE C – further phopspohorylation

Binding to calmodulin 2 Ca2+– activation of many other proteins e.g. CaM-kinases and trigger of phosphorylation of other proteins

REGULATION OF REGULATION OF PROTEIN PROTEIN SECRETIONSECRETION

Trigger for regulated exocytosis:1. Entry of Ca2+ through calcium channels2. Release of Ca2+ from intracellular stores (enoplasmic

reticulum)

Exocrine cellsEndocrine cellsRelease of neurotransmitters

BLOOD COAGULATION

Platelet aggregation becomes a definite clot due to fibrin. In the conversion of soluble protein fibrinogen to insoluble fibrin, the cascade of enzymatic reaction requires presence of Ca2+

CATHERIN ACTIVATION

Proteins in epitelial cells of jejunum that bind together in the presence of

Ca2+

plasma membrane cytoskeleton fibre

catherinbridging protein

cell 1 cell 2

BONE MINERALIZATION

The crystalline salts deposited in the organic matrix of bone are composed of calcium and phosphate in a form of hydroxyapatite

Ca10(PO4)6 (OH)2

The initial calcium salt to be deposited are not hydroxyapatite crystals but amorphous salts such as CaHPO4 .2H2O, Ca3(HPO4)2 . 3H2O. They convert into hydroxyapatite within weeks. These amorphous salts represent the readily exchangeable pool of calcium.

CALCIUM METABOLISMCALCIUM METABOLISM

From Guyton and Hall

Rapid exchange

PHOSPHATESPHOSPHATES

• 80% bones and teeth

• 10% blood and muscles

• 10% different chemical complexes

• Plasma (ECF) 0.65 – 1.62 mmol/L

• Cell (ICF) 65 mmol/L (including organic P)

PHOSPHATESPHOSPHATES

• calcium phosphate, hydroxyapatite (bone)

• inorganic anions: HPO32-, H2PO3

-

• organic: DNA, phospholipids

• ATP, cAMP, creatinphosphate molecules with metabolic significance

• Ca, P rates of intake 1g/day

BONEBONE COMPOSITION COMPOSITION

• Skeletal calcium: 99% of skeletal calcium forms stable bone (not exchangeable with the Ca2+ in

extracellular fluid) 1% is in the form of releasable pool of Ca2+

• Organic matrix : 90 – 95% collagen type 1 fibers and 5% homogenous ground substance

(extracellular fluids, proteoglycans - chondroitin sulphate, heparan sulphate, and hyaluronic acid)

• Bone forming cells: Osteoblasts - cells responsible for bone deposition

– Produce type 1 collagen, proteins (chondroitin sulphate heparan sulphate), hyaluronic acid, alkaline phposphatase.

– osteoblasts surrounded by mineralized matrix becomes osteocytes

• Osteoclasts – bone-consuming multinocleated cells that resorb the previously formed bone. They are cells of hematopoietic origin, derived from monocyte- macrophages.

BONE STRUCTUREBONE STRUCTURE

from Ganong

(cortical bone)

trabecular bone

3x more remodelling units than in cort. bone

BONE GROWTH AT THE EPIPHYSEAL PLATE

Tubular bones develop by endochondral ossification. As longitudinal growth occurs, the growth plate (physis) forms, consisting of 4 zones.

The zone of resting cartilage is adherent to epiphyseal bone.

The proliferating zone is characterized by vertical columns of chndrocytes which expand the plate until they enter the hypertrophic zone where they enlarge and polarize.

The hypertrophic chondrocytes, closest to the metaphysis, produce alkaline phosphatase promoting the formation of calcified cartilage.

STRUCTURE OF TRABECULAR AND COMPACT BONE

OSTEONS - concentric layers of mineralized collagen

HAVERSIAN CHANNEL

RESORPTION PORES

TRABECULAR BONE

(nutrients diffuse from ECF)

LACUNAE

COMPACT BONE

nutrient supply from Haversian channel

from Ganong

BONE REMODELLING

cycle 3 – 6 months

1. ostoclast differentiation

(M-CSF)

2. bone resorption (15 days)

3. reverse phase (ostoblasts fills the pores)

ACTIVITY ACTIVITY OF OSTEOBLASTS and OSTEOCLASTS OF OSTEOBLASTS and OSTEOCLASTS

activators of osteoblasts:TGF-ß (transforming growth factor - ßIGF-1 (insulin-like growth factor) secreted by liver but also byosteoblasts. IGF-1 is controled by pituitary growth hormone

activators of osteoclasts:osteoclast differentiation factor RANKL, interleukin-1ß,interleukin-6 tumor necrosis factor-α, macrophage colonystimulating factor (produced by osteoblasts)

other bone forming protein:trombospondine, osteopontin, osteocalcin

COUPLED OSTEOBLAST – OSTEOCLAST REGULATION

hematopoietic stem cell

Mesenchymal stem cells

chondrocytes

proosteoclast proosteoblast

osteoclast osteoblast

RANKLODF

OPG

macrophage colony stimulating factor

SIGNALING PATHWAY FOR NORMAL OSTEOCLASTOGENESIS

- osteoclast differentiation factorosteoprotegerin

adaptor protein

transcription factors

Boyce and Xing, Arth Res Ther, 2007

BONE REMODELLING

Osteoclastic resorption is coupled with new osteoblastic bone formation partly due to release of skeletal growth factors

BONE RESORPTION BY OSTOCLASTS

from Ganong

The osteoclasts send out villus-like projections toward the bone forming a ruffled border adjacent to the bone.The villi secrete proteolytic enzymes and several acids ( citric acid and lactic). The enzymes digest or the organic matrix of the bone, and the acids cause solution of the bone salts.The osteoclastic cells also imbibe by phagocytosis minute particles of bone matrix and crystals, eventually also dissoluting these and releasing the products into the blood.

WOLFF'S LAWWOLFF'S LAW

• Wolff's law is a theory developed by the German Anatomist/Surgeon Julius Wolff (1835-1902) in the 19th century that states that bone in a healthy person or animal will adapt to the loads it is placed under.

• If loading on a particular bone increases, the bone will remodel itself over time to become stronger to resist that sort of loading.

• The converse is true as well: if the loading on a bone

decreases, the bone will become weaker due to turnover as it is less metabolically costly to maintain and there is no stimulus for continued remodelling that is required to maintain bone mass.

EXAMPLES OF WOLFEXAMPLES OF WOLFF’S LAWF’S LAW

• The racquet-holding arm bones of tennis players become much stronger than those of the other arm. Their bodies have strengthened the bones in their racquet-holding arm since it is routinely placed under higher than normal stresses.

• Astronauts who spend a long time in space will often return to Earth with weaker bones, since gravity hasn't been exerting a load on their bones. Their bodies have reabsorbed much of the mineral that was previously in their bones.

• Weightlifters often display increases in bone density in response to their training.

SEX DIFFERENCESSEX DIFFERENCES

From Ganong

BONE MASS ACQUISITION AND LOSS

Recommended daily intake of Ca2+

adolescents :1200 – 1500 mg

adults: 1000 mg

pregnant women - 1500 mg

breastfeeding woman - 1500 mg

seniors – 1500 mg

HORMONAL HORMONAL REGULATION OF REGULATION OF CALCIUMCALCIUM METABOLISMMETABOLISM

1. PARATHYROID HORMONE (receptors on osteoblasts)

2. CALCITRIOL (receptors on osteoblasts)

3. CALCITONIN (receptors on osteoclasts)

THYROID AND THYROID AND PARATHYROID GLANDPARATHYROID GLANDSS

release PTH

(function?)

size: 6 x 4 x 2 mm

PARATHORMONPARATHORMON

• parathyroid glands• polypeptide of 84 amino acids• stimulus for secretion – low plasma calcium• function – to INCREASE plasma calcium

– activation of osteoblast - osteoclast interaction (resorption of calcium and phosphorus)

– decreases excretion of Ca2+ by kidney– increases excretion of phosphates by kidney– stimulates synthesis of calcitriol (hydroxylation of

25-(OH)D3 to 1,25-(OH)D3 in kidney

START HERE

Cholesterol 7-Dehydrocholesterol

sunlight (UV light)

CholecalcifeolVitamin D

Calciferdiol25-hydroxycholecarciferol

Liver enzyme

Kidney enzyme

Diet

Calcitriol1,25-hydroxycholecarciferol

CALCITRIOLCALCITRIOL

CALCITRIOLCALCITRIOL

vitamin D hormonevitamin D hormone Skin: preprovitamin D (7-dehydrocholesterol) UV irradiation (290 – 310 nm) - cholecalciferol (D3)

Liver microsomes: 25-hydroxylase - 25-(OH)D3 Kidney epitelial cell mitochondria : 1-hydroxylase - 1,25-(OH)2D3 ( mediated by PTH)

Function: INCREASE plasma calcium by absorption of Ca2+ in intestine via formation of calcium-binding protein in epithelial cells

Noncalcemic function:Regulation of immune functions, prevention of autoimmune diseases(rheumatoid arthritis, psoriasis, inflammatory bowel disease etc)

AA: Circulating concentrations of : Circulating concentrations of vitamin D after a single exposure to 1 vitamin D after a single exposure to 1 MED (minimal erythema dose ) of MED (minimal erythema dose ) of simulated sunlight, with a sunscreen simulated sunlight, with a sunscreen or a topical placebo cream. or a topical placebo cream.

B: Circulating concentrations of B: Circulating concentrations of vitamin D in response to whole-body vitamin D in response to whole-body exposure to 1 MED among healthy exposure to 1 MED among healthy young and elderly subjectsyoung and elderly subjects

EFFECT OF UV EXPOSURE ON CHOLECALCIFEROL LEVELS

Holick MF, Am J Clin Nutr, 2004

SUMMARY OF PLASMA Ca2+ REGULATION

uv light 290 -310 nm PARATYROIDEA

PTH

LIVER25-hydroxylase

25-(OH)D3

1-hydroxylase

DUODENUMupper jejunum

Ca2+ PO3-4

resorption

PO3-4

resorption

SKIN

D3

25-(OH)D3

1,25-(OH)2D3

Ca2+

resorption

BONE

KIDNEY

osteoblast (RANKL)

osteoclast

Ca2+PO3-4

release

Stimulation

inhibition

CALCITONINCALCITONIN

• Parafollicular cells of thyroid gland (C-cells)

peptide of 32 amino acids

• stimulus for secretion – high plasma calcium

• function – to DECREASE plasma calcium and

phosphates by inhibition of osteolysis (decreases absorption of Ca, P from bones)

RELATION RELATION - - PLASMA CONCENTRATIONPLASMA CONCENTRATION OF OF CA CA2+ 2+ X HORMONESX HORMONES

EFFECTS OF OTHER HORMONES

thyroxine - activate bone remodeling, hypersecretion activates osteoclastic activity

cortisol – inhibit maturation of osteoblasts

estrogens – stimulate osteoblast growrth by activation of TGF-β, inhibit interleukin production by osteoblasts resulting in lower osteoclast activity

testosterone – stimulate osteoblast activity

growth hormone – stimulates IGF-1 resulting in osteoblast maturation

LOSS OF CALCIUM HOMEOSTATLOSS OF CALCIUM HOMEOSTATAATIC TIC CONTROL CONTROL

Hypocalcemia

impaired calcitriol absorption, hypoparathyroidism, resistance to parathormone, autoantibody to parathormone

• increased neuromuscular irritability - carpopedal spasm

• dilatation of heart

• Increased cell membrane permeability, headache • Impaired blood clotting

• central nervous system irritability, depressions

CARPOPEDAL SPASMCARPOPEDAL SPASM

From Guyton and Hall

LOSS OF CALCIUM HOMEOSTATLOSS OF CALCIUM HOMEOSTATAATIC TIC CONTROL CONTROL

Hypercalcemia

excessive parathormone secretion by lack of feedbach by calcium, or by malignancy • depression of nervous system, reflex activity, sluggishness • impaired kidney function due to kidney overload - calciuria• formation of calcium phosphate crystalls in kidney• calcification of renal epitelium• primary arterial hypertension• muscle atrophy, hyporeflection• chronic fatigue • Decalcification of bones• Multiple fractures (Osteitis fibrosa cystica)

CHANGES IN CHANGES IN CALCITRIOLCALCITRIOL PLASMA LEVEL PLASMA LEVEL

LACK OF CALCITRIOL

• RICKETS (rachitis)– children• OSTEOMALACIA - adults

softening of bones resulting from lowered calcium absorption and enhanced phosphate excretion which gives rise to ineffective bone mineralization (aching bones and muscle discomfort) Attention! Osteoporosis is a decrease of bone mass (matrix and minerals) rickets normal rickets

OSTEOPOROSIS

the silent pandemic of the last century, about 60% of postmenopausal women are at risk of bone fracture

The osteoblastic activity is less than normal – consequence the bone deposition is depressed

Causes: lack of physical stress on the bones because of inactivity

Lack of calcitriol production – lowered Ca2+ reabsorption

lack of vitamin C

Postmenopausal lack of estrogen secretion, enhanced cytokine cascade – IL-1beta, TNFalpha, IL-6, diminished TGF-beta (which normally inhibits formation of new osteoclasts

At estrogen deficit the bone resorption is increased, the increse of calcium

results in PTH inhibition, which is followed by lowered availability of 1, 25(OH)D3

Treatment: calcitonin, parathormone, estrogens

CARTILAGEType of dense, connective tissue of chondrocytes, usuallycovers the surface of joints

The main stuctural entities include: aggrecan- glycosaminoglycans (chondroitin sulfate, keratan sulfate, hyaluronan), collagens (core collagen type I and type VI) plus collagen type IX, X, XI matrix proteins The proteoglycan aggrecan has a role in taking up load andresisting deformation. Type II collagen provides tensile properties and type VI collagen has a role in matrix assembly. Cartilage is not vascularized it has very low proliferative activity.

COLLAGEN ASSEMBLY IN CARTILAGE

Collagen fiber formation is influenced by matrix molecules

PROTEOGLYCAN AGGREGATE STRUCTURE AND ORGANIZATION

The chondroitin- and keratan sulfate molecules bind to a single large hyaluronan via G1 and G2 globe protein and link proteins forming large aggregates which resist deformation.