Lecture (1)

Biochemistry of bone tissues

Bone is a living connective tissue, structurally composed of solid matrix containing mixture of organic + inorganic materials. Organic component:1/3 rd of the matrix, including proteoglycans, non-collagen proteins and collagen fibers. The fibers produced by osteoblasts and then subsequently undergo mineralization , they are responsible for tensile flexibility of bones.The non-collagen proteins are mainly responsible for anchoring membranes of bone cells to matrix. Inorganic component of the matrix consists of various calcium salts, primarily hydroxyapatite (hydrated calcium phosphates) Ca10(PO4)6OH2, which are deposited in crystals within and between collagen fibers. Hydroxyapatite salts give bone its rigidity, hardness, and strength under compression conditions. The proper combination of the fibers and salts allows bones to be both strong and durable without being fragile. This mixture provides more strength for the bone than the strength enabled by either component present alone. Moreover, the strength-to-weight ratio of bone is very high compared to other non-living structural materials, such as aluminum and is half as strong as steel. Bone matrix proteins

Protein *Type I collagen *Fibronectin

Vitronectin & Tetranectin *Osteocalcin Bone sialoproteins Osteopontin *Alkaline phosphatase (* = important) Bone specific alkaline phosphatase contributes to the calcification process by

Function Support, rigidity, and integrity of bones Cell signaling (apoptosis, proliferation, differentiation) cell signaling (apoptosis, proliferation, differentiation) mineralization, bone resorption (inducting the release of proteases) Structural Hydroxyapatite-binding protein in bone remodeling Cell-matrix adhestion Bone resorption Bone formation

1-Liberating inorganic phosphate needed by organic phosphate compounds. 2-Breaking down pyrophosphate (ppi) to avoid the inhibition of calcification process Proteoglycans: (mucopolysaccharides) represent a special class of glycoprotein(sugar containing proteins) that are heavily glycosylated. They consist of a core protein with one or more covalently attached glycosaminoglycan chains (GAG chains) .The chains are long, linear, non-fibrpous gel-like heteropolysaccharide polymers. The heteroploysaccharide is made of repeating disaccharide units(x + y )n ,where x & y are non similar modified monosaccharides connected by glycosidic linkages. Chondroitin sulfate& Keratan sulfate are the two types of heteropolysaccharides present in bone matrix. They are negatively charged under physiological conditions, due to the presence of charged sulphate and uronic acid groups . Uronic acid is a modified glucose in which the terminal carbon 6 hydroxyl has been oxidized to carboxylic group.

: Keratan sulfate heteropolysaccharide

Chondroitin sulfate heteropolysaccharide The accumulation of highly negative charge on the surface GAG chains produces large repulsion power between negative charges, rendering these molecules more extended (non-compact) and occupy large surface volume. Also, these negative charges attract opposite cations like Na, K and Ca from the surrounding medium.The

osmotic effect of accumulating cations results in pulling large amounts of water molecules toward GAG structures ,so that highly hydrated and very viscous gel molecules build up to provide an efficient lubricant and shock absorbent insulators. Their high degree of viscosity and low compressibility makes GAG suitable lubricating materials for joints connection in other tissues.

Bone formation: (Osteogenesis) Formation and resorption of bones occur on osteons of bone surfaces . Bone formation is carried out by active osteoblasts that secrete collagen as deposited layers into the extracellular space. This uncalcified bone matrix (consisting mainly of type I collagen) is called osteoid. The next step in osteogenesis is calcification of the matrix. Calcification(minerilization) involves the deposition of hydroxyapatite [Ca10(PO4)6(OH)2] crystills within the matrix, which are surrounded and stabilized by the ground substance (collagens and proteoglycans) . This interaction of hydroxyapatite with collagenous fibers and proteoglycans brings about the hard matrix of bone tissue. Mineralization requires adequate Calcium and phosphate Dependent on Vitamin D Alkaline phosphatase and osteocalcin play roles in bone formation Their plasma levels are indicators of osteoblast activity Bone Modeling and Remodeling The processes of bone formation by osteoblasts and bone resorption by osteoclasts produce opposite results formation produces gain in bone mass and resorption reduces bone mass. These processes can work in two ways. 1.In modeling both processes work with different rates at the same time but on different surfaces; the net effect is an increase in bone size. This process is responsible for shaping the skeleton during growth. 2.In remodeling they act together on the same area but at different times to renew bone; the net effect is no change or net loss of bone size. This process is responsible for removing old bones and forming new ones. Calcium and phosphorus homeostasis. Calcium metabolism : Calcium metabolism or calcium homeostasis is the mechanism by which the body maintains adequate calcium levels in blood. Defects in this mechanism lead to hypercalcemia or hypocalcemia, which both can have important consequences on health. Calcium is the most distributed mineral in human body. The average adult body contains approximately a total of 1 kg of calcium. 99% is present in skeleton bone as calcium phosphate salts. The extracellular fluid (ECF) contains approximately 22.5 mmol, of which about 9 mmol is in the plasma. Approximately 500 mmol of calcium is exchanged between bone and the ECF over a period of twenty-four hours. The plasma level of calcium is closely regulated to keep a normal total calcium level of 2.2-2.6 mmol/L (8.5-10.5 mg/dL) and a normal ionized calcium of 1.1-1.4 mmol/L (4.5-5.6 mg/dL). The amount of total calcium varies with the level of plasma albumin,

a protein to which calcium is bound. The physiologic effect of calcium is determined by the amount of ionized calcium, rather than the total calcium. Ionized calcium does not vary with the albumin level, and therefore it is useful to measure the ionized calcium level when the serum albumin is not within normal ranges, or when a calcium disorder is suspected despite a normal total calcium level.

About 25 mmol of calcium enters the body in a normal diet. 40% of this amount (10 mmol) is absorbed in gut, and 5 mmol released in feces, leaving a net gain of 5 mmol calcium a day. Calcium is mainly regulated by the actions of Vitamin D, parathyroid hormone and to less extent by calcitonin.

Endocrine Control of Calcium and Phosphate Homeostasis

Most physiologic process in the body are affected by calcium. Therefore it is important to maintain blood calcium concentrations within a tight normal range. Deviations above or below the normal range frequently lead to serious disease. Hypocalcemia:is associated with muscle spasms, tetany cardiac dysfunction. and hypoparathyroidism Hypercalcemia: Normal concentrations of calcium and phosphate in blood and extracellular fluid are near the saturation point; therefore any elevations of calcium in extracellular fluids can cause diffuse precipitation of calcium phosphate in soft tissues, leading to widespread organ dysfunction and damage. As with calcium, the majority of body phosphate (approximately 85%) is present in the mineral phase of bone. The remainder of body phosphate is present in a variety of inorganic and organic compounds distributed within both intracellular and extracellular compartments. Normal serum phosphorus levels range from 3.0-4.5 mg/dL. Fluxes of Calcium and Phosphate Preventing hypercalcemia and hypocalcemia is largely the result of balanced endocrine control systems.Maintaining constant concentrations of calcium in blood requires frequent adjustments, which can be described as fluxes of calcium between blood and other body compartments. Three organs participate in supplying calcium to blood and removing it from blood when necessary: The small intestine is the site where dietary calcium is absorbed. Importantly, efficient absorption of calcium in the small intestine is dependent on expression of a calcium-binding protein in epithelial cells. Bone serves as a large reservoir of calcium. Stimulating net resorption of bone mineral releases calcium and phosphate into blood, and suppressing this effect allows calcium to be deposited in bone. The kidney is critically important in calcium homeostasis. Under normal blood calcium concentrations, almost all of the calcium that enters glomerular filtrate is reabsorbed from the tubular system back into blood, which preserves blood calcium levels. If tubular reabsorption of calcium decreases, calcium is lost by excretion into urine.

Hormonal control of blood calcium The regulation of normal blood calcium and phosphorus concentrations is managed through the concerted action of three hormones that control fluxes of calcium in and out of blood and extracellular fluid: Parathyroid hormone serves to increase blood concentrations of calcium by acting on bones,kidney and intestine. Mechanistically, parathyroid hormone preserves blood calcium by several effects: 1.Stimulates production of the biologically-active form of vitamin D within the kidney. 2.Facilitates mobilization of calcium and phosphate from bone. 3.Maximizes tubular reabsorption of calcium by the kidney. This activity results in minimal losses of calcium in urine. 4.It causes increased osteoclast-mediated bone resorption and movement of calcium into the extracellular fluid. Paradoxically, the receptors for PTH are on osteoblasts rather than osteoclasts. It is believed that PTH activation of osteoblasts results in their release of factor(s) that activate osteoclasts. Vitamin D Humans acquire vitamin D from two sources. a. Skin conversion of pro-vit D to active vit D3. b. Uptake through the diet . Cholesterol is converted into provitamin D (dehydrocholesterol) and stored in the skin. When the skin is exposed to sunlight, the provitamin is converted to vitamin D3. Also, he kidney produces the active form of vitamin D3 from the inactive precursor in presence of PTH . Vitamin D is not a classic hormone because it is not produced and secreted by an endocrine gland. Nor is it a true vitamin since it can be synthesized de novo from body cholesterol. However, its action is similar to hormones because it acts on distant target cells and binding to high affinity receptors to produce cellular changes. The main functions of vitamin D are: 1. Facilitates the absorption of dietary calcium from the small intestine 2.Similar to parathyroid hormone, vitamin D enhances the fluxes of calcium out of bone. Calcium from the intestine is usually used to replace the calcium that is withdrawn from the bones. Calcitonin is a hormone that functions to reduce blood calcium levels. It is secreted in response to hypercalcemia and has at least two effects:

1.In kidney: acts on distal tubule to decrease tubular reabsorption of both calcium and phosphate producing calcinuria and phosphatauria.Also inhibits the synthesis of VitD3 thus decreasing calcium absorption from intestine. May be more important in regulating bone remodeling than in Ca2+ homeostasis 2.In bones .It inhibits bone resorption by osteoclasts and therefore reducing mobilization of calcium and phosphate from bones to the circulation Calcitonin is produced by the thyroid gland as an antagonist of PTH. Also, it stimulates the inflex of phosphate in bones. Calcitonin release in the thyroid glands is subjected to negative feedback system, so that more hormone is secreted when the blood calcium level rises. A similar mechanism operating for PTH release from parathyroid glands but functions under hypocalcemia . Osteoporesis ( OP ) It is characterized by progressive atrophy (wasting away) of spongy bones, impaired mineralization, and high risk of bone fracture. The underlying problem in osteoporosis is an imbalance between bone resorption and bone formation, so that bone resorption becomes more dominant over bone formation resulting in a negative balance which may lead to net loss of bones. As estrogen hormone is more diminishing in postmenopausal women ,there is increased risk of net bone loss developing because bone resorption is higher than formation. The most common fragility fracture sites are the proximal femur (hip), spine (generally non-traumatic, or asymptomatic), and the distal forearm. Primary or idiopathic osteoporosis, by far the most common form of the disease, affects people in the second half of their lives. Though mechanisms in females and males are probably similar, the disease starts earlier in women, as estrogens in women decrease earlier than androgens in men. In women, the disease is termed postmenopausal osteoporosis. The main symptoms of osteoporosis are bone fractures. Typically, these affect the femoral neck or the vertebral bodies (impression fractures). In primary osteoporosis, several factors contribute to the development of osteoporosis Decrease in estrogen and testosterone concentrations Reduced physical activity Insufficient vitamin D and calcium intake Reduced UV exposure, resulting in lower endogenous production of vitamin D Reduced renal function secondary to diabetes and arteriosclerosis In the diagnostics of osteoporosis, the most measurable tests are bone density in addition to biochemical markers of bone formation and bone resorption. Bone density is usually determined by X-ray absorptiometry. Patients with osteoporosis usually are presented with low density. Bone formation can be assessed by measuring plasma osteocalcin, which is produced exclusively by osteoblasts. An additional marker of osteoblast activity is bone specific alkaline phosphatase . Bone resorption by osteoclasts in turn involves cleavage of cross-linked collagen products which appear in plasma under disease condition.