Study Guide for Calcium and Phosphate Metabolism

• The most important first messengers for Dental Biochemistry include parathyroid hormone, calcitonin, insulin, and glucagon

• Name common second messengers and how they operate

• Define calcium balance

• Describe the properties and mechanism of action of parathyroid hormone (PTH) including G-protein function

• Review the composition of bone, and know what bone mineral is

• What are the two main functions of parathyroid hormone in kidney?

• Describe the metabolic activation of vitamin D

• What are the actions of vitamin D?

• What is osteomalacia? Rickets? Hyperparathyroidism? Pseudohypoparathyroidism?

• What is osteoporosis?

• Describe physiological ways to establish and maintain maximum bone density

• What foods are good sources of dietary calcium?

Elements of the Body (Table 2-1)

OverviewCalcium (Ca2+) Characteristic Phosphate (HPO4

2-)

99% Body total in bone/teeth 85%

1.3 mM (ionized) Plasma concentration of ionic form

1.3 mM

Carefully controlled Normal variation of ionic form

Within 50%

104-105 [extracellular]/[intra-cellular]

10

Cell signaling and 2nd messenger

Neurotransmitter and hormone release

Exocytosis of proteins

Muscle contraction

Blood clotting

Biomineralization

Regulatory roles

Important conclusion, calcium is an

important regulator

Nil

Regulation of Calcium Metabolism• Minerals; serum concentration

– Calcium (Ca2+); 2.2-2.6 mM (total)– Phosphate (HPO4

2-); 0.7-1.4 mM– Magnesium (Mg2+); 0.8-1.2 mM

• Organ systems that play an import role in Ca2+ metabolism– Skeleton– GI tract– Kidney

• Calcitropic Hormones– Parathyroid hormone (PTH)– Calcitonin (CT)– Vitamin D (1,25 dihydroxycholecalciferol)– Parathyroid hormone related protein (PTHrP)

Sutherland Second Messenger Hypothesis

Understand this key concept

Second Messengers

Fig. 19-4

More Second Messenger

Three Forms of Circulating Ca2+

Calcium Balance• Intake = output• Negative calcium

balance: Output > intake– Neg Ca2+ balance

leads to osteoporosis

• Positive calcium balance: Intake > output– Occurs during

growth

• Calcium is essential, we can’t synthesize it

Calcium and the Cell

• Translocation across the plasma membrane• Translocation across the ER and mitochondrion;

Ca2+ ATPase in ER and plasma membrane

Anatomy and Feedback Inhibition

Parathyroid Hormone Structure• Synthesized in

the 4 para-thyroid glands

• PreProPTH • 115 aa precursor

giving a 90 aa prohormone

• Cleaved at -6/-7 84 residues in the mature peptide

• Regulator of Ca2+ homeostasis

Parathyroid Hormone Biosynthesis

Regulation of PTH Secretion and Biosynthesis

• Extracellular Ca 2+ regulates secretion of PTH– Low Ca 2+ increases– High Ca 2+ decreases

• Ca2+ also regulates transcription

• High levels of 1,25 dihydroxyvitamin D3 inhibit transcription

Calcium Sensing Receptor (CaSR)

• Parathyroid chief cells contain a Ca2+ sensing receptor (CaSR)– 7 transmembrane segments (We will see a lot of 7 TM receptors)– mM affinity for Ca2+

– GPCR of the GPLC and GI varieties• Generates inositol 1,4, 5-trisphosphate which increases

intracellular Ca2+

• There are two paradoxes– The receptor responds to decreasing concentrations of

agonist– Low extracellular Ca2+ increases intracellular Ca2+

– Also found in thyroid C cells (calcitonin), kidney, and brain

Circulating Forms of PTH

• Intact, active PTH of 84 aa• Inactive carboxyterminal fragments lack the 1-34 active

domain• PTH t1/2 (half life) is 2-3 min• Liver (2/3rds) and kidney (1/3rd) are major sites of

fragmentation

Actions of Parathyroid Hormone• Fine tunes Ca2+ levels in blood

– It increases Ca2+

– It decreases Pi

• Parathyroid hormone acts directly on bone to stimulate resorption and release of Ca2+ into the extracellular space (slow)– Gs protein-coupled receptors in osteoblasts increase cAMP and activate PKA– Inhibits osteoblast function– This occurs when PTH is secreted continuously; the opposite occurs when it is

given once daily by injection

• Two effects in kidney– Parathyroid hormone acts directly on kidney to increase calcium reabsorption

and phosphate excretion (rapid)• Gs protein-coupled receptors• Parathyroid hormone acts on distal tubule • Calcitonin inhibits

– Stimulates transcription of 1-alpha hydroxylase for Vitamin D activation in kidney

• Vitamin D increases calcium and phosphate absorption

Parathyroid Hormone Receptor• 7 TM

• GPCR

G-Protein Cycle (Fig. 19-10)

Regulation of Adenylyl Cyclase(Fig. 19-11)

7 and 12 TM segments

Cyclic AMP Metabolism (Fig. 19-12)

Know each step involved in the generation of cAMP by PTH (words, not structures)

Bone• Inorganic (67%)

– Hydroxyapatite 3 Ca10(PO4)6(OH)2

– There is some amorphous calcium phosphate

• Organic (33%) component is called osteoid– Type I collagen (28%)– Non-collagen structural proteins (5%)

• Proteoglycans• Sialoproteins• Gla-containing proteins (gamma carboxyglutamate)• Phosphoproteins• Bone specific proteins: osteocalcin, osteonectin

– Growth factors and cytokines (Trace)

• Bone undergoes continuous turnover or remodeling throughout life– About 20% of bone is undergoing remodeling at any one time

Bone Composition

Calcium and the Skeleton

• A, absorption is stimulated by Vit D; S, secretion• GF, glomerular filtration; TR, tubular reabsorption

of Ca2+ is stimulated by PTH

Osteoblast and Osteoclast Function

• Osteoblasts

• Bone formation

• Synthesis of matrix proteins– Type I collagen

– Osteocalcin

– Others

• Mineralization

• Activation of osteoclasts via RANKL production

• Osteoclasts• Bone resorption

– Degradation of proteins by enzymes

– Acidification

• RANK is activated by RANKL, and this leads to cells differentiation to osteoclasts

Bone Remodeling• Osteoclasts dissolve

bone– Large multinucleated

giant cells

• Osteoblasts produce bone– Have receptors for PTH,

CT, Vitamin D, cytokines, and growth factors

– Main product is collagen

• When osteoblasts become encased in bone, they become osteocytes

PTH and Osteoblastogenesis

Osteoclast Mediated Bone Resorption

Osteoclastogenesis: RANKL, RANK, and OPG

• Osteoblasts activate osteoclasts, formation of a multinuclear cell• The molecular participants in this pathway are the membrane-associated

protein named RANKL (receptor activator of nuclear factor kappa B ligand,) a member of the tumor necrosis factor family of cytokines

• Its cognate receptor is RANK; TRAF, TNF receptor associated factors– Mediates activation of NF-kappa-B by unknown mechanism

• OPG (osteoprotegerin) is a soluble "decoy" receptor for RANKL• RANKL is expressed on the surface of osteoblastic stromal cells • By binding to RANK, its receptor, on osteoclast precursors, RANKL

enhances their recruitment into the osteoclastogenesis pathway in the physiology of bone metabolism

• RANKL also activates mature osteoclasts to resorb bone• RANKL is a factor through which osteoblasts regulate osteoclasts, and

bone formation is coupled to bone resorption

RANK and RANKL

Osteoclastogenesis

PTH and Kidney

PTH acts on the distal tubule

Calcitonin• Product of

parafollicular C cells of the thyroid

• 32 aa

• Inhibits osteoclast mediated bone resorption – This decreases serum

Ca2+

• Promotes renal excretion of Ca2+

Calcitonin

• Probably not essential for human survival

• Potential treatment for hypercalcemia

• 7 transmembrane segment receptor

• Stimulates cAMP production in bone and kidney

Vitamin D Metabolism

Transport and Metabolic Sequence of Activation of Vitamin D

Proposed Mechanism of Action of 1,25-DihydroxyD3 in Intestine

Vitamin D-dependent Ca2+ Absorption

• Duodenum>jejunun>ileum

• Absorption is greater at low pH– The pH of the stomach is about 2– Peak absorption at the beginning of the

duodenum

Vitamin D Deficiency: Rickets• Inadequate intake and absence of sunlight• The most prominent clinical effect of Vitamin D

deficiency is osteomalacia, or the defective mineralization of the bone matrix

• Osteoblasts contain the vitamin D receptor• Vitamin D deficiency in children produces rickets• A deficiency of renal 1α-hydroxylase produces

vitamin D-resistant rickets– Sex linked gene on the X chromosome

– Renal tubular defect of phosphate resorption

– Teeth may be hypoplastic and eruption may be retarded

Rickets

Vitamin D-Resistant Rickets

• Above: Hypoplastic teeth

• Below: Minimal caries can produce pulpitis; periapical abscesses are thus common

• Lack 1-hydroxylase in kidney• Rx: Respond well to 1, 25-

dihydroxyD3

PTHrP; Parathyroid Hormone related Protein

• It is synthesized as 3 isoforms as a result of alternative splicing (139, 141, 173 aa)

• Can activate the PTH receptor• Plays a physiological role in lactation, possibly as a

hormone for the mobilization and/or transfer of calcium to the milk

• May be important in fetal development• May play a role in the development of hypercalcemia of

malignancy– Some lung cancers are associated with hypercalcemia– Other cancers can be associated with hypercalcemia

PTHrP; Parathyroid Related Protein

Causes of HypocalcemiaHypoparathyroid Nonparathyroid PTH Resistance

Postoperative Vitamin D deficiency

Pseudo-hypoparathyroidism

Idiopathic Malabsorption

Post radiation Liver disease

Kidney disease

Vitamin D resistance

Sequence of Adjustments to Hypocalcemia

Pseudohypoparathyroidism• Symptoms and signs

– Hypocalcemia– Hyperphosphatemia– Characteristic physical appearance: short stature, round face, short

thick neck, obesity, shortening of the metacarpals– Autosomal dominant

• Resistance to parathyroid hormone• The patients have normal parathyroid glands, but they fail to respond

to parathyroid hormone or PTH injections• The rise in urinary cAMP after parathyroid hormone fails to occur• The cause of the disease is a 50% deficiency of Gs in all cells• Symptoms begin in children of about 8 years

– Tetany and seizures– Hypoplasia of dentin or enamel and delay or absence of eruption

occurs in 50% of people with the disorder• Rx: vitamin D and calcium

Pseudohypoparathyroidism

Elfin facies, short stature, enamel hypoplasia

Signs and Symptoms of Hypercalcemia

• Neurologic– Lethargy, drowsiness, depression, confusion– Can lead to coma and death

• Neuromuscular– Muscle weakness, hyptonia, decreased reflexes

• Cardiac– Arrhythmias

• Bone– Ache, pain, fracture

Causes of Hypercalcemia

Common Uncommon

Malignant disease, e.g. some lung cancers

Renal failure

Hyperparathyroidism Sarcoidosis

Vitamin D toxicity (excessive intake)

Multiple myeloma

Causes of Hypercalcemia• Primary hyperparathyroidism

– Most people are asymptomatic

– Classically affects skeleton, kidneys, and GI tract

• Triad of complaints: bones, stones, and abdominal groans

– Renal stones are most common single presenting complaint

– Usually due to an adenoma (tumor)

Hyperparathyroidism• The disorder is characterized by hypercalcemia,

hypercalcuria, hypophosphatemia, and hyperphosphaturia

• Parathyroid hormone causes phosphaturia and a decrease in serum phosphate

• Calcium rises and it is also secreted in the urine• Most common complication are renal stones made of

calcium phosphate– Stone chemistries: calcium, phosphate, urate

• Most serious complication is the deposition of calcium in the kidney tubules resulting in impaired renal function

Primary Hyperparathyroidism• Calcium excretion > calcium intake• Large regions of bone are replaced by connective

tissue• Two lesions: maxilla and forehead

Hyperparathyroidism

• Left: Giant Cell Granuloma

• Right: Loss of lamina dura, pathognomonic oral change in hyperparathyroidism

Lamina Dura

• Tooth sockets are bounded by a thin radiopaque layer of dense bone• Lamina dura: “hard layer”

Congenital Hypoparathyroidism

• Hypoplasia of the teeth, shortened roots, and retarded eruption

Hypercalcemia of Malignancy

• Treatment improves quality of life when Ca2+ is elevated but not yet life threatening

• Treat with bisphosphonates– Inhibits osteoclastic activity

• When serum Ca2+ > 3.00 mM treat with NaCl IV

Bisphosphonates

Osteoporosis

• Osteoporosis is characterized by a significant reduction in bone mineral density compared with age- and sex-matched norms

• There is a decrease in both bone mineral and bone matrix

• Osteoporosis is the most common metabolic bone disease

• Affects 20 million Americans and leads to 1.3 million fractures in the US per year

• Women lose 50% of their trabecular bone and 30 % of their cortical bone

• 30% of all postmenapausal women will sustain an osteoporotic fracture as will 1/6th of all men

• The cost of health care and lost productivity is $14 billion in the US annually

Normal and Osteoporotic Bone

Factors that Affect Peak Bone Mass• Gender (M>F), males have greater PBM than females• Race (Blacks >Whites)• Genetics (osteoporosis runs in families and this may be the

predominant factor)– Estrogen receptor gene

– Type I collagen gene

– Vitamin D receptor gene

• Gonadal steroids (estrogen and testosterone increase bone mass)

• Growth hormone (increases bone mass)• Calcium intake (supplements work)• Exercise (increases bone mass)

Sequelae of Osteoporosis

Osteo-porosis

Bone Density as a Function of Age

FDA Approved Rx’s for Osteoporosis• Bisphosphonates (alendronate and risedronate),

calcitonin, estrogens, parathyroid hormone and raloxifene are approved by the US Food and Drug Administration (FDA) for the prevention and/or treatment of osteoporosis

• The bisphosphonates (alendronate and risedronate), calcitonin, estrogens and raloxifene affect the bone remodeling cycle and are classified as anti-resorptive medications

• Teriparatide, a form of parathyroid hormone, is a newly approved osteoporosis medication. It is the first osteoporosis medication to increase the rate of bone formation in the bone remodeling cycle

Treatments (Continued)• Exercise, activity

• Calcium intake should be 1000-1500 mg/day

– Postmenapausal women take in less than 500 mg/day

– Males and females should take in 1000-1500 mg/day

– All adults greater than 65 years should take 1500 mg/day

– Three glasses of milk or three cups of yogurt per day provide 1000-1500 mg/day

• Estrogen treatment

– Estrogen inhibits osteoclastic activity

– Bone density increases 3-5% per year for the first three years after menopause

– This therapy needs to be individualized• Estrogen may increase the incidence of breast cancer, heart attacks, stroke, blood

clots

• That it may exacerbate cardiovascular disease is controversial

– All the data are not in yet, and estrogen treatment is under review; for more information go to http://www.fda.gov/bbs/topics/NEWS/2003/NEW00863.html

Treatments (Continued)• Raloxifene (Brand name Evista) is a selective estrogen receptor

modulator• Decreases in estrogen levels after menopause lead to increases in bone

resorption and bone loss. Bone is initially lost rapidly because the compensatory increase in bone formation is inadequate to offset resorptive losses. This imbalance between resorption and formation is related to loss of estrogen, and may also involve age-related impairment of osteoblasts or their precursors

• Raloxifene reduces resorption of bone and decreases overall bone turnover. These effects on bone are manifested as increases in bone mineral density (BMD)

• Raloxifene’s biological actions, like those of estrogen, are mediated through binding to estrogen receptors. This binding results in the modulation of expression of multiple estrogen-regulated genes in different tissues

Treatments (Continued)• Bisphosphonates inhibit osteroclasts

– Alendronate (Brand name Fosamax)

– Risedronate (Brand name Actonel)

• Calcitonin (Brand name Miacalcin )

– From salmon

– Given intranasaly

– Probably least effective Rx

• Vitamin D

– Most Americans consume less than recommended amount

– 800 IU per day seems safe and not enough to cause vitamin D toxicity

Treatments (Continued)• Parathyroid hormone (Brand name Forteo)

– Teriparatide, a form of parathyroid hormone, is approved for the treatment of osteoporosis in postmenopausal women and men who are at high risk for a fracture

– Chronically elevated PTH leads to bone loss; however, intermittent PTH (once daily bolus injection) leads to new bone synthesis

– Must be injected daily, a major disadvantage– Cost about $7000 per year

• Future Rx’s• Sodium fluoride

– Considered a possibility for years– Adoption seems unlikely

• Strontium ranelate– NEJM 350 (2004) 459-468.

Calcium Content of Foods

• http://www.nal.usda.gov/fnic/foodcomp/Data/SR16/wtrank/wt_rank.html

Selected Web Sites

Properties of Parathyroid Hormone• It is a small protein hormone• It acts on its 7 transmembrane segment receptor

– Gs protein-coupled receptors in osteoblasts increase [cAMP] and activates protein kinase A

– Parathyroid hormone acts directly on bone to stimulate resorption and release of Ca2+ into the extracellular space (slow)

– Parathyroid hormone acts directly on kidney to increase calcium reabsorption and phosphate excretion (rapid)

– Parathyroid hormone acts on distal tubule and stimulates calcium resorption

– Parathyroid hormone stimulates transcription of 1-alpha hydroxylase for vitamin D activation in kidney

• Know how 7 transmembrane segment receptors activate or inhibit adenylyl cyclase via the G-protein cycle

• Know the pathway for the formation of active vitamin D

Transport and Metabolic Sequence of Activation of Vitamin D

PTH Biosynthesis

• PTH is co-secreted with chromogranin A, a protein; significance unknown

Sequence of Adjustments to Hypocalcemia