Bone and Mineral Metabolism

1. Bone and Mineral Metabolism Lecture 8

2. Introduction • The skeletal system is one of the largest organs in the body. • It is the storehouse for 98% to 99% of the body’s 1 kg of calcium. • Bones are mineralized connective tissue in which type I collagen forms a network of flexible fibers. • Mineralization of this network, or matrix, with calcium salts is required to produce the rigid skeleton. • Bone is a living tissue that is constantly being remodeled by degradation of old tissue and replacement with new bone matrix.

3. Introduction • Osteoclasts and osteoblasts are the bone cells mainly responsible for remodeling. • Osteocytes are important regulators of bone cell activity that can be estimated by laboratory methods. • Calcium is required for mineralization of bone and is a key regulator of many body processes. • Calcium ions play critical roles in: • intracellular signaling, • in regulation of events at the plasma membrane, • and in the function of extracellular proteins such as those involved in blood coagulation. • The circulating concentration of calcium ions is kept constant under the control of parathyroid hormone (PTH) and metabolites of vitamin D.

4. Introduction • Deviations of the concentration of free (unbound) calcium outside its very narrow reference interval can cause morbidity and mortality. • The importance of the tight regulation of free calcium is underscored by the recognition that skeletal health is allowed to suffer markedly to allow physiologic processes in other organs to be maintained. • Phosphate is also important in bone mineralization and is a component of high-energy molecules. • Fibroblast growth factor 23 (FGF23) is involved in regulation of phosphate in combination with PTH and vitamin D metabolites. • Bone is increasingly recognized as having endocrine functions playing an important role in regulating metabolic processes.

5. OVERVIEW OF SKELETAL METABOLISM • Bone is composed primarily of an extracellular mineralized matrix with a smaller cellular fraction. • Bone is a dynamic tissue that is under continuous turnover or remodeling, which enables bone to repair damage and adjust strength. • Osteoclasts and osteoblasts are two main types of bone cells located on bone surfaces. • Osteoclasts resorb bone, osteoblasts lay down new bone at a site of previous bone resorption. • Osteocytes, the most abundant cells in mature bone, are located in lacunae within the bone matrix. • Osteocytes nourish the skeleton and regulate bone cell activity

6. OVERVIEW OF SKELETAL METABOLISM

7. OVERVIEW OF SKELETAL METABOLISM • Circulating mononuclear osteoclast precursors are recruited, proliferate, and fuse to form giant multinucleate osteoclasts that resorb bone by producing hydrogen ions to mobilize minerals and lysosomal enzymes to digest the organic matrix. • Deep folds of the plasma membrane (ruffled border) are in contact with the bone surface, forming the osteoclastic bone-resorbing compartment. • The resorption lacuna contains degradative enzymes such as collagenases, cathepsin K, and several matrix metalloproteinases (MMPs). • After resorption ceases, a cement line is deposited in the resorption cavity.

8. OVERVIEW OF SKELETAL METABOLISM • Acidification of the vacuole leads to activation of tartrate-resistant acid phosphatase (TRACP) and cathepsin K (Cat K).

9. OVERVIEW OF SKELETAL METABOLISM • Stromal lining cells differentiate to osteoblasts. • Osteoblasts form bone by synthesizing the organic matrix, including type I collagen, and participating in the mineralization of newly synthesized matrix. • The development of the osteoblast phenotype has been divided into three consecutive phases, each with its typical gene-expression patterns. • Osteoblasts/osteoprogenitor cells that are trapped within the bone matrix can develop into osteocytes with mechanosensory properties that can communicate with other bone cells via a network of dendritic processes within canaliculi.

10. OVERVIEW OF SKELETAL METABOLISM

11. OVERVIEW OF SKELETAL METABOLISM • The organic matrix of bone is primarily type I collagen (90%) combined with lesser amounts of a large number of non-collagenous proteins, some of which are found only in bone. • Type I collagen is a product of two genes—COL1A1 on chromosome 17 and COL1A2 on chromosome 7—that encode two chains of the collagen molecule, called α1(I) and α2(I) chains. • The organic matrix is mineralized by the deposition of inorganic calcium and phosphate in small crystals with lesser amounts of carbonate, magnesium, sodium, potassium, and various other ions.

12. OVERVIEW OF SKELETAL METABOLISMCell Signaling in Bone • Two key signaling pathways are known as RANK/RANKL/OPG and Wnt.

13. BIOCHEMICAL MEASUREMENTSIN METABOLIC BONE DISEASE • There are several analytes that can be measured in biologic fluids that give important information on bone and calcium metabolism enabling diagnosis of abnormalities and disease.

14. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium • Distribution of Calcium in the Body • In blood, virtually all of the calcium is found in the plasma, which has a mean calcium concentration of 9.5 mg/dL (2.38 mmol/L). • Calcium exists in three physicochemical states in plasma: • 50% is free (ionized), • 40% is bound to plasma proteins, and • 10% is complexed with small diffusible inorganic and organic anions, including bicarbonate, lactate, phosphate, and citrate

15. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium • The free calcium fraction is the biologically active form. • Its concentration in plasma is tightly regulated by the calcium regulating molecules PTH and 1,25(OH)2D. • Synthesis and secretion of PTH by the parathyroid glands is controlled via a calcium-sensing receptor (CaSR), which is a transmembrane receptor on the surface of parathyroid gland cells. • A decrease in circulating free calcium is detected at the CaSR, resulting in the chief cells releasing PTH to increase free calcium acting via: • the kidneys (calcium reabsorption), • the gut (calcium absorption), and • skeleton (bone resorption releasing calcium)

17. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium • About 80% of protein-bound calcium is associated with albumin, with the remaining 20% associated with globulins. • Because calcium binds to negatively charged sites on proteins, its binding is pH dependent. • Alkalosis leads to an increase in the negative charge of proteins increasing binding, resulting in a decrease in free calcium; • conversely, acidosis leads to a decrease in negative charge, decreasing binding and resulting in an increase in free calcium. • In vitro, for each 0.1-unit change in pH, approximately 0.2 mg/dL (0.05 mmol/L) of inverse change occurs in the serum free calcium concentration.

18. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium

19. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium: Hypocalcemia • Low total plasma calcium may be due to a reduction in albumin-bound calcium, the free fraction of calcium, or both. • Hypoalbuminemia is the most common cause of apparent hypocalcemia on a standard biochemical profile, particularly in hospitalized patients, because 1 g/dL (1 g/L) of albumin binds approximately 0.8 mg/dL (0.02 mmol/L) of calcium. • Common clinical conditions associated with low plasma albumin include: • chronic liver disease, nephrotic syndrome, congestive heart failure, malignancy, malnutrition, and postsurgical volume replacement with saline or colloidal solutions. • In these conditions, the concentration of free calcium typically is maintained within its physiologic reference interval.

20. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium: Hypocalcemia • The most common causes of hypocalcemia are: • Chronic renal failure • In CKD, hypoproteinemia, hyperphosphatemia, low plasma 1,25(OH)2D (caused by reduced renal synthesis), and skeletal resistance to PTH can all contribute to hypocalcemia. • Hypoparathyroidism • Hypomagnesemia • Magnesium deficiency can also lead to hypocalcemia through several mechanisms, including: • impairment of PTH secretion by interfering with the fusion of intracellular vesicles containing PTH with the chief cell membrane, and decreased responsiveness of target organs to PTH action (end-organ resistance)

21. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium: Hypocalcemia • Less common causes of hypocalcemia include: • Pseudohypoparathyroidism and activating mutations of the CaSR. • In pseudohypoparathyroidism, patients have an inherited resistance to PTH and, as a result, increased circulating concentrations of PTH. • The molecular basis for the most common form, pseudohypoparathyroidism type 1 (Albright’s hereditary osteodystrophy [AHO]), is an inactivating mutation in the gene coding for the stimulatory guanine nucleotide-binding protein in the adenylate cyclase complex, resulting in an inability to produce the second-messenger cyclic adenosine monophosphate (cAMP).

22. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium: Hypocalcemia • Hypoparathyroidism • is caused most commonly by parathyroid gland destruction or removal during neck surgery (90%), less commonly by autoimmune endocrine disorders, and rarely by genetic causes.

23. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium: Hypercalcemia • Hypercalcemia often results when the influx of calcium into the extracellular fluid compartment from the skeleton, intestine, or kidney is greater than the efflux as, for example: • When excessive resorption of bone mineral occurs in malignancy. Hypercalciuria often develops in such situations. • When the capacity of the kidney to excrete filtered calcium is exceeded, hypercalcemia develops; where renal failure is present and calcium excretion is decreased, hypocalciuria may paradoxically be present.

24. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium: Hypercalcemia • Hypercalcemia can be caused by increased intestinal absorption (eg, vitamin D intoxication [rare]), increased renal retention (eg, thiazide diuretics), increased skeletal resorption (eg, immobilization), or a combination of mechanisms (eg, PHPT).

25. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium: Hypercalcemia • PHPT is the most common pathologic cause in outpatients, whereas malignancy is more common in hospitalized patients. • Vitamin D (cholecalciferol or ergocalciferol) or vitamin D analog therapy has become one of the most common causes of hypercalcemia detected in laboratory practice. • Together, these causes account for 90% to 95% of all cases of hypercalcemia. • PHPT is often characterized by increased secretion of PTH that results in hypercalcemia. • It is most often due to a solitary adenoma (80% to 85% of cases), less frequently to hyperplasia involving all glands (~15%), and infrequently to parathyroid carcinoma (<1%).

26. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium: Hypercalcemia

27. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASECalcium: Hypercalcemia

28. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASE Measurement of Calcium • Total Calcium Adjusted for Albumin (Adjusted or Corrected Calcium) • Wide variation in the concentrations of compounds that bind calcium in blood may be noted; this variation will affect the measured total calcium concentration without changing the free calcium fraction. • Several types of calculation have been suggested to “adjust” the measured calcium concentration. • In practice, only adjustments based on albumin are commonly used.

29. BIOCHEMICAL MEASUREMENTS IN METABOLIC BONE DISEASE Measurement of Calcium • Adjusted calcium is calculated from total calcium and albumin by first calculating a correction factor by multiplying the deviation of plasma albumin from the mean of its reference interval by the slope of the regression of total calcium against albumin. • The following two equations are frequently used for results expressed as mg/dL and mmol/L, respectively:

30. PHOSPHATE • An adult has about 600 g or approximately 20 mol of phosphorus in inorganic and organic phosphates, Distribution of Phosphate in the Body

31. PHOSPHATE: Regulation of phosphate • Regulation of phosphate is a complex process involving the kidneys, intestine, and skeleton. • Plasma phosphate in adults is maintained in the range of 0.7 to 1.4 mmol/L. • Throughout 24 hours, plasma phosphate concentration demonstrates a diurnal variation with a significant increase after the evening meal, a peak in the early hours of the morning, and a decrease to nadir in the early morning.

32. PHOSPHATE: Regulation of phosphate • 1,25(OH)2D can promote phosphate and calcium absorption via the intestine and can increase phosphate mobilization from the bone by stimulating osteoclastic resorption of bone mineral containing hydroxyapatite [(Ca10(PO4)6 (OH)2] as the storage form of phosphate. • PTH can have effects on phosphate via the kidneys. • Type IIa sodium-phosphate cotransporter (NPT2a) Na+/Pi activities are inhibited by PTH. PTH leads to the disappearance of both cotransporters from the apical membrane, and that both cotransporters are rapidly degraded in response to PTH. • PTH action at the tubules promotes phosphaturia and will lower circulating phosphate indirectly.

33. PHOSPHATE: Regulation of phosphate • FGF23 is a mid- to long-term modulator of phosphate homeostasis. • FGF23 increases fractional excretion of phosphate by the kidneys. • It also decreases production of the active form of vitamin D, 1,25(OH)2D, by decreasing the activity of the enzyme responsible for its formation (25-hydroxyvitamin D 1-alpha-hydroxylase). • Both of these key activities lead to decreased plasma phosphate concentrations. • FGF23 is secreted by bone cells (osteocytes, osteoblasts, and osteoclasts) in response to sustained increased plasma phosphate or increased plasma 1,25(OH)2D. • In CKD, FGF23 increases in a compensatory mechanism to counter increasing plasma phosphate.

34. PHOSPHATE: Regulation of phosphate

35. PHOSPHATE: Regulation of phosphate The central role of FGF23 in the regulation of phosphate balance. • Fibroblast growth factor (FGF23) secretion from bone is stimulated by phosphate loading as a consequence of higher dietary phosphate intake or reduced phosphate excretion in the glomerulus (due to low estimated glomerular filtration rate (eGFR)). FGF23 induces internalization of phosphate transporters, such as NaPi2a, in the kidney , which reduces tubular phosphate reabsorption. The consequent increase in renal phosphate clearance (phosphaturia) restores phosphate balance. Both parathyroid hormone and active vitamin D promote FGF23 secretion and, in a negative feedback loop, are suppressed by FGF23. In addition, vitamin D can also increase phosphate absorption in the gut by inducing the expression of NaPi2b, thus contributing to phosphate loading and FGF23 secretion. Vitamin D and parathyroid hormone (PTH) also influence the concentrations of calcium and phosphate, and these minerals also have direct effects on these hormonal axes. Both hyperphosphataemia and hypocalcaemia stimulate PTH release. Hypocalcaemia also stimulates direct activation of vitamin D, whereas this activation is inhibited by hyperphosphataemia.

36. PHOSPHATE: Hypophosphatemia • Hypophosphatemia, defined as the concentration of inorganic phosphate in the serum below the reference interval (usually <2.5 mg/dL [<0.81 mmol/L]), is relatively common in hospitalized patients (≈2%). • Hypophosphatemia is not necessarily associated with intracellular phosphate depletion. • Hypophosphatemia may be present when cellular concentrations are normal, and cellular phosphate depletion may exist when plasma concentrations are normal or even high. • Hypophosphatemia or phosphate depletion in blood may be caused by: • A shift of phosphate from extracellular to intracellular spaces; • Renal phosphate wasting; • Decreased intestinal absorption; and • Loss of intracellular phosphate.

38. PHOSPHATE: Hyperphosphatemia • The most common cause of hyperphosphatemia is inability of the kidneys to excrete phosphate. • Hyperphosphatemia is a major clinical problem in CKD. • In acute kidney injury and CKD, a decrease in glomerular filtration rate reduces the renal excretion of phosphate, resulting in hyperphosphatemia. • Moderate increases in plasma phosphate occur in individuals with low PTH (hypoparathyroidism), PTH resistance (pseudohypoparathyroidism), or acromegaly (increased growth hormone) caused by an increased renal phosphate threshold. • Growth hormone contributes to the increased renal phosphate threshold and the higher phosphate concentrations observed in children. • EDTA therapy has also been associated with hyperphosphatemia.

39. MAGNESIUM • Magnesium is the fourth most abundant cation in the body and the second most prevalent intracellular cation. • The total body magnesium content is about 25 g (~1 mol).

40. MAGNESIUM: Regulation of magnesium • Vitamin D status has minimal effect on magnesium absorption. • Passive absorption occurs paracellularly through a favorable electrochemical gradient and solvent-driven cellular uptake. • Active absorption is via two magnesium transporters in the large intestine: transient receptor potential melastatin 6 (TRPM6) and TRPM7. • It has been suggested that TRPM6 plays an important role in epithelial magnesium uptake. • This receptor is expressed along the whole length of the large intestine, whereas TRPM7 is more ubiquitous in its expression and is thought to be involved in cellular magnesium homeostasis.

41. MAGNESIUM: Regulation of magnesium • The kidneys play a major role in regulating magnesium balance.

42. MAGNESIUM: Hypomagnesemia/ Magnesium Deficiency • Hypomagnesemia is common in hospitalized patients. • Ten percent of patients admitted to city hospitals and as many as 65% of patients in intensive care units may be hypomagnesemic. • Moderate or severe magnesium deficiency is usually due to loss of magnesium from the gastrointestinal tract or kidneys.

43. MAGNESIUM: Hypomagnesemia/ Magnesium Deficiency

44. MAGNESIUM: Hypermagnesemia • Magnesium intoxication is not a frequently encountered clinical problem, although a mild to moderate increase in the serum magnesium concentration may be noted in as many as 12% of hospitalized patients. • Symptomatic hypermagnesemia is almost always caused by excessive intake, resulting from administration of antacids, enemas, and parenteral fluids containing magnesium. • Many of these patients have concomitant renal failure, thereby limiting the ability of the kidneys to excrete excess magnesium. • Magnesium used to treat preeclampsia and eclampsia may cause magnesium intoxication in mothers and their neonates.

45. MAGNESIUM: Hypermagnesemia

46. HORMONES REGULATING BONEAND MINERAL METABOLISM • PTH and 1,25(OH)2D are the primary hormones regulating bone and mineral metabolism. • FGF23 is recognized as an important molecule controlling phosphate homeostasis. • Calcitonin has pharmacologic actions, but a physiologic role has not been established in adults. • Parathyroid hormone–related peptide (PTHrP) is the principal mediator of HHM, but it also has physiologic functions in fetuses and in women during pregnancy and lactation. • Sclerostin inhibits the Wnt signaling pathway that plays a major role in osteoblast development and function and so results in decreased bone formation and turnover.

47. HORMONES REGULATING BONE AND MINERAL METABOLISMParathyroid Hormone • PTH is synthesized and secreted by the parathyroid glands. • The chief cells are responsible for synthesizing, storing, and secreting PTH. • The concentration of PTH in blood is determined by the rates of its synthesis and secretion by the parathyroid glands and of its metabolism and clearance by the liver and kidneys. • The primary regulators of PTH secretion are free calcium, 1,25(OH)2D, and phosphate. • PTH acts directly on bone and the kidneys, and indirectly on intestine via 1,25(OH)2D, to increase the plasma concentration of free calcium and ultimately decrease the plasma concentration of phosphate.

48. HORMONES REGULATING BONE AND MINERAL METABOLISMParathyroid Hormone • The concentration of free calcium in blood or extracellular fluid is the primary acute physiologic regulator of PTH synthesis, metabolism, and secretion. • Free calcium is sensed by a G-protein–coupled CaSRin the plasma membrane of parathyroid cells; intracellular signal transduction pathways following activation of the receptor involve release of free calcium from intracellular stores and opening of plasma membrane calcium channels. • 1,25(OH)2D, phosphate, and magnesium also influence the synthesis and secretion of PTH. • 1,25(OH)2D interacts with vitamin D receptors in the parathyroid glands to chronically decrease PTH secretion by suppressing PTH gene transcription and subsequent secretion.

49. HORMONES REGULATING BONE AND MINERAL METABOLISMParathyroid Hormone • Hyperphosphatemia and hypophosphatemia increase and decrease PTH synthesis and secretion, respectively, and the hyperphosphatemia of CKD leads to parathyroid hyperplasia and hyperparathyroidism. • Magnesium probably does not play an important role in PTH secretion except at the extremes of magnesium concentration. • Chronic severe hypomagnesemia, such as that occurring in alcoholism, has been associated with impaired PTH secretion, whereas acute hypomagnesemia may stimulate secretion. • Chronic hypomagnesemia can cause resistance to the effects of PTH. • Hypermagnesemia suppresses PTH secretion via the CaSR, although not as effectively as does calcium.

50. HORMONES REGULATING BONE AND MINERAL METABOLISMParathyroid Hormone: Biologic Actions • PTH influences calcium and phosphate homeostasis directly through its actions on both bone and kidneys and indirectly through its actions on the intestine through 1,25(OH)2D. • The hormone exerts its actions by interacting with type 1 PTH receptors (PTH/PTHrP receptors) located in the plasma membranes of target cells. • In the kidneys, PTH has several functions. • It induces 25-hydroxyvitamin D-1α-hydroxylase, increasing the production of 1,25(OH)2D, which, in turn, stimulates intestinal absorption of both calcium and phosphate; • it increases calcium reabsorption in the distal convoluted tubules; it decreases reabsorption of phosphate by the proximal tubules;