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Nussey S, Whitehead S. Endocrinology: An Integrated Approach. Oxford: BIOS Scientific Publishers; 2001.

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Endocrinology: An Integrated Approach.

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Chapter 5The parathyroid glands and vitamin D

Chapter objectives

Knowledge of

1.

Physiology of the regulation of serum calcium and phosphate concentrations

2.

Causes and clinical features of hypo- and hypercalcemia

3.

Investigation and treatment of hypo- and hypercalcemia

4.

Causes and clinical features of vitamin D deficiency

5.

Investigation and treatment of vitamin D deficiency

6.

Physiology of bone formation and its remodeling

7.

Causes of osteoporosis, their investigation and treatment

8.

Paget's disease and its treatment

“Children, you are very little,

And your bones are very brittle;

If you would grow great and stately,

You must try to walk sedately.”

A Child's Garden of Verses XXVII Good and Bad Children, Robert Louis Stevenson.

The calcium ion (Ca2+) plays a fundamental role in a number of physiological functions including bone formation, muscle contraction, secretion, enzyme co-factor, stabilization of membrane potentials, blood coagulation and stimulus-response coupling (Box 5.1). Many of these effects are brought about by the binding of Ca2+ to proteins altering their structures and, thus, functions.

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Box 5.1

Functions of calcium and phosphate. Bone growth and remodeling Secretion (exocytosis)

Calcium is common, forming approximately 3.5% of the earth's crust and biosphere. In the adult human body there is about 1–2 kg calcium, 99% of which resides in teeth and bone as hydroxy-apatite crystals. Of the remainder, approximately 1% is intracellular and a tiny fraction, less than 0.1%, is extracellular (Box 5.2). It is this small extracellular fraction of Ca2+ that is homeostatically regulated by hormones and that determines calcium balance within the body.

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Box 5.2

Distribution of body calcium.

Phosphate forms about 0.09% of the earth's crust and biosphere. In the body at physiological pH, inorganic phosphate exists as HPO42– and H2PO4 (collectively referred to as Pi). Like calcium, it is critically important in physiological processes. Phosphate is an essential component of: high-energy compounds (such as ATP and creatinine phosphate), second messengers (such as cAMP and inositol phosphates) and fundamental molecules including DNA, RNA and phospholipids. It is also an important intracellular anion to balance the charge of the cations K+ and Mg+ and enzymatic phosphorylation of proteins alters their structure and function (Box 5.1). Bone contains 85% of the Pi in the body with just under 5% in the intracellular compartment and less than 0.03% in the serum. Since the extracellular concentration of Pi is inversely related to that of Ca2+, and is also regulated by the same hormones, calcium and phosphate homeostasis are considered together.

Multiple symptoms arise when disease leads to hyper- or hypocalcemia and Clinical Case 5.1 illustrates features of hypercalcemia.

Clinical Case 5.1

A 45-year-old woman presented to her primary care physician with general malaise, aches and pains, anorexia and weight loss of more than 3 months duration. Her only other symptoms had been polyuria and polydipsia, which, together with the weight loss, had suggested the diagnosis of DM. Her physician had, therefore, arranged for some biochemical tests to be performed. The fasting serum glucose concentration was normal (4.1 mmol/l, NR <3–6.0 mmol/l) but he was concerned when the laboratory staff phoned the surgery to report that her total serum Ca2+ was high at 4.1 mmol/l (NR 2.2–2.6 mmol/l) with an albumin of 38 g/l (NR 38–40 g/l). Her serum urea and electrolyte concentrations were normal as were her routine hematological investigations.

Clinical Case 5.1 is a middle-aged woman suffering from the consequences of marked hypercalcemia, presenting with symptoms of polydipsia and polyuria. These, together with the weight loss, made her primary care physician suspect DM. However, this patient had anorexia whilst DM is much more commonly associated with a good appetite, despite weight loss. The polyuria resulted from the effects of hypercalcemia antagonizing the action of arginine vasopressin on the distal tubule and collecting ducts of the kidney (see Box 7.42). The resulting diuresis stimulated thirst and led to polydipsia. Her anorexia was due to the effects of hypercalcemia acting on the brain to reduce appetite.

To understand the pathophysiological mechanisms by which Clinical Case 5.1 developed hypercalcemia it is necessary to discuss the regulation of whole body calcium balance and factors controlling serum Ca2+ concentration.

Calcium and phosphate in serum and its measurement

Serum calcium ranges between 2.2–2.6 mmol/l although only about 1 mmol/l exists as free ionized Ca2+ (Box 5.2). Serum proteins bind about 45% (mainly albumin and a smaller proportion to globulin) and approximately 10% is associated with inorganic anions such as lactate, phosphate and bicarbonate. The bound forms of calcium are in equilibrium with ionized Ca2+ and so factors that affect calcium binding in the serum such as serum protein concentration, pH or phosphate affect serum Ca2+ concentration. For example, a reduction in serum proteins reduces Ca2+, as does alkalosis, whilst acidosis increases serum Ca2+ concentrations. The total phosphate concentration in serum is normally between 0.8 and 1.45 mmol/l. Approximately 50% exists in free form (mainly HPO42–), the remainder being bound to serum proteins (<10%), combined with Na+ (30%), Ca2+ or Mg2+ (approximately 6%).

Serum calcium concentration is usually measured, as it was in Clinical Case 5.1, by a method developed some 30 years ago. This involves the addition of a chemical that displaces calcium from protein-binding sites. The total calcium concentration is then measured colorimetrically by its effect on an added dye (such as cresolphthalein). The ionized calcium (i.e. Ca2+) concentration is not measured by all laboratories and no cheap automated assay exists. Various ‘factors’ have been used to ‘correct’ the serum concentrations of Ca2+ when there are changes in serum albumin or, indeed, blood pH. Like calcium, the serum concentration of Pi is also measured photometrically by the complex of phosphate ions with ammonium molybdate (and usually expressed in terms of phosphorus content).

Intracellular calcium concentration

The intracellular cytosolic concentration of Ca2+ is generally quoted as approximately 0.1 μmol/l, some four orders of magnitude lower than the extracellular concentration. This does not represent the total concentration because much of the intracellular calcium is sequestered in intracellular organelles such as the endoplasmic reticulum, sarcoplasmic reticulum (in skeletal muscle) and mitochondria. In response to a stimulus, the release of Ca2+ from these intracellular stores, or its entry into the cells through Ca2+ channels in the membrane, rapidly increases the intracellular concentration of Ca2+ some 10–100 fold. Such a transient increase in intracellular Ca2+ concentration is a ubiquitous signal transduction mechanism producing, for example, muscle contraction, secretion or enzyme activation. The latter includes the activation of a calcium-binding protein, calmodulin, that subsequently activates (by phosphorylation) protein kinase C, analogous to cAMP and diacylglycerol activating other specific protein kinases (Box 5.3). Protein kinase C may then activate cytoplasmic enzymes or affect gene transcription by phosphorylating further kinases such as those in the MEK—MAPK signalling pathway (see Box 1.10). Thus, intracellular Pi is also important in signal transduction processes. As with Ca2+, its actions are effected by its binding to intracellular proteins.

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Box 5.3

The role of calcium as a second messenger. Intracellular concentrations of Ca2+ increase as a result of either the opening calcium channels in the cell membrane or the release from intracellular stores such as that sequestered in the endoplasmic reticulum. (more...)

Calcium and phosphate balance

The daily turnover of calcium and phosphate for an adult is shown in Box 5.4. The net balance depends not only on the absorption of these minerals from the gut, their retention or excretion by the kidneys and loss in the feces, but also on bone turnover. The recommended intake of calcium is approximately 1000 mg/day although physiological states such as lactation and pregnancy increase requirements; these are also higher during childhood growth. The tendency to reduce the consumption of dairy products as a result of concern about serum cholesterol concentration or strict vegetarianism has led to a reduction in the daily intake of the vital element in some sections of the population.

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Box 5.4

Daily turnover of calcium and phosphate in the human with a dietary intake of 1000 mg/day. Numbers are milligrams/day with those for calcium in squares and for phosphate in ovals.

The daily intake of Pi is normally between 800–1500 mg, comfortably exceeding homeostatic minimum requirements. Homeostasis is primarily maintained by the kidneys which deal with the filtered Pi by regulated reabsorption of approximately 80% of the total load. Any increase in the filtered load leads to Pi excretion. Transport of Pi across the luminal tubule membrane is mediated by Na+-PO43– co-transporters, of which there are three families; two are expressed almost exclusively in the kidney. Transfer of Pi across the basolateral tubule cell membrane is passive but regulated by an anion exchange mechanism. The maximal rate for Pi reabsorption is variable; it decreases with a high-phosphate diet and increases with a low-phosphate diet. This variation in the rate of reabsorption is independent of parathyroid hormone (PTH, see below). Patients with chronic renal failure cannot excrete Pi and, since gut absorption continues, Pi accumulates in the body. The excess Pi consequently complexes with Ca2+ thus lowering ionized calcium concentrations. PTH secretion is, therefore, stimulated.

Hormonal control of serum Ca2+ and Pi concentrations

Vitamin D, synthesized in the skin or obtained from the diet, and PTH, secreted by the parathyroid glands, increase serum Ca2+ concentrations via actions on the gut, kidney and bone (Box 5.5). Calcitonin (secreted by the parafollicular cells of the thyroid gland) reduces serum Ca2+ concentrations in experimental animal models. In the human, however, a marked reduction in the circulating concentration of this hormone (after, for example, total thyroidectomy) has no demonstrable effect on serum Ca2+. Indeed, when its marked excess occurs in a clinical situation (see Clinical Case 5.8) it is notable for its lack of effect on calcium homeostasis.

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Box 5.5

Overview of the hormonal control of calcium and phosphate homeostasis by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25-(OH)2D). PTH stimulates the release of Ca2+ from bone by increasing osteolysis and resorption, increases Ca2+ reabsorption (more...)

Sources, metabolism and transport of vitamin D

The dietary source of vitamin D (D3 cholecalciferol and D2 ergocalciferol) was the first to be recognized and, as a result, it was classified as a vitamin. However, vitamin D3 is undoubtedly a secosteroid prohormone (see website) for steroid hormone terminology) because even though it is not secreted by a classical endocrine gland, the active form of the hormone is released from the kidney and acts at distant sites, bone and kidney. The major source of vitamin D is synthesis from 7-dehydrocholesterol in the keratinocytes of the skin. Synthesis is stimulated by sunlight (Box 5.6) although the effect of near ultraviolet wavelengths (230–313 nm) may be reduced by melanin skin pigment.

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Box 5.6

Vitamin D synthesis. Vitamin D3 synthesis in the skin is stimulated by sunlight and subsequent hydroxylations in the liver (at C-25) and kidney (at C-1) produce the active form of the hormones. In the circulation vitamin D3 is bound to transcalciferin (more...)

The prohormones vitamin D2 and vitamin D3 have virtually no biological activity. However, they are hydroxylated in the liver at the carbon-25 (C-25) position and subsequently at C-1 in the kidney (and also in the placenta when this is present) to generate the active hormones 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 (Box 5.6). Alternatively, these 25-hydroxylated products may be C-24 or C-26 hydroxylated in the kidney to give 24,25-dihydroxyvitamin D2 or 24,25-dihydroxyvitamin D3 or the 25,26-dihydroxy equivalents. Whether these forms of vitamin D have any biological activity remains controversial, particularly in light of recent data from mice in which the 24-hydroxylase gene has undergone targeted disruption (‘knock out’). Some argue that they are simply inactive alternatives to the 1,25-dihydroxyvitamin D hormone. Thus, this pathway may be compared with the hepatic alternatives of 5´-outer ring or 5-inner ring deiodination of thyroxine to give active tri-iodothyronine (T3) or inactive reverse T3 (see Box 3.29).

Four important points should be emphasized regarding the synthesis of active metabolites of vitamin D. The first is that the hydroxylations progressively increase the polarity of the hormone so that it becomes more water soluble and less lipid soluble. The second is that C-25 hydroxylation in the liver is determined almost entirely by the concentrations of the precursors. A clinical consequence of this is that measurements of serum 25-hydroxyvitamin D are good indicators of body vitamin D status. The third is that the activity of the C-1 hydroxylase in the kidney is regulated by changes in the serum concentrations of PTH, Pi and Ca2+. Thus, a reduction in serum Ca2+ concentration and the consequent increase in PTH secretion both independently stimulate C-1 hydroxylase activity, as does a decrease in serum Pi concentration. Note also that 1,25-dihydroxy-cholecalciferol regulates its own synthesis by decreasing the transcription of the C-1 hydroxylase enzyme. The fourth is that C-1 hydroxylation of 25-hydroxycholecalciferol occurs in other cell types (monocytes/macrophages and lymphocytes) but in these it is not regulated by the same factors.

Like all steroid and thyroid hormones, 1,25-dihydroxyvitamin D circulates bound to a globulin that is synthesized in the liver (in this case transcalciferin). A small proportion of vitamin D remains in a free form in the circulation and has a serum t1/2 of about 5 h. Transcalciferin preferentially binds 25-hydroxylated molecules and so non-hydroxylated molecules are stored in adipose tissue. This has some clinical utility (see Box 5.29). Vitamin D is rapidly cleared by the liver and biliary metabolites of 1,25-dihydroxyvitamin D are more polar than the native hormone. These glucuronides and sulfates undergo an entero-hepatic circulation being absorbed from the gut and resupplied to the liver.

Classical actions of vitamin D on intestine and bone

Vitamin D acts via receptors that dimerize with other receptors, notably the retinoic acid receptor. In this form, the ligand-bound dimerized receptor attaches to a specific region of DNA. With the help of other transcription co-activators or co-repressors, gene expression is either stimulated or inhibited (Box 5.7). As with other steroid and thyroid hormones, there is also evidence that vitamin D exerts actions via a non-genomic pathway via membrane receptors.

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Box 5.7

Actions of vitamin D on target tissues. In circulation vitamin D is bound to the globulin, transcalciferin (TC), with only a small fraction existing in free form. The free form crosses the cell membrane and the nuclear membrane where, in target cells, (more...)

The most important action of 1,25-dihydroxy-vitamin D is to increase the active absorption of Ca2+ from the intestinal lumen of the gut (Box 5.8). Calcium is absorbed from the gut by several processes. The best studied involves: the active uptake of Ca2+ from the luminal brush border of the enterocytes; the binding of Ca2+ to a calcium-binding protein (CaBP); translocation of the complex across to the basolateral surface of the cell; active extrusion of Ca2+ by an ATP-dependent calcium pump that pushes Ca2+ out of the cell in exchange for Na+. This pump is maintained by a Na+/K+ exchanger (pumping Na+ back out of the cell) retaining a favorable sodium gradient. In the gut lumen, ionization of calcium occurs at low pH. Exposure of food to gastric acid and substances that form soluble complexes with Ca2+ (e.g. amino acids and bile salts) increases absorption whilst those forming insoluble complexes (e.g. oxalate and long-chain fatty acids) decrease absorption.

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Box 5.8

Absorption of Ca2+ from the gut. Image dclcc1.jpg Ca2+ enters the microvilli of the brush border through calcium channels, the opening and closing of which are regulated by the concentration of unbound Ca2+ in the microvilli. The ion is transferred across the enterocyte (more...)

Ca2+ may also be absorbed from the gut by endocytosis and fusion of the particles with lysosomes containing a CaBP. At the basolateral surface, the calcium is released by a process of exocytosis that involves the opening of calcium channels. Another mechanism that has been proposed is via a transcellular route whereby calcium, bound to a CaBP, is transported through gap junctions between the enterocytes (Box 5.8).

The rate of calcium absorption across the duodenum is proportional to the number of CaBPs and 1,25-dihydroxyvitamin D increases the expression of CaBPs. In addition to stimulating CaBP production in the gut, vitamin D increases the permeability of the brush border to Ca2+, increases the number of Ca2+/Na+ exchange pumps in the basolateral membrane and may open Ca2+ channels via activation of a membrane-bound receptor. Some of these effects may account for the rapid effects of vitamin D on calcium absorption before an increase in CaBP is observed. Finally, it should also be noted that vitamin D also increases uptake of Pi and Mg2+ from the gut.

Within bone, 1,25-dihydroxyvitamin D has an effect synergistic with that of PTH stimulating bone resorption and, thereby, raising circulating Ca2+ concentrations. The precise actions of 1,25-dihydroxyvitamin D on bone have not been clearly defined. It is thought that it induces differentiation of monocytic cells (originating in bone marrow) into osteoclasts and activates these bone-resorbing cells by stimulating the release of a paracrine signal from neighboring osteoblasts (see Box 5.32). Vitamin D also plays an important role in bone mineralization with clinically very important effects, discussed in relation to Clinical Case 5.4. Whether this is due to an increase in the extracellular supply of calcium and phosphate or the stimulation of bone formation by a direct effect is not defined.

Parathyroid glands and PTH synthesis

The parathyroid glands develop embryologically from the 3rd and 4th branchial arches (see Box 3.21). Typically, there are four parathyroid glands, one lying behind each of the upper and lower poles of the thyroid gland. Supernumerary glands in the neck or mediastinum (particularly within the thymus) are not uncommon and may cause considerable clinical problems in the search for sources of excessive PTH secretion. Six glands have been reported in approximately 2.5% of the normal population with even seven or eight glands in a few people. Each gland weighs approximately 30–50 mg and is supplied by blood from the thyroid arteries; these can easily be disrupted during thyroid surgery.

PTH is initially synthesized as a larger preprohormone and subsequently cleaved to a biologically active 84 amino acid peptide (molecular weight 9500, Box 5.9). This synthesis occurs in the more numerous chief cells of the parathyroid glands (Box 5.10). The less numerous oxyphil cells that appear at puberty secrete PTH only in certain pathological conditions. Full biological activity resides in the first 34 amino-terminal amino acids of the PTH molecule and cleavage of both the amino- and carboxy-terminals of the peptide leads to the production of truncated peptides with little or no biological activity. Cleavage of the first two amino acids from the amino terminal of the peptide markedly reduces bioactivity of PTH but leaves the ability of the hormone to bind to receptors unaltered. PTH does not have a serum-binding protein and the t1/2 of circulating PTH is about 4 min; it is rapidly cleared by the liver and kidney.

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Box 5.9

Structure and synthesis of PTH. PTH is synthesized from the precursor molecule pre-pro-PTH containing 115 amino acids. As the peptide length increases inside the rough endoplasmic reticulum, the signal sequence is removed in two steps leaving pro-PTH (more...)

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Box 5.10

Histology of the parathyroid gland. The gland has a connective tissue capsule and contains numerous chief cells (CC) and fewer oxyphil (OC) cells. The latter are larger with more cytoplasm. With age fat cells (FC) become more numerous. This image is available (more...)

Since the carboxyl terminal fragment of PTH is biologically inactive, assays that only measure the carboxyl terminal portion of the molecule may give aberrant results, especially in renal failure when there is accumulation of the truncated peptides. Modern assays use two different antibodies, one to recognize the amino-terminus and another the carboxyl-terminus and the principle of these two-site immunoradiometric assays has been described in detail (Box 3.25).

Control of PTH secretion

Changes in circulating Ca2+ concentrations are detected and alter PTH secretion via a negative feedback system. The chief cells detect circulating concentrations of Ca2+ by a unique G-protein-linked calcium receptor. An increase in Ca2+ binding stimulates phospholipase C and inhibits adenylate cyclase and the resultant rise in phosphatidylinositol trisphosphate (PI3) and reduction in cAMP concentrations reduces PTH release. A decrease in the activation of this receptor reduces the generation of IP3 and increases the generation of cAMP leading to an increase in PTH secretion (Box 5.11). As a result, PTH secretion is inversely proportional to serum calcium concentration (Box 5.12) and the ‘set point’ for PTH secretion is around 1.3 mmol/l. Maximal rates of secretion are achieved at a serum Ca2+ concentration of about 1.15 mmol/l. However, the secretion of PTH is never fully suppressed and hypercalcemia arises in the presence of hyperplastic parathyroid glands (Box 5.16).

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Box 5.11

Control of PTH secretion. The circulating concentration of Ca2+ is detected by a unique G-protein-linked calcium receptor (CaR) on the cell surface of the chief cells. When the Ca2+ concentration is high the α-subunit of the G-protein preferentially (more...)

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Box 5.12

Relationship between serum Ca2+ concentrations and PTH secretion.

In addition to this negative feedback control of PTH secretion, an increase in vitamin D concentration not only reduces transcription of the C-1 hydroxylase gene but also that of the PTH gene. Thus, vitamin D not only regulates its own conversion to its active metabolite but also the synthesis of PTH.

Actions of PTH

PTH acts on osteoblasts in bone and tubular cells within the kidney via G-protein-linked receptors that stimulate adenylate cyclase production of cyclic AMP (Box 5.13). In bone, within 1 or 2 hours, PTH stimulates a process, known as osteolysis, in which calcium in the minute fluid-filled channels (canaliculi/lacunae) is taken up by syncytial processes of osteocytes and transferred to the external surface of the bone and, thence, into the extracellular fluid (Box 5.33). Some hours later, it also stimulates resorption of mineralized bone; a process that releases both Ca2+ and Pi into the extracellular fluid. The Pi is rapidly removed from the circulation because the most dramatic effect of PTH on the kidney is to inhibit reabsorption of Pi in the proximal tubule and markedly increase its excretion. At the same time, PTH also enhances Ca2+ reabsorption in the ascending loop of Henlé and the distal convoluted tubule by increasing the active uptake of calcium by Ca2+-ATPase and a Na+-Ca2+ antiporter. Calcium excretion rate is reduced. As noted previously, PTH also stimulates the C-1 hydroxylation of 25-hydroxy-vitamin D within the kidney, thus indirectly stimulating Ca2+ reabsorption by the gut.

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Box 5.13

Actions of PTH. Parathyroid hormone (PTH) acts via a G-protein (G) linked receptor (R) to activate adenyl cyclase (AC) which converts ATP to cAMP. This second messenger stimulates signal transduction processes that activate various functions on bone cells (more...)

Hypercalcemia and primary hyperparathyroidism

It is clear from the foregoing that an excess of PTH or vitamin D leads to hypercalcemia and that the resulting symptoms will be multiple (Box 5.14). Clinical Case 5.1 underwent radiological investigation of the bones in which she suffered aches and pains (Box 5.15). This showed marked abnormalities in her humerus and also her hands. At the time her serum Ca2+ was 4.1 mmol/l her serum PTH concentration was inappropriately elevated (35 pmol/l, NR 1–6 pmol/l), indicating primary hyperparathyroidism.

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Box 5.14

Clinical features of hypercalcemia.

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Box 5.15

X-rays of the left arm and hands of Clinical Case 5.1. Left humerus. The arrows delineate a large osteolytic area. The most common cause of this appearance is metastatic tumor. In this case it is due to a ‘brown’ tumor or collection of (more...)

This is the most common cause of hypercalcemia with an annual incidence of about 45 per 100 000 (Box 5.16). It occurs approximately 2.5 times more frequently in women than in men and its incidence increases with age. Unlike that of Clinical Case 5.1, many cases are asymptomatic. In about 80% of the cases it results from a benign parathyroid adenoma and in about 15% a primary hypertrophy of the gland. Parathyroid carcinoma is rare (<0.5%).

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Box 5.16

Causes of hypercalcemia. Primary hyperparathyroidism (~99% of ambulant patients) Single adenoma (~80%)

The ability to measure total serum Ca2+ routinely, simply and cheaply in blood samples means that cases such as Clinical Case 5.1 with severe hypercalcemia and marked symptoms are now rarely seen in the clinic. In this patient, radiological studies showed a large osteolytic lesion in the head of the humerus and marked erosion of the terminal phalanges, together with cyst formation. Such appearances are due to PTH-induced resorption of bone. The osteolytic lesion is caused by the PTH-induced localized accumulation of osteoclasts within the bone, termed a brown tumor because of the color when seen by the naked eye.

Hyperparathyroidism and multiple endocrine neoplasia (MEN)

The finding of hyperparathyroidism should always suggest the possibility of multiple endocrine neoplasia because of the implications for affected families. Detailed enquiry into the family history of patients is, therefore, important. Hyperparathyroidism is the most common feature of MEN-1, being present in virtually all gene carriers by the age of 40 years, and is usually the first manifestation of the disease process. MEN-1 is due to loss of function mutations in the putative tumor suppressor gene menin and is characterized by tumors of the parathyroids (in ~90%), pituitary gland (~60%) and the pancreas (~70%). Hyperparathyroidism is also seen in approximately 10–30% of families with MEN-2a that is due to loss of function mutations in the putative tumor suppressor gene ret. Since MEN-2a is associated with the development of pheochromocytoma, such patients should undergo measurements of urinary or serum catecholamines (see Clinical Cases 5.8 and 4.7). Families with MEN should be followed in specialized multi-disciplinary clinics with regular, structured screening programs.

Hypercalcemia and vitamin D excess

Vitamin D excess is usually due to an excessive intake of the vitamin. As the C-1 hydroxylation is tightly regulated, it is more often seen as a result of accidental therapeutic overdose with hydroxylated pharmaceutical products rather than as a result of vitamin supplementation with non-hydroxylated prohormones; unless, of course, the prohormones are taken in very large doses (>50 000 U/d). It is also seen in about 10% of patients with sarcoidosis, tuberculosis and other granulomatous disorders. This is due to extra-renal conversion of 25-hydroxyvitamin D to the active 1,25-dihydroxyvitamin D by the granulomata, a process that is not regulated by PTH. The characteristic clinical feature of such hypercalcemia is that it responds to high doses of immunosuppressant glucocorticoid steroid such as prednisolone.

Hypercalcemia and malignancy

Clinical Case 5.2

A 63-year-old male factory worker was referred to the clinic with weight loss, general malaise and a cough. He drank four pints of beer and smoked 20 cigarettes a day. There was no history of excessive intake of vitamin supplements and he took no regular medication. Blood tests revealed normal renal and liver function but a serum Ca2+ of 3.8 mmol/l (NR 2.2–2.6 mmol/l) with an albumin of 37g/l (NR 38–48 g/l). The serum PTH concentration was below the assay detection limits. In view of his cough and smoking history, a chest X-ray was performed (Box 5.17). X-rays of his skeleton were normal.

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Box 5.17

Chest X-ray of Clinical Case 5.2. The arrows delineate a mass in the left lung that in the presence of a strong history of cigarette smoking is very suggestive of a lung cancer. This image is available on the website.

Clinical Case 5.2 clearly differs from Clinical Case 5.1 in having appropriately suppressed PTH concentrations and there was no history of excess vitamin D intake. His smoking history and the chest X-ray appearances clearly suggested an underlying malignant neoplasm, probably lung cancer.

Malignancy is the second most common cause of hypercalcemia overall, but in hospitalized patients it is the most common cause. Malignancy causes hypercalcemia via two main mechanisms. Hematological malignancies (e.g. myeloma) and those that metastasize to bone (e.g. breast or prostate cancer) produce local factors that act in a paracrine manner to activate osteoclasts (Box 5.18). Others, particularly squamous tumors of the lung and head and neck, produce a hormone, PTH-related peptide (PTHrp), that acts at PTH receptors. Patients with tumors secreting PTHrp, as occurred in Clinical Case 5.2, have appropriately suppressed PTH levels due to feedback effects of the hypercalcemia on the parathyroid glands. For the sake of completeness, note that exceptionally rarely tumors may secrete PTH itself and that some lymphomas may have increased (and unregulated) C-1 hydroxylase activity.

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Box 5.18

Bone resorption and local effects of malignant cells. Osteoclasts arise from hematopoietic precursors of the monocyte lineage. Their differentiation is stimulated by factors such as macrophage colony-stimulating factor (M-CSF), cytokines such as IL-6 (more...)

Parathyroid hormone-related peptide (PTHrp)

PTHrp is synthesized in various tissues including keratinocytes, lactating mammary tissue, the placenta and fetal parathyroid glands and has actions similar to PTH. It is synthesized from a gene considered to have evolved from a common ancestor of the PTH gene. Alternate splicing of the primary transcript gives rise to three similar products that range in length from 139–173 amino acids.

The amino terminal fragments of these peptides show striking homology to the amino terminal fragment of PTH (eight of the first 13 amino acids are identical) and PTHrp binds to PTH receptors (Box 5.19). It was originally discovered in patients like Clinical Case 5.2 with cancers of squamous cell origin in whom it caused hypercalcemia. However, PTHrp does not, like PTH, increase renal C-1 hydroxylase enzyme activity and, thus, patients with hypercalcemia due to PTHrp do not have the raised concentrations of 1,25-dihydroxyvitamin D seen in patients with hyperparathyroidism. The role of PTHrp in the adult is uncertain but there is evidence for its importance in regulating Ca2+ fluxes between fetal and maternal circulations, Ca2+ concentrations in breast milk and a role in fetal development. Recently, it has been suggested that there are secreted forms of the mid-region PTHrp viz PTHrp38–94, PTHrp38–95 and PTHrp38–101 that appear to act via a separate receptor as does the carboxyl terminal peptide PTHrp107–139.

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Box 5.19

Parathyroid hormone-related peptide. Comparison of the sizes and also the amino terminal 13 amino acid sequences of PTH and PTHrp

Treatment of hypercalcemia

Fluids

The initial treatment of hypercalcemia is the same irrespective of its cause (Box 5.20). As illustrated by Clinical Case 5.1, the inhibitory effect of hypercalcemia on the action of arginine vasopressin leads to polyuria. Thus, the initial step is assessment of a patient's fluid balance and initiation of fluid therapy (that may need to be given intravenously). Once fluid balance has been restored, excretion of calcium may be further enhanced by a saline diuresis (because Na+ and Ca2+ reabosorption parallel each other in the loop of Henle) using, for example, the loop diuretic furosemide and additional intravenous fluids.

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Box 5.20

Treatment of hypercalcemia. General Fluid replacement that may need to be given intravenously ± a diuretic active on the loop of Henle such as furosemide

Subsequent treatment depends on the cause (Box 5.20).

Bisphosphonates

Bisphosphonates were developed in the detergent industry at Port Sunlight near Liverpool as anti-foaming agents. The nucleus of the molecule contains two phosphate groups attached to a carbon and the different members of the family differ in the nature of the side chains attached to the carbon. The negative charges on the phosphate groups give the bisphosphonates their affinity for bone mineral. They are incorporated into newly formed bone. It was originally thought they reduced osteoclast action by a physicochemical action on hydroxyapatite crystals. However, it is clear that bisphosphonate absorbed to bone surface is taken up into osteoclasts and interferes with several biochemical processes. Those bisphosphonates most resembling pyrophosphate (e.g. etidronate) become incorporated into non-hydrolyzable ATP analogs inhibiting function and leading to cell death. Those containing nitrogen (e.g. alendronate) interfere with other reactions such as the mevalonate pathway affecting signalling functions and thus inhibiting osteoclastic bone resorption. Bisphosphonates are available for both intravenous and oral use (Box 5.20).

Parathyroid surgery

The embryology of the parathyroid glands (leading to considerable anatomical variation), together with their small size, makes parathyroid surgery a specialized field. The most important question is whether a single gland has become adenomatous (the most common cause of primary hyperparathyroidism) or whether there is hyperplasia of all glands. The magnitude of the increase in serum PTH concentration does not help distinguish these possibilities, though it is said that very high concentrations of PTH are typical of the rare carcinomas. A variety of imaging techniques has been used to localize parathyroids prior to surgery, but none offers the desired degree of specificity and sensitivity most clinicians require (Box 5.21). Accordingly, many physicians do not use imaging techniques, relying on the skill of experienced surgeons who generally have success rates >95%. A recent development has been the use of minimally invasive surgery using modified laparoscopic techniques.

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Box 5.21

Localization of the parathyroid glands*.

It is incumbent on a surgeon to identify all four glands. In most cases, a single adenoma will be found and the other glands will be small (i.e. <50 mg in weight and <5 mm in greatest dimension). Biopsies of the normal glands (with rapid frozen-section histology) may be required if there is any doubt. Occasionally (i.e. approximately 1% of cases), two adenomas are present. However, the greatest problems are caused by parathyroid gland hyperplasia. In these cases, the unknown stimulus to hyperplasia may have also acted on a 5th, 6th or intrathymic gland so that the patient remains hypercalcemic despite the removal of four glands.

Furthermore, in this situation, the surgeon has to face the dilemma of whether to remove all glands (leaving the patient requiring vitamin D treatment life-long), or to remove all but part of one leaving the patient at risk of redeveloping hypercalcemia and requiring further surgery on the neck. Some surgeons remove all the glands from the neck and transplant part of one into the forearm so that only minor surgery is required if hypercalcemia returns.

The situation is different if surgery fails. It is clear that this usually arises because one or more parathyroids were not found at the first operation either because of anatomical variation or because there were more than four parathyroid glands. Selective venous catheterization of the veins in the neck and mediastinum coupled with assays of PTH has been used to localize the source of PTH prior to a second (or subsequent) operation.

Malignancy

The treatment of malignant hypercalcemia involves the same initial generic therapy followed by bisphosphonates and treatment of the malignancy. The specific treatments for the latter are under constant review and are not covered here. The most common tumors metastasizing to bone are prostate, breast and lung. The tumors most likely to secrete PTHrp are squamous cancers of the lung, head and neck and esophagus although breast, renal and bladder cancers may also do so. Hypercalcemia resulting from an excess of PTHrp secreted by malignant tumors is best treated with bisphosphonates. Metastases to bone may be treated by local radiotherapy.

Hypervitaminosis D

When this has resulted from an excess oral intake, treatment after initial generic treatment of hypercalcemia is simply to withhold the source of the excess, though glucocorticoid steroids such as prednisolone may also be used. As noted above, vitamin D excess associated with sarcoidosis and tuberculosis is treated with steroids and additional therapy according to the underlying diagnosis.

Mutations of the Ca2+ or PTH receptors

It is apparent that the normal function of the parathyroid gland Ca2+ receptor (Box 5.11) is crucial to the regulation of serum Ca2+ concentration. Situations in which its function is decreased (loss of function mutations) falsely signal to the parathyroid gland chief cells a low serum concentration and hyperparathyroidism will result, leading to hypercalcemia. This is seen in two clinical conditions, familial benign hypercalcemia and neonatal severe hyperparathyroidism (Box 5.22). To some extent these vary in severity according to whether the patients are heterozygous or homozygous and, therefore, have one or two copies of the mutation.

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Box 5.22

Clinical conditions associated with mutations of the Ca2+ receptor. Familial hypocalciuric hypercalcemia (FHH) Autosomal dominant

Similarly, if the PTH receptor were to contain mutations leading to increased biological activity (gain of function mutations) that are independent of serum PTH concentrations then hypercalcemia would also result. This occurs in the very rare, dominantly inherited condition Jansen-type metaphyseal chondrodysplasia. This is characterized clinically by short-limbed dwarfism, hypercalcemia and hyper-calciuria. Whilst PTH secretion is suppressed by the hypercalcemia and, hence, serum concentrations of PTH are low, there is evidence of increased bone resorption. In both cases, the ‘set points’ for the control of PTH secretion have been shifted.

Hypocalcemia and its treatment

Hypocalcemia, as judged by routine analysis of serum (i.e. total) calcium concentrations, is quite common, particularly in a hospitalized population in which a low serum albumin concentration is frequently seen. A low serum ionized (i.e. Ca2+) concentration is much less common. It is clear from the foregoing that hypocalcemia arises because of the inability of the body to respond to low serum calcium concentrations. Thus, chronic hypocalcemia is likely to be the result of a deficiency of PTH or vitamin D or a resistance to one or other hormone. It is important to note, however, that in the presence of normal parathyroid glands, low vitamin D concentrations (of whatever cause) will result in compensatory secondary hyperparathyroidism; severe hypocalcemia is likely, therefore, to be due to hypoparathyroidism. The causes of hypocalcemia (and to some extent the clinical symptoms and signs) differ according to the age of the patient. The next clinical case illustrates the presentation in early life.

Clinical Case 5.3

A 3-week-old girl of Indian parents had been noted by her mother (who had brought her to the Emergency Room) to have intermittent twitching of her left-hand-side limbs over the previous 4 days. The girl had been born normally at 39 weeks of gestation and was being breast-fed. Examination revealed nothing abnormal. Investigations were performed into the cause of these symptoms. As a result of these, the baby was found to have a low total serum calcium concentration of 1.39 mmol/l (NR 2.2–2.75 mmol/l) a high serum phosphate of 2.87 mmol/l (NR (1.55–2.0 mmol/l) and normal albumin of 38 g/l (NR 38–48 g/l).

For the reasons given above, hypoparathyroidism is the most common cause of hypocalcemia (Box 5.23). In adults, this is usually caused by the surgeon's scalpel related to the fact that thyroid disease is common. Clinical Case 5.3 presented very soon after birth suggesting that she had congenital hypoparathyroidism. As might be expected from the embryology of parathyroid gland development (see Box 3.21), congenital absence of the parathyroid glands is likely to be associated with maldevelopment of the 3rd, 4th and 5th branchial arches giving rise to defective thymus and cardiac development (known as Di George syndrome). Familial congenital hypo-parathyroidism may also be inherited as an X-linked or recessive condition. These are, however, exceedingly rare and Clinical Case 5.3 was phenotypically normal.

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Box 5.23

Causes of symptomatic hypocalcemia. Common (~99%) Neonatal Maternal disorder e.g. pre-eclampsia, diabetes, placental insufficiency, hyperparathyroidism Prematurity Adulthood Post-surgical damage

To understand fully the neonatal presentation, it is important to appreciate fetal—maternal calcium balance outlined in Box 5.24. It is clear that maternal hyperparathyroidism leading to hypercalcemia may be translated by the placenta into fetal hypercalcemia suppressing PTH secretion by the fetal parathyroid glands. In the extrauterine environment, the fetal (now neonatal) system (essentially the kidneys) clears the hypercalcemia but the parathyroid glands, having been suppressed fetally, take time to recover. Thus, a neonate subjected to fetal life in a hypercalcemic environment may present with symptoms and signs of hypocalcemia (Box 5.23). Noteworthy is the high serum phosphate measured in the infant. This is because in hypoparathyroidism the effect of PTH on Pi excretion by the kidney is lost and thus hyperphosphatemia develops.

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Box 5.24

Materno—fetal calcium balance. The average neonate contains ~20 g of calcium, most of which is accumulated during the 3rd trimester (weeks 28–40) Maternal serum Ca2+, Pi and PTH concentrations are within the non-pregnant normal range throughout (more...)

The serum calcium in the mother of Clinical Case 5.3 was measured and found to be high (2.9 mmol/l, NR 2.2–2.6 mmol/l) with an albumin of 39 g/l (NR 38–48 g/l) and phosphate of 1.11 mmol/l. The mother's serum PTH was reported to be 23.9 pmol/l (NR 1–6 pmol/l) whilst that of the baby was 5.7 pmol/l. These values were inappropriately high and low respectively for the serum calcium concentrations. The daughter's serum calcium corrected spontaneously over a number of weeks; the mother underwent removal of a single parathyroid adenoma prior to undertaking a second pregnancy.

Hypoparathyroidism also occurs in rare syndromes that have associated features suggesting an autoimmune etiology (Box 5.25). The targets of the autoimmunity are as yet unknown; autoantibodies blocking functions have been suggested in some studies.

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Box 5.25

Autoimmunity and the parathyroid gland. Characterized by the triad of clinical features: Mucocutaneous candidiasis (~100%)

Whilst endocrine deficiencies are usually treated with replacement therapy of the deficient hormone (e.g. insulin, T4, glucocorticoids), PTH deficiency is treated with vitamin D because it is cheap and orally active (see Box 5.29). PTH produced by recombinant DNA technology is currently being developed for use in osteoporosis (see below). In acute situations, as occurred in the patient in Clinical Case 5.3, intravenous Ca2+ can be used.

Pseudohypoparathyroidism

The first hormonal resistance syndrome to be described was that of PTH resistance. In 1942, Fuller Albright and his colleagues described patients who were hypocalcemic and hyperphosphatemic with a typical phenotype of short stature, short neck, brachydactyly (short fingers especially affecting the 4th and 5th metacarpals), obesity, and subcutaneous calcification. When injected with PTH they did not show the normal responses of increased serum Ca2+ concentrations and increased Pi and cAMP concentrations in the urine. As would be expected of a resistance syndrome, such patients have high serum concentrations of PTH.

The disease was termed pseudohypoparathyroidism, a cumbersome term that remains in widespread use. A better term would, perhaps, be PTH resistance. Since that time pseudohypoparathyroidism has been divided into several types depending on the stage at which PTH signal transduction is affected. In some, the typical phenotypic features described (now known as Albright's hereditary osteodystrophy, AHO) are not present even though there is resistance to PTH.

In type Ia pseudohypoparathyroidism, there is an approximately 50% reduction in the activity of the stimulatory G-protein linked to the PTH receptor and typical features of AHO. Different mutations of the Gs protein are seen in different families and these are inherited in an autosomal dominant manner. Some family members of patients with type Ia pseudohypoparathyroidism show features of AHO and reduced Gs activity although they show a normal urinary phosphate and cAMP response to PTH administration. This abnormality has been termed pseudo-pseudohypoparathyroidism (an inelegant term) and is paternally transmitted. However, when the abnormal Gs gene is maternally transmitted, patients tend to exhibit PTH resistance (no kidney response) as well. This suggests that other factors/genes are involved in PTH resistance other than mutations in the Gs gene.

Other types of pseudohypoparathyroidism, classified as type Ib, show features of tissue-specific resistance to PTH with hypocalcemia, hyperphosphatemia and secondary hyperparathyroidism, yet they lack features of AHO or abnormal Gs activity. These cases tend to occur sporadically and it is thought that the resistance is caused by an abnormal receptor or an abnormality in the transduction of the signal after activation of the G-protein.

Vitamin D deficiency

As has been emphasized, lack of vitamin D rarely leads to symptomatic hypocalcemia because the secondary increase in PTH compensates. Vitamin D deficiency has marked effects on bone as illustrated in Clinical Case 5.4, chosen for its particularly eloquent additional demonstration of the non-classical actions of the hormone.

Clinical Case 5.4

A 28-year-old Asian woman from East Africa was admitted for investigation of suspected aplastic anemia. She was barely able to walk and was brought by wheel chair to the ward. A history revealed that she had delivered a normal baby a few months previously. She was thought to have aplastic anemia and had been treated with repeated blood transfusions. She complained of aches in her hips and was noted to have a severe proximal myopathy (weakness of the muscles around her hips). Her serum calcium was 1.96 mmol/l (NR 2.2–2.6 mmol/l), serum phosphate 0.66 mmol/l (NR 0.8–1.4 mmol/l) and albumin of 37 g/l (NR 38–48 g/l). Her serum creatinine was normal but she was noted to have a marked hyperchloremic acidosis (serum chloride 114 mmol/l (NR 99–109 mmol/l), serum bicarbonate 19 mmol/l)). The serum concentration of the bone isoform of the enzyme alkaline phosphatase (that is involved in mineralization) was markedly elevated at 1239 IU/l (NR 30–120 IU/l). X-rays of her hips showed marked deformation of the hip joint sockets on both sides and those of the lumbar spine osteopenia and osteoporosis. Detailed history taking revealed that she had become so weak during pregnancy that she had been unable to leave the house. Her only other symptom was of recurrent loose bowel motions that had become more marked during pregnancy.

The clinical features were dominated by the weakness due to a proximal myopathy and aches and pains in bones. The anemia was severe and bone marrow examination showed a marked lack of blood-forming cells (Box 5.26). The hypocalcemia was mild and the serum biochemistry dominated by the hypophosphatemia and hyperchloremic acidosis. The latter resulted from PTH-mediated inhibition of renal reabsorption of phosphate, bicarbonate and sodium thereby increasing their urinary excretion.

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Box 5.26

The bone marrow appearances of Clinical Case 5.4. The pelvis X-ray shows the molding of the hip joint (arrows) that has occurred as a result of the loss of the tensile strength of bone. The low power photomicrograph of the bone marrow trephine shows the (more...)

Clinical Case 5.4 had a serum 25-hydroxy vitamin D concentration of 6 nmol/l (NR 20–100 nmol/l) and a diagnosis of osteomalacia (loss of bone mineralization) due to vitamin D deficiency was made. The causes of vitamin D deficiency are given in Box 5.27 and the clinical features in Box 5.28. The diagnostic question in this patient was the cause of the vitamin D deficiency. In the UK (with substantially less sun than East Africa), lack of sun exposure and poor dietary intake (or absorption) lead to a much higher prevalence in high risk populations including Asians and the elderly. In this case dietary intake was regarded as adequate.

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Box 5.27

Causes of vitamin D deficiency. Deficient intake or cutaneous synthesis e.g. dietary deficiency or reduced sunlight (particularly seen in the UK in the elderly and Indian subcontinent Asian populations) Deficient absorption e.g. celiac disease or pancreatic (more...)

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Box 5.28

Clinical features of vitamin D deficiency. Rickets Bony deformity e.g. bowing of long bones Image dclcc1.jpg , widening of cartilage at growth plate Image dclcc2.jpg

The only symptom to give a clue to the cause came from the gut, suggesting the failure to absorb dietary vitamin D. The patient had symptoms of diarrhea without those such as loss of blood or mucus to suggest large bowel involvement. This suggested the possibility of a small bowel disease such as celiac disease. This is an immune-mediated condition leading to loss of intestinal villi and, therefore, impaired absorption of a number of important dietary materials. Duodenal biopsy was performed and confirmed the diagnosis of celiac disease. This, together with her lack of exposure to sunlight, had caused her vitamin D deficiency.

The treatment of vitamin D deficiency is, naturally, hormone replacement. The principles of oral replacement are given Box 5.29. In Clinical Case 5.4, it was recognized that oral vitamin D may not be absorbed and intramuscular vitamin D was given. At the same time, she was given dietary advice to avoid foods containing wheat that contains the protein gluten to which there is hypersensitivity in celiac disease. Once this was done and a repeat duodenal biopsy showed recovery, she was treated with oral vitamin D. The bone marrow showed marked recovery (see website).

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Box 5.29

Principles of the treatment of hypocalcemia. In the acute situation with severe symptoms (e.g. neuromuscular irritability), intravenous calcium will usually be required. Oral calcium (1–3 g daily) supplementation is usual. In clinical situations (more...)

Non-classical actions of vitamin D

The widespread distribution of vitamin D receptors (VDRs) in the body suggests functions for the hormone far beyond that of the regulation of Ca2+ and Pi (Box 5.30). VDRs have been located in at least 30 different tissues and over 70 genes are regulated by the VDR. These include genes associated with mineral homeostasis, cell differentiation and proliferation, oncogenes, metabolism and signal transduction proteins. The antiproliferative and maturational effects of vitamin D on keratinocytes have been put to clinical use; vitamin D is used to treat psoriasis, a proliferative skin disorder. In the bone marrow, vitamin D acts to suppress the production of cytokines by megakaryocytes (the precursors to platelets) and, in the absence of the hormone, the cytokines produced inhibit normal marrow function (as seen in Clinical case 5.4).

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Box 5.30

Genomic actions of vitamin D. Increased calcium binding proteins Increased calcium pumps in basolateral surface of intestinal villus and crypt cells

Vitamin D is also involved in immunomodu-lation. It stimulates the differentiation of promonocytes to monocytes and macrophages (believed to be precursors of osteoclasts that resorb bone), reduces the production of cytokines by immune cells and decreases the proliferation of T and B lymphocytes. Thus, vitamin D increases non-specific immunity but suppresses antigen-specific immunity. No major immune defect, however, is notable in individuals who lack vitamin D receptors. In contrast, life-long alopecia (hair loss) is frequently associated with loss of VDRs indicating that the vitamin is important in the maturation of the hair follicle.

Vitamin D resistance and rickets

As with other hormones, resistance to vitamin D also occurs, but it is rare. As inherited defects, they present in childhood with clinical features of the bone disease rickets. There are two forms of resistance. Type 1, also known as pseudovitamin D deficiency, is not actually a true resistance but is caused by a defect in its C-1 hydroxylation. Hence, the active form of the hormone is not produced and bone formation is impaired. It is easily treated by oral administration of the active 1,25-dihydroxylated hormone.

Type 2 vitamin D resistance, otherwise known as hereditary vitamin D resistant rickets (VDRR), is due to a true end-organ resistance and is inherited in an autosomal recessive manner. Several different mutations in the vitamin D receptor have been documented. Some decrease the binding affinity for the hormone whilst others affect the subsequent processes involved in the transcription of genes normally controlled by the hormone receptor complex. Rickets is discussed further in the context of Clinical Case 5.5, a rare case chosen to introduce bone disease.

Clinical Case 5.5

An 18-month-old child first walked at 14 months of age. She was noted to have an odd gait and deformity of the legs. Dietary vitamin D deficiency rickets was diagnosed at another hospital and she was treated with vitamin D2. By the age of 2 years her deformity was more marked (Box 5.31). She was reinvestigated. When taking her vitamin D2, her serum calcium was 2.32 mmol/l (NR 2.2–2.6 mmol/l) with a phosphate of 0.58 mmol/l (NR 1.16–1.91 mmol/l). The serum PTH concentration was 12 pmol/l (NR 1–6 pmol/l).

The child was diagnosed as having dietary vitamin D deficiency but the clinical features deteriorated while taking apparently adequate treatment; either the family had not been administering the treatment or the diagnosis was incorrect (or both). Family frictions made it difficult to obtain a full family history but, after lengthy discussions, the child's mother later admitted to having been treated for rickets in childhood and blamed her own mother for her childhood difficulties associated with this disease.

A diagnosis of X-linked dominant hypophosphatemic rickets was made and the child was treated with 4-hourly oral phosphate supplements and 1,25-dihyydroxyvitamin D. The dramatic radiological and clinical benefits of treatment can be seen (Box 5.31). Hypophosphatemic rickets remains to be fully understood. It is characterized by a defect in proximal renal tubule function resulting in phosphate wasting plus low or inappropriately normal concentrations of serum 1,25-dihydroxyvitamin D and defective bone mineralization. Studies in hyp mice, a model of the disease, have shown defects in the C-1 hydroxylation of 25-hydroxyvitamin D in the same cells that have a defect in phosphate absorption. It appears to be due to a mutation in the PHEX gene — a PHosphate-regulating gene with homologies to Endopeptidases located on the X-chromosome. It has been suggested that PHEX codes for a membrane enzyme that cleaves the putative hormone phosphatonin that is involved in regulating Pi absorption. Inadequate cleavage of phosphatonin leads to increased circulating concentrations of the protein, decreased expression of renal Na+/PO43– co-transporters and, thus, Pi wasting.

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Box 5.31

Clinical features of Clinical Case 5.5. Clinical photograph of the lower legs at presentation. Note the marked bowing. Clinical photograph of the lower legs after 7 years treatment with regular oral phosphate and vitamin D. A vivid demonstration of the (more...)

Hormones and the skeleton

Clinical Case 5.5 (Box 5.31) shows the spectacular ability of bone to remodel and correct deformities and the requirement of vitamin D for bone mineralization. Clinical Cases 5.1 and 5.3 have demonstrated the importance of vitamin D and PTH in regulating Ca2+ and Pi concentrations. There are, in addition, several endocrine conditions that can give rise to skeletal abnormalities in the absence of changes in serum calcium concentration, and without alterations in vitamin D or PTH status. Since many hormones can affect bone formation and remodeling (Box 5.32) and so give rise to skeletal abnormalities it is appropriate to discuss these processes in more detail.

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Box 5.32

Bone remodelling and hormonal control of bone resorption. On bone surfaces osteoblasts secrete collagen into the extracellular space and subsequently calcium and phosphate are deposited in this osteoid. Eventually the osteoblasts become surrounded by (more...)

Structure, formation and function of bone

Bone is composed of an organic matrix (primarily collagen) impregnated with hydroxyapatite crystals (Ca10[PO4]6[OH]2). Other minerals are also present (e.g. magnesium) and bone is an important store of these. There are two types of bone, cortical and trabecular, the former constituting approximately 80% of the total bone mass. In the long bones of the skeleton, cortical or compact bone predominates and is characterized by an outer layer of circumferential rings of bone surrounding columns of concentric rings of bone (Box 5.33). Each column surrounds a Haversian canal that contains blood and lymph vessels and nerves. Inside this thick hard shell is the trabecular bone that is made up of spicules of bone or trabeculae arranged in lamellae. Between these spicules lie the bone marrow elements and connective tissue cells, as well as blood and lymphatic vessels. In the axial skeleton (skull, ribs, vertebrae etc.) there is only a relatively thin layer of circumferential cortical bone with a much greater mass of trabecular or spongy bone. Since trabecular bone has five times as much surface area as compact bone — i.e. the surfaces within bones exposed to the ECF — it is far more important than compact bone in phosphate and calcium homeostasis.

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Box 5.33

Generalized structure of bone and the process of osteocytic osteolysis. A schematic diagram of cortical and trabecular bone is shown in the upper figure together with the osteons where bone remodeling occurs. Surface osteoblasts become osteocytes as they (more...)

Bone formation and resorption (remodeling) occurs on bone surfaces of microscopic units called osteons (Box 5.32). Formation is carried out by active osteoblasts that extrude collagen into the extracellular space. Subsequently (about 10 days later), calcium and phosphate are deposited in this osteoid and mature hydroxyapatite crystals are slowly formed. Alkaline phosphatase is required for this mineralization process as well as adequate concentrations of calcium and phosphate. Vitamin D is essential and this is why lack of, or resistance to, this hormone leads to impaired mineralization as seen in Clinical Case 5.5.

As the osteoblasts become surrounded by mineralized bone these cells lose their activity and become interior osteocytes. However, they remain in contact with the bone surfaces, and hence the ECF, through long cytoplasmic processes (syncytial processes) that extend through the fluid-filled lacunae traversing the bone lamellae. The syncytial processes form gap junctions with surface osteoblasts. It is through these connections that calcium can be released from bone — a process termed osteocytic osteolysis and under the control of PTH (Box 5.33).

Bone resorption also occurs on bone surfaces and is carried out by giant multinucleate cells (up to 100 μm diameter) formed by the fusion of several progenitor cells (Box 5.18). These osteoclasts literally tunnel their way into mineralized bone. The actions of numerous enzymes break down the hydroxyapatite crystals and destroy the osteoid leaving craters on the bone surface. These pits are subsequently filled in with new bone by osteoblasts so bone resorption and formation are coupled.

Under normal circumstances, bone formation and resorption are co-ordinated processes occurring in osteons and responsible for the continual remodeling of bone. In any one year, it is estimated that there is a turnover of approximately 25% of trabecular and 3% of cortical bone. It should be noted that peak bone mass is considered to be reached at about the age of 30 years and studies in twins have indicated that approximately 80% of the variation in bone mineral density is due to genetic factors. It is clear that up to this age bone formation must exceed resorption and both bone formation and its remodeling are controlled by a vast array of hormones. These include growth factors and cytokines (many better known for roles played in the immune system), some of which stimulate bone formation or resorption, others inhibiting these processes.

Osteoporosis

Osteoporosis is one of the most common endocrine diseases. It differs from osteomalacia (in vitamin D deficiency) in that collagen as well as mineral is lost from bone. It occurs when bone resorption exceeds formation. Normally these bone remodeling processes are tightly co-ordinated by a wide variety of hormones and growth factors, allowing compensation for any change. If, for example, the primary action of a hormone is to stimulate bone resorption, this can be partially balanced by a secondary increase in formation. Thus, the net effect of any endocrine abnormality depends on the degree of compensatory coupling.

The most common form of osteoporosis is age related and there is a gradual loss of bone from the age of 30–40 years onwards. In women, bone loss is accelerated in the postmenopausal years due to the loss of estrogens and this occurs in both men and women at any age when there is a deficiency of sex hormones or other defined diseases. Osteoporosis is also a major problem in patients confined to chronic bed rest because immobilization increases bone resorption as does space flight due to the loss of gravity.

Osteoporosis may occur as a result of a reduced peak bone mass or a mismatch between bone formation and resorption. It has been calculated that lifetime risk of osteoporotic fracture over the age of 50 years is 40% for females and 13% for males. These fractures cost the Health Service many hundreds of millions of pounds per year. To emphasize that the disease is not restricted to elderly women, Clinical Case 5.6 is an unusual example of a young man with severe osteoporosis.

Clinical Case 5.6

A 26-year-old man had been under long-term follow up for hypopituitarism as a result of the treatment of a very large 3rd ventricular brain tumor presenting at the age of 15 years. He had been treated by the insertion of a ventricular—peritoneal shunt to reduce hydrocephalus and 5500 cGy of craniospinal irradiation. Over the course of his 26th year, it was noted that his standing height decreased by 6 cm. His serum biochemistry was normal. In particular, the serum calcium was 2.45 mmol/l (NR 2.2–2.6 mmol/l), the Pi 1.1 mmol/l (NR 0.8–1.4 mmol/l) and the albumin 38 g/l (NR 38–48 g/l) and his replacement endocrine medication of hydrocortisone, thyroxine and mixed testosterone esters was unchanged. X-rays of his spine reveal marked osteoporosis (Box 5.34) that was confirmed on bone biopsy. Bone mineral density by dual X-ray absorptiometry was markedly reduced.

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Box 5.34

Clinical Case 5.6. Clinical photographs of Clinical case 5.6 age 26 years. Note the shortening and marked antero-posterior curvature (termed kyphosis) of the spine, particularly noticeable in the thoracic region. This arises as a result of the combination (more...)

Osteoporosis arises when bone resorption exceeds its formation and its major causes are given in Box 5.35. In Clinical Case 5.6 there is a number of possible etiologies. Whilst hypogonadism is well recognized to cause osteoporosis, he was being treated with intramuscular androgens and there was no evidence of a past or present deficiency. Osteoporosis is also a feature of Cushing's syndrome and high doses of exogenous glucocorticoids. Again, in this case, there was no evidence that his glucocorticoid replacement was instrumental in its development. Radiotherapy also gives rise to osteoporosis but in this case bones in non-irradiated areas were also affected. It seems likely that a deficiency of pituitary somatotrophin (growth hormone) played a role in causing the bone disease.

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Box 5.35

Definition and causes of osteoporosis. Definition: A disease characterized by low bone mass and microarchitectural deterioration of bone tissue leading to enhanced bone fragility and a consequent increase in fracture risk. Diagnostic categories: Non-invasive (more...)

Somatotrophin provides a constant stimulus to bone formation and this stimulus can be promoted by insulin-like growth factors and other local growth factors. Cortisol inhibits bone formation and stimulates resorption, whilst the sex steroids have opposite effects. Cytokines generally stimulate bone resorption. Thus, the osteoporosis seen in Clinical Case 5.6 was likely to be caused by the loss of somatotrophin stimulation on bone formation and that this loss could not be compensated for by other bone-promoting hormones and growth factors. The syndrome of adult somatotrophin deficiency and its treatment is discussed in more detail on the website. The treatments for osteoporosis are given in Box 5.36.

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Box 5.36

Treatment of osteoporosis. General A number of recommendations have been made to reduce the risk of the development of osteoporosis. These include adequate calcium and vitamin D intake, regular weight-bearing exercise, moderate alcohol intake and not (more...)

Paget's disease (osteitis deformans)

Clinical Case 5.7

A 78-year-old widow presented to the Rheumatology clinic with a several year history of deformity and discomfort in the lower right leg (Box 5.37). Her serum Ca2+ was 2.76 mmol/l (NR 2.2–2.6 mmol/l) with a Pi of 1.03 mmol/l (NR 0.8–1.4 mmol/l) and an alkaline phosphatase of 335 IU/l (NR 30–100 IU/l). She was treated with an oral bisphosphonate. Nine years later she tripped on a pavement fracturing her right tibia (Box 5.37). At that time the serum Ca2+ was 2.57 mmol/l with a Pi of 1.00 mmol/l and an alkaline phosphatase of 684 IU/l. She was treated with a plaster of Paris cast and the fracture healed over the subsequent 6 months. Seventeen years after presentation, she complained of deteriorating hearing and was referred to the audiology department. At the age of 95 years her serum Ca2+ was 2.42 mmol/l with a Pi of 0.92 mmol/l and an alkaline phosphatase of 513 IU/l.

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Box 5.37

Clinical Case 5.7. Clinical photograph of the patient's right tibia on presentation. X-rays of the lower leg of the patient before and after the fracture. Note the bowing and marked irregularity of the cortex of the bone of the tibia (arrowed) compared (more...)

Clinical Case 5.7 vividly illustrates the origin of the name of the condition osteitis deformans and it is clear that Paget's disease is a disorder of bone remodeling. It is prevalent in Northern Europe, affecting up to 4% of people over the age of 40 and up to 10% over the age of 60. It is not known why large, somewhat bizarre-looking osteoclasts, with up to a hundred nuclei, stimulate bone metabolism and initiate bone remodeling in a disorganized way. An infectious etiology has been implicated (e.g. paramyxovirus or canine distemper) and other studies have suggested a genetic trait. The result of the disorganized remodeling is focal areas of highly vascular and cellular bone, with either a mosaic pattern of lamellar bone or woven bone. The disease can affect any bone but the most common sites are the sacrum and spine, femur, skull and the pelvis.

In the initial stages of the disease, osteoclastic activity usually predominates so that bones become soft and deformed and may fracture as vividly illustrated by Clinical Case 5.7. Later increased osteoblastic activity results in thickened deformed bone. This can trap nerves or leave abnormal bone in the joint areas. Thus, neurological (such as deafness as in Clinical Case 5.7) and rheumatological symptoms are associated with Paget's disease. The worst complication is the progression to bone sarcoma, but this occurs rarely (<1%). In the main, this disorder is asymptomatic and diagnosed coincidentally from radiological investigation performed for another purpose.

Biochemical investigations of this disease show that serum calcium is usually in the normal range whilst serum alkaline phosphatase is raised. This is a useful marker of disease activity and for assessing the response to bisphosphonate therapy used to inhibit bone resorption. Indications for treatment with bisphosphonates include deformity, pain and the involvement of bones that are prone to lead to complications (e.g. weight bearing or the base of the skull).

Clinical Case 5.7 was used to illustrate the presentation of Paget's disease and some of its complications but also because she demonstrates the chronic course of hyperparathyroidism in many patients. At presentation, when the serum Ca2+ was 2.76 mmol/l, she was noted to have a serum PTH concentration of 8.2 pmol/l (NR 1–6 pmol/l). It has been suggested that patients with Paget's disease have an increased prevalence of primary hyperparathyroidism and it may be these patients that are particularly prone to develop marked hypercalcemia when immobilized after, for example, a fracture. As she had normal renal function, no kidney stones and no evidence of parathyroid bone disease, parathyroid surgery was not considered appropriate.

Calcitonin and calcitonin gene-related peptide

At the beginning of this chapter it was noted that Ca2+ homeostasis was unaffected by the absence of calcitonin from the thyroid gland. Thus, its physiological role (at least in man) remains problematic even though circulating concentrations rise when serum Ca2+ concentrations increase. However, there are some important clinical and physiological aspects of this hormone and calcitonin gene-related peptide (CGRP). Both these hormones are synthesized from the same gene and different post-transcriptional processing of exons and introns give rise to two different hormones (Box 5.38). This contrasts with the synthesis of PTH and PTHrp which are coded from different genes evolved from a common ancestral gene.

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Box 5.38

Structure and synthesis of calcitonin and calcitonin gene related peptide.

Diseases in which calcitonin features are much less common than those in which PTH plays a major role and the next clinical case exemplifies the lack of effect of calcitonin on Ca2+ homeostasis in the human.

Clinical Case 5.8

A 27-year-old woman was seen in the clinic complaining of a lump in her neck that had increased in size over the previous year. She was clinically and biochemically euthyroid and the lump had not caused any problems from its size or position. Her serum calcium was normal. She had been adopted and no family history of thyroid disease was available. There were no abnormal findings on examination apart from a 1 × 2 cm mass in the left lobe of the thyroid gland.

To diagnose the cause of the lump in her neck, she underwent a fine needle aspiration cytological investigation (see Box 3.33) and the diagnosis of medullary cell carcinoma was made (Box 5.39). This is a tumor of calcitonin-secreting interstitial C-cells in the thyroid gland and it usually presents as a thyroid mass. Additional symptoms such as diarrhea are unusual and suggest metastatic disease and large tumor bulk. Treatment of this carcinoma is total thyroidectomy and lymph node dissection and therapeutic success can be monitored using serum calcitonin assays (two-site IRMA) with or without stimulation with a calcium infusion or pentagastrin.

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Box 5.39

Medullary cell carcinoma of the thyroid gland. Color image available on website. This is a multicentric neoplasm of the C cells of the thyroid gland.

A diagnosis of medullary cell carcinoma should, like that of hyperparathyroidism, always suggest the possibility of multiple endocrine neoplasia (MEN, Box 5.40). Whilst hyperparathyroidism is usually present in MEN type 1 syndrome, it only occurs in about 30% cases of MEN-2a. Evidently the ret oncogene is less important for parathyroid growth than for interstitial C-cells of the thyroid gland. Thus, although the serum calcium concentration was normal in Clinical Case 5.8 this does not exclude MEN type 2a. It later transpired that this patient's biological mother had died suddenly aged 45, suggesting the possibility of a pheochromocytoma. Since she had recently married and was planning to have children she underwent genetic investigation for the ret oncogene.

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Box 5.40

Multiple endocrine neoplasia. Autosomal dominant with incomplete penetrance. Loss of function mutations in the menin gene on chromosome 11.

Clinical case questions

Clinical Case Study Q5.1

A 6-yearold boy was seen in the Emergency Room with 18 h severe nausea and vomiting associated with a decrease in conscious level. He was seen initially by the Duty Surgical team who referred him to the Duty Medical Team. In the past, there was a poorly documented history of recurrent abdominal pain and polyuria. There was no history of therapeutic or ‘over-the-counter’ medication or vitamin supplementation. When admitted, he had a serum Na+ of 143 mmol/l (NR 135–145 mmol/l), K+ 3.6 mmol/l (NR 3.5–4.5 mmol/l), urea 16.6 mmol/l (NR 2.5–8.0 mmol/l), creatinine 51 μmol/l (NR 60–110 μmol/l). The serum Ca2+ was 5.14 mmol/l (NR 2.2–2.6 mmol/l) with a Pi of 1.75 mmol/l (NR 0.8–1.4 mmol/l) and an albumin of 49 g/l (NR 38–48 g/l) and an alkaline phosphatase of 231 IU/l (NR 30–100 IU/l).

Question 1: List the possible causes of hypercalcemia and outline what investigations you would perform.

Question 2: How should he be treated?

Clinical Case Study Q5.2

A 53-year-old woman was seen in the Outpatient Clinic complaining of muscle weakness and, in particular, of difficulty going upstairs. Her past medical history was noteworthy for the fact that she had undergone jejuno-ileal bypass surgery 25 years previously for morbid obesity (Box Q5.2). Over the previous 3 years, she had suffered from several episodes of renal colic due to kidney stones that on analysis were shown to be predominantly of calcium oxalate. She was 1.63 m tall and prior to the bypass operation she had weighed 125 kg. At the time of her regular outpatient clinic visit, she weighed 78 kg. Direct questioning revealed that she had tripped over several times recently and that she had stopped driving her car at night. Serum biochemical tests revealed normal serum concentrations of Na+, K+, urea and creatinine. The serum Ca2+ was 2.03 mmol/l (NR 2.2–2.6 mmol/l) with a Pi of 0.8 mmol/l (NR 0.8–1.4 mmol/l), albumin of 38 g/l (NR 38–48 g/l), alkaline phosphatase of 143 IU/l (NR 30–100 IU/l).

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Box Q5.2

Jejuno-ileal bypass surgery for severe obesity. In this operation, the amount of small intestine available for digestion and absorption is markedly reduced. Fat malabsorption is nearly universal.

Question 1: Outline the synthesis and metabolism of vitamin D. How are her current symptoms related to her previous surgery?

Question 2: What other investigations would you perform?

Question 3: How would you treat her?

Clinical Case Study Q5.3

A 56-year-old previously well pub owner was seen in the Emergency Room with a 5 h history of central ominal pain and vomiting. He smoked 20 cigarettes and drank half a bottle of whisky a day during the course of his duties. He was placed ‘nil by mouth’, admitted under the surgeons and treated with intravenous fluids and nasogastric tube drainage of the stomach. The serum Na+ was 138 mmol/l (NR 135–145 mmol/l), the K+ 3.9 mmol/l (NR 3.5–4.5 mmol/l), the urea 11.0 mmol/l (NR 2.5–8 mmol/l) and creatinine 110 μmol/l (NR 50–110 μmol/l). The serum amylase was 1098 (NR<200 IU/l), the Ca2+ 2.43 mmol/l (NR 2.2–2.6 mmol/l), the Pi 1.1 mmol/l (NR 0.8–1.4 mmol/l) and the albumin 40 g/l (NR 38–48 g/l). A diagnosis of acute pancreatitis, probably secondary to alcohol, was made. Ultrasound examination of the abdomen revealed no signs of gall stones but evidence of pancreatic inflammation. Three days after admission, he complained of paresthesiae (‘tingling’) in his extremities accompanied by painful cramps in his hands. Trousseau's sign was positive (Box Q5.3). The serum Ca2+ was 1.45 mmol/l and the albumin 29 g/l.

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Box Q5.3

Trousseau's sign. In this test the sphygmomanometer cuff is inflated to 300 mmHg for 3 minutes. The carpopedal spasm is painful.

Question 1: List the causes of hypocalcemia. What process occurred over the first 3 days of the patient's admission to cause the precipitous fall in serum Ca2+ concentration?

Question 2: Which further investigations would you perform and how would you treat this patient?

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