http://informahealthcare.com/lab ISSN: 1040-8363 (print), 1549-781X (electronic) Crit Rev Clin Lab Sci, Early Online: 1–13 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10408363.2014.970266
REVIEW ARTICLE
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Laboratory approaches for the diagnosis and assessment of hypercalcemia Qing H. Meng and Elizabeth A. Wagar Department of Laboratory Medicine, MD Anderson Cancer Center, The University of Texas, Houston, TX, USA
Abstract
Keywords
Calcium, the fifth most common element in the body, plays major physiological functions. Measurement of blood calcium is one of the most commonly ordered laboratory tests in assessments of calcium homeostasis and disease diagnosis. Hypercalcemia is an increased level of calcium in the blood. This disorder is most commonly caused by primary hyperparathyroidism and malignancy. However, other less common causes of elevated calcium levels need to be considered when making a differential diagnosis. This review is intended to provide readers with a better understanding of calcium homeostasis and the causes and pathophysiology of hypercalcemia. Most importantly, this review describes useful approaches for laboratory scientists and clinicians to appropriately diagnose and assess hypercalcemia.
Calcium, diagnosis, hypercalcemia, hyperparathyroidism, malignancy History Received 19 August 2014 Revised 10 September 2014 Accepted 24 September 2014 Published online 17 October 2014
Abbreviations: 1,25-(OH)2D: 1,25-dihydroxyvitamin D; 25(OH)D: 25-hydroxyvitamin D; AA: amino acid; cAMP: cyclic adenosine monophosphate; ECF: extracellular fluid; FBH: familial benign hypercalcemia; FHH: familial hypocalciuric hypercalcemia; HHM: humoral hypercalcemia of malignancy; MEN: multiple endocrine neoplasia; MTC: medullary thyroid carcinoma; PTH: parathyroid hormone; PTH-rP: parathyroid hormone-related protein; UCCR: urinary calcium/creatinine ratio
Introduction Calcium, the fifth most common element in the body, plays major physiological functions. Measurement of blood calcium is one of the most commonly ordered laboratory tests when assessing calcium homeostasis and diagnosing a disease. Hypercalcemia is an increased level of calcium in the blood and is most commonly caused by primary hyperparathyroidism and malignancy due to inappropriately elevated levels of parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D) and PTH-related protein (PTH-rP), and to local osteolysis. Other causes of elevated calcium are less common but should be considered when generating a differential diagnosis. The prevalence of hypercalcemia depends on the population studied. In the general population, the prevalence of hypercalcemia is approximately 1%; however, this rate can increase to 3% in hospitalized patients1,2. Hypercalcemia affects almost every organ system in the body, but it particularly affects the central nervous system, cardiovascular Referee Dr. Geoffrey Baird, Department of Laboratory Medicine, University of Washington, Seattle WA USA Address for correspondence: Dr. Qing H. Meng, Department of Laboratory Medicine, The University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030-4009, USA. E-mail: [email protected]
system, bones and kidneys. Severe hypercalcemia can be lifethreatening. Hypercalcemia needs to be investigated thoroughly and managed appropriately. This review is intended to provide readers with a better understanding of calcium homeostasis and the causes and pathophysiology of hypercalcemia, and, most importantly, to present a useful approach for laboratory scientists and clinicians to appropriately diagnose and assess hypercalcemia.
Calcium biochemistry and homeostasis The average human body contains approximately 1 kg of calcium3,4. It is a fundamental element that is the main constituent in bone, acts an essential cofactor for many enzymes, and is necessary to form electrical gradients across membranes. Under normal physiologic conditions, the concentration of calcium in serum and in cells is tightly controlled. Calcium is distributed to three main compartments: the skeleton, soft tissues and extracellular fluid (ECF)5. Approximately 99% of the body’s calcium is found in the skeleton, whereas soft tissues and ECF contain 1.0% and 0.1%, respectively, of the total calcium. Physiologically, calcium is distributed intracellularly and extracellularly. Under normal physiological conditions, plasma concentration is between 2.15 and 2.55 mmol/L (8.6–10.2 mg/dL).
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Figure 1. Distribution of calcium. Calcium
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Extracellular fluid 2.15-2.55 mmol/L (8.6-10.2 mg/dL)
Protein bound 40%
Albumin 80%
The intracellular cytosolic concentration of calcium is only around 0.1 mmol/L, which is less than 1/1000 of that in the ECF4. In plasma, calcium exists in three states: free or ionized calcium, which accounts for approximately 50% of total plasma calcium, protein-bound calcium (40% of total plasma calcium) and calcium complexes with small anions (10% of total plasma calcium)4 (Figure 1). Only ionized calcium is considered physiologically active. Previous studies have suggested that ionized calcium is more sensitive and specific than total or corrected calcium for the diagnosis of hypercalcemia6. Overall, there is a good correlation between ionized calcium and total calcium in determining calcium homeostasis. Thus, plasma total calcium is usually used for determining calcium homeostasis. Of the protein-bound calcium, 80% is bound to albumin and 20% is bound to globulins7,8. Approximately 10% of plasma total calcium forms a complex with small diffusible inorganic and organic anions, including bicarbonate, lactate, phosphate and citrate. Because calcium is bound by serum proteins, total calcium levels are greatly affected by protein concentrations, especially albumin, while the ionized calcium concentration may be normal8. For every 10 g/L decrease in serum albumin below 40 g/L, measured serum calcium decreases by 0.20 mmol/L (0.8 mg/dL). Therefore, to correct for an albumin level of less than 40 g/L, one should add 0.20 mmol/L (0.8 mg/dL) to the measured value of calcium for each 10 g/L decrease in albumin. With this correction, an abnormally low serum calcium level may appear to be normal. The following equation is usually used for total corrected (adjusted) calcium: corrected calcium (mmol/L) ¼ measured total calcium (mmol/L) + 0.02 (40 albumin [g/L]), or corrected calcium (mg/dL) ¼ measured total calcium (mg/dL) + 0.8 (4 albumin [g/dL])4. As Endres indicated, this correction considers only the albumin concentration; other factors that can alter the binding are not included6. Therefore, the utility of this equation is limited. Indeed, the majority of laboratories in the United States and Canada do not report total corrected calcium. Some European laboratories do provide the
Intracellular fluid cytosol 0.1 µmol/L, ionized calcium 50-100 nmol/L
Ionized 50%
Complexed 10%
Globulin 20%
corrected calcium, where it is usually reported at each physician’s discretion6. As calcium binds to negatively charged sites on proteins, its binding is pH-dependent. Acidosis leads to a decrease in binding and an increase in ionized calcium. With each 0.1-unit increase in pH, there will be approximately a 0.05 mmol/L (0.2 mg/dL) increase in ionized calcium. Alkalosis, on the other hand, increases the binding of calcium to proteins and decreases ionized calcium. Changes in calcium ions are usually accompanied by changes in the total calcium in the ECF. Thus, calcium states in plasma can be changed based on the albumin concentration, the number of small anionic molecules and the acid–base status.
Regulation of calcium homeostasis The serum ionized calcium concentration is regulated by PTH, 1,25(OH)2D, calcitonin, and several other calciumregulating hormones. The primary mediator of the intracellular effects of calcium is the calcium-binding regulatory protein, calmodulin. Serum ionized calcium is maintained despite its expansive movements across the gut, bone, kidney and cells. The serum ionized calcium concentration is tightly regulated by PTH and 1,25(OH)2D9. In brief, three major hormones [PTH, 1,25(OH)2D and calcitonin] and three organs (bone, kidneys and intestine) are involved in calcium metabolism (Table 1, Figure 2). PTH and calcium homeostasis PTH is produced and secreted by the parathyroid glands (chief cells). The hormone is synthesized as a 115-amino acid (AA) precursor (pre-pro-PTH) cleaved to pro-PTH (90-AA) and then to the intact PTH (84-AA) in the circulation. The N-terminal region (1-34 AA) of intact PTH possesses the biological activity. The half-life of intact PTH is less than 5 min. PTH is stimulated by ionized calcium that is low by as little as 0.025 mmol/L (0.1 mg/dL) and inhibited by hypercalcemia via the binding of ionized calcium to
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Table 1. Regulation of calcium homeostasis. Hormones
Effect on bones
PTH
"Bone resorption
1,25 (OH)2 D
Weak effect on bone resorption #Bone resorption
Calcitonin
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Effect on intestine
"Ca reabsorption #PO4 reabsorption Weak effect on "Ca2+ reabsorption
"Ca2+ #PO4 "Ca2+, "PO4
No direct effects
#Ca2+, #PO4 reabsorption
#Ca2+, # PO4
↑ PTH ↑ PTH ↑ Bone resorption ↑Renal Ca2+ reabsorption
↑1,25(OH)2D
↑ Ca2+
↑ Ca2+
↑ Ca2+
↑ Serum Ca2+ Figure 2. PTH and calcium homeostasis.
calcium-sensing receptors on the surface of parathyroid cells9. The overall action of PTH is to (1) increase plasma Ca2+ levels and decrease plasma phosphate levels; (2) stimulate bone resorption so as to release calcium and phosphate from bones (PTH can directly activate PTH receptors on surface osteocytes or indirectly act on osteoclasts); (3) increase kidney distal tubule reabsorption of calcium and inhibit tubular reabsorption of phosphate; and (4) stimulate the conversion of 25-hydroxyvitamin D [25(OH)D] to 1,25(OH)2D in kidneys by activating the mitochondrial P450 enzyme 1-a-hydroxylase. Increased production of 1,25(OH)2D enhances intestinal absorption of calcium and phosphate, and to a small extent, bone resorption9.
1,25(OH)2D and calcium homeostasis Vitamin D is acquired from two sources: the skin (via ultraviolet radiation) and the diet. Vitamin D is also not a true ‘‘vitamin’’ because it can be synthesized de novo. On the other hand, vitamin D is not a classic hormone because it is not produced and secreted by an endocrine gland. Indeed, vitamin D is a true hormone that acts on calcium-binding proteins (calbindin) to facilitate calcium uptake by intestinal cells10. 1,25(OH)2D regulates calcium homeostasis in three
2+
Blood level
"1,25(OH)2D Indirectly "Ca2+, "PO4 absorption "Ca2+, "PO4 absorption
↓ Serum Ca2+
↑ GI Ca2+ absorption
Effect on kidneys
ways: (1) it increases calcium and phosphate absorption by the small intestine; (2) it weakly increases the resorption of calcium and phosphate from the skeleton through direct binding and activation of osteoblasts and indirect activation of osteoclasts; and (3) it weakly promotes the reabsorption of calcium by the kidney cells. In addition, 1,25(OH)2D stimulates osteocytic osteolysis and promotes bone calcification and mineralization11.
Calcitonin and calcium homeostasis Calcitonin, a 32-AA peptide, is produced by parafollicular cells of the thyroid gland (C-cells). Overall, the physiological role of calcitonin in normal Ca2+ control is not clearly understood. Calcitonin is secreted in direct response to serum hypercalcemia. The main function of calcitonin is to reduce plasma calcium and phosphate by inhibiting bone resorption, stimulating bone mineralization, reducing the reabsorption of calcium and phosphate in kidneys and antagonizing the effect of PTH on bones4. However, in comparison to PTH and 1,25(OH)2D, the role of calcitonin in the regulation of serum calcium levels in humans is minor. Measurements of serum calcitonin levels are, therefore, not useful in the diagnosis of disorders of calcium homeostasis. Chronic excess of calcitonin does not produce hypocalcemia, and the removal of parafollicular cells of the thyroid gland does not cause hypercalcemia. It is generally accepted that physiological calcium homeostasis is mainly regulated by PTH and 1,25(OH)2D4. Calcitonin is used clinically to treat hypercalcemia and certain bone diseases to suppress bone resorption. It is considered more important in regulating bone remodeling than in Ca2+ homeostasis4. The calcium level is also regulated by PTH-rP, gonadal and adrenal steroids, thyroid hormones, prostaglandins, somatomedins, various growth factors and cytokines. In certain pathological disorders, these mechanisms may become dominant4. Increased expression of fibroblast growth factor 23 has been reported in certain tumors12. Fibroblast growth factor 23 inhibits renal tubular phosphate reabsorption and renal conversion of 25(OH)D to 1,25(OH)2D. This may cause secondary hyperparathyroidism12,13. Circulating calcium is excreted via glomerular filtration and reabsorbed in the proximal tubule. Calcium reabsorption in the proximal tubule is affected by the tubular sodium concentration, whereas PTH induces calcium uptake in the distal tubule and the collecting duct. Excess calcium is excreted in the urine and feces. As PTH increases renal tubular reabsorption of calcium, one would expect patients
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with hyperparathyroidism to be hypocalciuric. However, high urinary calcium/creatinine ratios are found in most patients with primary hyperparathyroidism in the presence of overall highly elevated serum calcium, and less frequently in patients with hypercalcemia resulting from other causes.
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Physiology and pathophysiology Intracellular calcium has key roles in many important physiologic functions, including cell signaling as a second messenger, neural transmission, muscle contraction, enzymatic co-factor, hormone secretion, glycogen metabolism and cell division14. Extracellular calcium provides calcium ions for the maintenance of intracellular calcium, bone mineralization, blood coagulation and plasma membrane potential. Calcium stabilizes the plasma membranes and influences permeability and excitability. A decrease in the serum-free calcium concentration causes increased neuromuscular excitability and tetany while an increased concentration reduces neuromuscular excitability. Hypercalcemia is an abnormally high level of calcium in the blood. The cutoff value for hypercalcemia is not well defined. It is generally defined as a serum calcium concentration of 2.65 mmol/L (10.6 mg/dL) or higher on two occasions15. This disorder is most commonly caused by primary hyperparathyroidism or malignancy. Hypercalcemia of malignancy occurs in approximately 10% of patients with advanced cancers and is the most common life-threatening metabolic disorder during the late stages of cancer. The occurrence of hypercalcemia may rise to as high as 40% in some types of cancer, including breast cancer, lung cancer, multiple myeloma or other tumors with extensive metastasis to the bone. It may also occur in patients with head and neck cancer, cancer of unknown primary origin, lymphoma, leukemia, kidney cancer and gastrointestinal cancer. Individuals with mild or moderate hypercalcemia may have no symptoms or mild non-specific symptoms15–17. The symptoms may vary depending on the organ system(s) affected16,18. The gastrointestinal effects include nausea, vomiting, abdominal pain and constipation. Cardiac effects may include hypertension, bradycardia, shortened QT interval and enhanced sensitivity to digitalis. Renal effects include polyuria, nocturia, polydipsia, dehydration, nephrocalcinosis, renal stones and renal failure. Neurological manifestations include fatigue, lethargy, weakness, confusion, stupor and coma. Bone-related effects include bone pain, arthritis, osteoporosis and osteitis fibrosa cystica. Depending on the preliminary disease and clinical progression, the symptoms can vary but may present as one of ‘‘stones, bones, abdominal moans and psychic groans’’. These symptoms may be due directly to the hypercalcemia, to increased calcium and phosphate excretion, or to skeletal changes. With higher levels of hypercalcemia, patients may have anxiety, depression, personality changes and confusion. Those with more sudden onset and severe hypercalcemia may experience dramatic conditions that include confusion and coma and that may possibly lead quickly to death15,18. Serum calcium levels greater than approximately 3.75 mmol/L (15 mg/dL) are usually considered to be a medical emergency and must be treated aggressively.
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Hypercalcemia due to pre-analytical, analytical and post-analytical factors A number of pre-analytical factors can cause falsely elevated levels of plasma calcium. Patient preparation and specimen collection can significantly affect both free and total calcium measurements. Prolonged tourniquet application and venous occlusion are associated with increased protein-bound calcium and total calcium caused by the efflux of water from the vascular compartment19–22. This can lead to an increase in total calcium of 0.25 mmol/L (1.0 mg/dL)6. Repeated fist clenching or forearm exercise decreases blood pH and thus may increase ionized calcium23. Hyperventilation induces respiratory alkalosis and increases the binding of calcium to albumin and thus reduces the level of ionized calcium20,21,24. In contrast, exercise raises the level of serum ionized calcium because of the change in blood pH20,21,24. Changes in standing posture decrease intra-vascular water and increase protein, and thus increase total calcium concentrations19,25. Prolonged immobilization and bed rest increase both total and free calcium levels as a consequence of increased bone resorption26. Contamination with calcium from corks, glassware, tubes, drywall and water supply pipes can increase measured levels of calcium27. Although false-positive interferences with calcium are rare compared with false-negative interferences, potential analytical interference could occur depending on the methodology28,29. Reference intervals and discrepancies between measured total calcium, corrected total calcium and ionized calcium may affect result interpretations. Clinical causes of hypercalcemia Medical disorders associated with hypercalcemia are listed in Table 2. Of those, primary hyperparathyroidism and cancer account for 90% of cases of hypercalcemia30. In outpatients, approximately 90% of the cases are due to primary hyperparathyroidism, whereas malignancy is the most common cause of hypercalcemia in hospitalized patients6,30. Other common and rare causes are also listed in Table 2 and will be described briefly31–35. Primary hyperparathyroidism The excessive secretion of PTH due to parathyroid adenoma accounts for 80% of cases of primary hyperparathyroidism18,30. Parathyroid gland hyperplasia due to multiple endocrine neoplasia type 1 (MEN 1), MEN 2A, familial hyperparathyroidism, familial benign hypercalcemia (FBH) and parathyroid carcinoma (51%) also lead to increased secretion of PTH and hypercalcemia35–37. Both excess PTH and concomitant excessive production of 1,25(OH)2D are involved in the pathophysiology of primary hyperparathyroidism-induced hypercalcemia38,39. Excess PTH causes hypercalcemia by increasing bone resorption and renal calcium reabsorption. It also indirectly increases intestinal calcium absorption by increasing the synthesis of 1,25(OH)2D16,18. Research has shown that the incidence of hypercalcemia is higher in women than in men, at an approximately 3:1 ratio. Post-menopausal women have five times high incidence compared to the general population, which is about 0.4%40. Primary hyperparathyroidism is diagnosed based on the criteria indicated in Table 3 38,39.
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Table 2. Causes of hypercalcemia. Parathyroid hormone Primary hyperparathyroidism (adenoma, hyperplasia, carcinoma) Sporadic, familial, multiple endocrine neoplasia 1 (MEN 1) or MEN 2A Tertiary hyperparathyroidism Coexisting malignancy and primary hyperparathyroidism Ectopic PTH in malignancy Malignancy Humoral hypercalcemia of malignancy Parathyroid hormone-related protein (PTHrP) Local osteolysis of multiple myeloma Cancer: Breast cancer, lung cancer, hematological malignancy Vitamin D intoxication or related diseases Granulomatous disease: sarcoidosis, tuberculosis, berylliosis, coccidioidomycosis Vitamin D intoxication Vitamin D supplements, vitamin D metabolites or analogues Lymphoma Renal failure Chronic renal failure with treatment with calcium and 1,25(OH)2D or vitamin D analogs Recovery phase of rhabdomyolysis and acute renal failure Renal transplant Other endocrine diseases Hyperthyroidism (thyrotoxicosis) Adrenal insufficiency Acromegaly Pheochromocytoma Medications Thiazide diuretics Lithium-related release of PTH Milk-alkali syndrome (calcium and antacids) Vitamin A intoxication Theophylline Other disorders Immobilization with high bone turnover (e.g. Paget’s disease, bedridden child) Familial hypocalciuric hypercalcemia (FHH) Williams Beuren syndrome Hyperphosphatemia Acute hypomagnesemia
Table 3. Diagnosis of primary hyperparathyroidism. High serum calcium Elevated intact PTH Serum phosphate in the low-normal to mildly decreased range Serum ALP increased Hyperchloremic metabolic acidosis Urine calcium normal to slight increase 24-h urine calcium excretion Used to rule out FHH Values below 2.5 mmol/d (100 mg/24 h) or a calcium creatinine clearance ratio of 50.01 are suggestive of FHH
Hypercalcemia of malignancy Hypercalcemia occurs in 5–30% of patients with cancer41,42. Hypercalcemia associated with malignancy is the second most common cause of hypercalcemia overall and the most common cause of hypercalcemia in hospitalized patients2,31,43. Two types of cancer-associated hypercalcemia have been identified: local osteolytic hypercalcemia and humoral hypercalcemia. Osteolytic hypercalcemia, which accounts for 20% of all malignancy-related hypercalcemia, results from direct bone destruction by a primary or metastatic tumor. Humoral hypercalcemia caused by secretion
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of PTHrP is the most common cause of malignancy-related hypercalcemia. Humoral malignancy-related hypercalcemia accounts for approximately 80% of patients with hypercalcemia-associated malignancy43. The fundamental causes of malignancy-related hypercalcemia are increased bone resorption, bone destruction (osteolysis) and inadequate ability of the kidneys to resolve higher calcium levels. Humoral hypercalcemia occurs commonly in patients with squamous (head and neck, esophageal, lung or cervical), renal, bladder, ovarian and breast carcinomas. PTHrP is considered to be the critical mediator of humoral hypercalcemia of malignancy (HHM). Three forms of PTHrP have been identified, and all are about twice the size of native PTH. PTHrP has marked structural homology with PTH and can bind to the same receptor for PTH on target organs and tissues43–45. Thus, PTHrP reproduces the full spectrum of PTH activities. PTHrP causes biochemical abnormalities including hypercalcemia, hypophosphatemia and increased urinary cyclic AMP excretion, similar to the biological functions of PTH. However, patients with PTHrP-induced hypercalcemia have lower levels of PTH and 1,25(OH)2D, metabolic alkalosis instead of hyperchloremic metabolic acidosis, reduced distal tubular calcium reabsorption and uncoupling of bone formation4. Local osteolysis occurs most commonly in breast cancer, multiple myeloma, lymphoma and leukemia, and is mediated by cytokines and chemokines. Increased synthesis of 1,25(OH)2D by lymphomas also leads to hypercalcemia. Primary hyperparathyroidism can coexist with cancer in some cases46. In such cases, patients can have hypercalcemia and elevated PTH levels. Nearly all individuals with elevated PTH levels have coexisting primary hyperparathyroidism, as PTH is rarely produced ectopically43. Normally, healthy kidneys are able to filter out large amounts of calcium from the blood. However, patients with renal failure are unable to excrete the excess calcium, and this result in hypercalcemia31,47. Patients with chronic renal failure usually have secondary hyperparathyroidism due to reduced inorganic phosphate excretion and consequent phosphate retention. Elevated phosphate levels and hypocalcemia stimulate PTH synthesis and the cellular mass of the parathyroid glands to maintain calcium at low or normal levels. However, hypercalcemia may develop because of treatment with calcium, phosphate binders or vitamin D metabolites and analogues. In chronic renal failure, hypercalcemia occurs mainly due to tertiary hyperparathyroidism18,48, which develops in patients with longstanding secondary hyperparathyroidism; the secondary hyperparathyroidism stimulates parathyroid hyperplasia, which progresses to autonomous hyperparathyroidism. One indication of tertiary hyperparathyroidism is intractable hypercalcemia and/or an inability to control osteomalacia despite vitamin D therapy. The characteristic biochemical changes of primary, secondary and tertiary hyperparathyroidism are listed in Table 4. Because of tertiary hyperparathyroidism and the restoration of 1,25(OH)2D produced by the transplanted kidney, blood calcium levels are elevated following kidney transplantation. Blood calcium levels can also become elevated during the recovery phase of acute renal failure due to rebound increases in levels of PTH and 1,25(OH)2D.
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Table 4. The characteristic biochemical changes of primary, secondary and tertiary hyperparathyroidism. Type Primary HPT Secondary HPT Tertiary HPT
Calcium
Phosphate
PTH
1,25(OH)2D
" #"
# " "
" "" ""
" # #
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MEN Primary hyperparathyroidism is a common feature of the rare MEN syndromes types 1 and 2A49. MEN is a group of syndromes involving two or more tumor types of the endocrine system. These tumors can be either benign or malignant. MEN 1 occurs in 1 in 30 000 persons. Approximately 95% of patients present with hyperparathyroidism, which is the most common and earliest endocrine manifestation. Other endocrine manifestations include gastrinoma (45%), pituitary tumor (25%) or facial angiofibroma (85%). MEN 1 usually involves multiple glands including the pituitary, parathyroid and pancreas. Postoperative hypoparathyroidism may develop due to extensive surgery. On the other hand, 50% of patients who undergo successful subtotal parathyroidectomy have recurrent hyperparathyroidism within 10 years50. MEN 2A generally involves endocrine organs such as the thyroid, parathyroid and adrenal glands. The most common clinical disorders associated with MEN 2A are medullary thyroid carcinoma (MTC; 95%), pheochromocytoma (50%) and primary hyperparathyroidism (20%). MEN 2A is primarily caused by the RET gene mutation (98%). The prevalence of MEN 2A is approximately 1 in 30 000–50 000 people.
Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant condition characterized by lifelong hypercalcemia, hypocalciuria and inappropriately elevated PTH levels. It is caused by loss-of-function mutations in the calcium-sensing receptor gene that needs higher serum calcium to shut off PTH secretion and increase renal tubular calcium reabsorption18,57,58. FHH is a benign condition, and patients with FHH are usually asymptomatic. It is easily misdiagnosed as primary hyperparathyroidism because both entities can manifest as hypercalcemia with an inappropriately normal or elevated level of PTH59,60. FHH must be differentiated from primary hyperparathyroidism, and this differentiation is further discussed below. Multiple myeloma associated hypercalcemia is mainly attributed to widespread tumor-induced bone destruction and osteoclastic bone resorption (osteolysis) mediated through lymphotoxin, interleukin-6, hepatocyte growth factor, receptor activator of nuclear factor-kB ligand, macrophage inflammatory protein-1a and tumor necrosis factors a and b61. Hypercalcemia occurs in most granulomatous disorders, including sarcoidosis, tuberculosis, leprosy, histoplasmosis, berylliosis, eosinophilic granuloma and silicone-induced granuloma. High serum calcium levels are seen in about 10% of patients with sarcoidosis; hypercalciuria is about three times more frequent. Other conditions such as fungal granulomas and lymphomas are also associated with disorders of calcium metabolism. These abnormalities in calcium metabolism are due to increased ectopic production of 1,25-(OH)2D3 (calcitriol) by activated macrophages in pulmonary alveoli and granulomatous inflammation and infection; calcitriol in turn increases intestinal calcium absorption18,32,34,62,63.
Non-parathyroid-related diseases Patients with thyrotoxicosis usually have mild hypercalcemia33. Thyrotoxicosis can occur in patients with primary hyperthyroidism, with hypothyroidism treated with thyroid hormones, and with thyroid cancer treated with thyroid hormones. Excessive osteoclast activity and thyroid hormonemediated bone resorption during thyrotoxicosis can result in hypercalcemia. However, once the thyroid function is corrected, calcium levels return to normal. Patients with pheochromocytoma can present with hypercalcemia; this is mainly attributed to the production of PTHrP and coexisting primary hyperparathyroidism in MEN 2A51. Addison’s disease can be the cause of hypercalcemia. Multiple factors appear to contribute to the hypercalcemia, including increased bone resorption, volume contraction, hemoconcentration, decreased glomerular filtration, increased proximal tubular calcium reabsorption and perhaps increased binding of calcium to serum proteins52,53. Cortisol administration reverses the hypercalcemia within several days. Hypercalcemia has also been reported in patients with secondary adrenal insufficiency due to lymphocytic hypophysitis54. Vasoactive intestinal polypeptide hormone-producing tumors can coexist with primary hyperparathyroidism in MEN 155. The typical features of patients with vasoactive intestinal polypeptide-producing tumors include large-volume diarrhea, hypokalemia and hypercalcemia56. Plasma vasoactive intestinal polypeptide levels are usually markedly elevated in these patients.
Medications Hypercalcemia caused by medications is also seen clinically. Thiazide diuretics can cause hypercalcemia by increasing renal tubular reabsorption of calcium64. Other changes such as metabolic alkalosis, increased intestinal calcium absorption and hemoconcentration that are associated with thiazide diuretics may also contribute to an increase in serum calcium. Hypercalcemia in primary hyperparathyroidism can be exacerbated by thiazide. Increased intake of calcium and calcium-containing antacids can cause hypercalcemia, metabolic alkalosis and renal insufficiency (milk-alkali syndrome)65–67. The metabolic alkalosis augments the hypercalcemia by directly stimulating calcium reabsorption in the distal tubule, thereby diminishing calcium excretion. During the past decade, vitamin D has become a hot topic both publically and medically. There is an increasing concern and debate on inappropriate vitamin D supplementation. High vitamin D levels increase intestinal calcium absorption and bone resorption. Hypercalcemia can be caused by vitamin D intoxication due to excessive exogenous intake or ingestion of vitamin D, its metabolites or vitamin D analogues68–70. Recently, a rare case of hypercalcemia was caused by elevated 1,25(OH)2D due to a mutation in CYP24A1 that failed to metabolize 1,25(OH)2D71. Hypercalcemia can be occasionally caused by taking excessive amounts of vitamin A72–74. Increased intestinal
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calcium absorption and increased interleukin-6-mediated bone resorption are potential mechanisms of vitamin A-associated hypercalcemia. Theophylline possesses a beta-adrenergic effect and induces the release of cyclic adenosine monophosphate (cAMP), which can stimulate the release of PTH and cause hyperparathyroidism. Theophylline can also cause dehydration. Together, these effects contribute to high serum calcium levels75. Patients receiving chronic lithium therapy often develop mild hypercalcemia, most likely due to increased secretion of PTH and decreased urinary calcium excretion76–78. Lithium can also unmask previously unrecognized mild hyperparathyroidism. Conversely, lithium can increase serum PTH concentrations without raising serum calcium concentrations. In emergency medicine, patients who have overdosed on calcium channel blockers may be given large amount of calcium to raise their ionized calcium concentrations more than two-fold. In such case, high calcium can be iatrogenic, and in fact helpful. Other rare conditions Several rare conditions are also linked to hypercalcemia. Williams–Beuren syndrome is a genetic disease associated with hypercalcemia79. This syndrome results from the deletion of contiguous genes on chromosome 7; the mechanisms are not known, but increased sensitivity to vitamin D is thought to play a role. Jansen’s metaphyseal chondrodysplasia, a rare form of dwarfism, is associated with asymptomatic but significant hypercalcemia and hypophosphatemia; the primary defect in this condition is a mutation in the PTH-PTH-rP receptor gene, resulting in continuous activation of the receptor at normal or low levels of PTH secretion80. Hypercalcemia has been described in infants with congenital lactase deficiency81; the hypercalcemia may be due to an increase in calcium absorption in the ileum in the presence of non-hydrolyzed lactose, and it resolves rapidly after the institution of a lactose-free diet. Recently, hypercalcemia was reported to be associated with posterior reversible encephalopathy syndrome in a 38-year-old woman who presented with altered mental status82. Factors increasing bone resorption, including immobilization, Paget’s disease and the administration of estrogen, can cause hypercalcemia4,18,34. Hypercalcemia can also develop during the recovery phase of rhabdomyolysis, when calcium that was deposited in the injured muscles is mobilized back to the extracellular space83. Hyperphosphatemia or acute hypomagnesemia stimulates PTH secretion, resulting in hypercalcemia. Hypercalcemia can occur in patients with hyperalbuminemia due to severe dehydration or with multiple myeloma who have a calcium-binding paraprotein. This phenomenon is called pseudohypercalcemia (or factitious hypercalcemia), since the patient has a normal ionized serum calcium concentration4. Hypercalcemia can be classified as mild, moderate or severe based on adjusted serum calcium concentrations15. Mild hypercalcemia is an adjusted serum calcium concentration of 2.65–3.00 mmol/L (10.6–12.0 mg/dL). Moderate hypercalcemia is an adjusted serum calcium concentration of 3.01–3.40 mmol/L (12.04–13.6 mg/dL). Severe hypercalcemia is a serum calcium concentration of greater than
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3.40 mmol/L (13.6 mg/dL). Hypercalcemic crisis represents a life-threatening emergency84. It is usually defined as a serum calcium concentration of 3.5 mmol/L (14.0 mg/dL) or higher85. The pathogenesis of hypercalcemic crisis is poorly understood. In some cases, the crisis has been ascribed to infarction of a parathyroid adenoma, usually with severely elevated serum calcium levels (43.8 mmol/L or 15.2 mg/dL) and a PTH concentration that is 20 times the upper limit of normal. Most patients experience cardiovascular, neurologic, gastrointestinal and renal manifestations of hypercalcemia. Immediate and effective therapy directed toward the pathophysiology of hypercalcemia is essential.
Laboratory assessment and diagnosis of hypercalcemia Clinically, an investigation into hypercalcemia should start with its underlying causes. Hypercalcemia usually develops in the context of known disease, which is the apparent cause. Sometimes, there may be multiple causes, the details and clinical features of which are described in the preceding sections. Although the symptoms and signs of hypercalcemia are similar, the clinical findings of the associated primary disorders are different and can be the key for differentiating the etiology of hypercalcemia. For instance, chronic hypercalcemia in an asymptomatic, post-menopausal woman may suggest the diagnosis of primary hyperparathyroidism. Patients with malignancy-associated hypercalcemia often have rapidly increasing concentrations of calcium and apparent symptoms. Laboratory testing plays a pivotal role in the assessment of hypercalcemia to confirm the diagnosis of hypercalcemia and to differentiate between primary hyperparathyroidism, FHH and non-PTH mediated hypercalcemia. International societies have issued guidelines and recommendations on the diagnosis and differential diagnosis of primary hyperparathyroidism9,86–91. A laboratory investigation algorithm, based on published evidence, was developed in our laboratory for the assessment of hypercalcemia (Figure 3). Measurement of blood calcium Methods used for quantifying total and ionized calcium levels in the blood are well established and readily available in clinical laboratories4. Ionized calcium is usually measured on blood gas analyzers via calcium ion-selective electrodes. Ionized calcium is the biologically active fraction of blood calcium; it is tightly regulated by PTH and 1,25(OH)2D, and thus is the best indicator of calcium status. Ionized calcium measurements have been recommended because these measurements avoid the consequences of delayed treatment and the cost of working up patients with misleading total calcium results6,92. Various methods have been described for total calcium measurement. At present, only photometry, calcium ion-selective electrodes and, very rarely, atomic absorption spectrophotometry are used in clinical laboratories for measuring serum and urine total calcium4. Current methods for measuring both total and ionized calcium are subject to analytical interference and errors. The analytical accuracy and the degree of analytical interference vary with methodologies. Variations in the patient’s biology, preparation and specimen
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Hypercalcemia (Serum calcium >10.2 mg/dL) Clinical evaluation and exclusion of medication (thiazide, lithium etc.)
Hypercalcemia confirmed (Serum calcium, albumin, ionized calcium)
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Intact PTH VitD/vitA excess, Cancer, Milkalkali syndrome Adrenal insufficiency hyperthyroidism
Low
Normal
PTHrP
24-hour urine calcium /creatinine ratio
Low or normal
1,25(OH)2D
Low
Cancer
High
Primary hyperparathyroidism
High
Cancer
24-h urine calcium Ratio0.02 High>250mg
Primary HPT, MEN 1 or 2a, Tertiary HPT
Lymphoma, sarcoidosis
Low
REVIEW ARTICLE
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Laboratory approaches for the diagnosis and assessment of hypercalcemia Qing H. Meng and Elizabeth A. Wagar Department of Laboratory Medicine, MD Anderson Cancer Center, The University of Texas, Houston, TX, USA
Abstract
Keywords
Calcium, the fifth most common element in the body, plays major physiological functions. Measurement of blood calcium is one of the most commonly ordered laboratory tests in assessments of calcium homeostasis and disease diagnosis. Hypercalcemia is an increased level of calcium in the blood. This disorder is most commonly caused by primary hyperparathyroidism and malignancy. However, other less common causes of elevated calcium levels need to be considered when making a differential diagnosis. This review is intended to provide readers with a better understanding of calcium homeostasis and the causes and pathophysiology of hypercalcemia. Most importantly, this review describes useful approaches for laboratory scientists and clinicians to appropriately diagnose and assess hypercalcemia.
Calcium, diagnosis, hypercalcemia, hyperparathyroidism, malignancy History Received 19 August 2014 Revised 10 September 2014 Accepted 24 September 2014 Published online 17 October 2014
Abbreviations: 1,25-(OH)2D: 1,25-dihydroxyvitamin D; 25(OH)D: 25-hydroxyvitamin D; AA: amino acid; cAMP: cyclic adenosine monophosphate; ECF: extracellular fluid; FBH: familial benign hypercalcemia; FHH: familial hypocalciuric hypercalcemia; HHM: humoral hypercalcemia of malignancy; MEN: multiple endocrine neoplasia; MTC: medullary thyroid carcinoma; PTH: parathyroid hormone; PTH-rP: parathyroid hormone-related protein; UCCR: urinary calcium/creatinine ratio
Introduction Calcium, the fifth most common element in the body, plays major physiological functions. Measurement of blood calcium is one of the most commonly ordered laboratory tests when assessing calcium homeostasis and diagnosing a disease. Hypercalcemia is an increased level of calcium in the blood and is most commonly caused by primary hyperparathyroidism and malignancy due to inappropriately elevated levels of parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D) and PTH-related protein (PTH-rP), and to local osteolysis. Other causes of elevated calcium are less common but should be considered when generating a differential diagnosis. The prevalence of hypercalcemia depends on the population studied. In the general population, the prevalence of hypercalcemia is approximately 1%; however, this rate can increase to 3% in hospitalized patients1,2. Hypercalcemia affects almost every organ system in the body, but it particularly affects the central nervous system, cardiovascular Referee Dr. Geoffrey Baird, Department of Laboratory Medicine, University of Washington, Seattle WA USA Address for correspondence: Dr. Qing H. Meng, Department of Laboratory Medicine, The University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030-4009, USA. E-mail: [email protected]
system, bones and kidneys. Severe hypercalcemia can be lifethreatening. Hypercalcemia needs to be investigated thoroughly and managed appropriately. This review is intended to provide readers with a better understanding of calcium homeostasis and the causes and pathophysiology of hypercalcemia, and, most importantly, to present a useful approach for laboratory scientists and clinicians to appropriately diagnose and assess hypercalcemia.
Calcium biochemistry and homeostasis The average human body contains approximately 1 kg of calcium3,4. It is a fundamental element that is the main constituent in bone, acts an essential cofactor for many enzymes, and is necessary to form electrical gradients across membranes. Under normal physiologic conditions, the concentration of calcium in serum and in cells is tightly controlled. Calcium is distributed to three main compartments: the skeleton, soft tissues and extracellular fluid (ECF)5. Approximately 99% of the body’s calcium is found in the skeleton, whereas soft tissues and ECF contain 1.0% and 0.1%, respectively, of the total calcium. Physiologically, calcium is distributed intracellularly and extracellularly. Under normal physiological conditions, plasma concentration is between 2.15 and 2.55 mmol/L (8.6–10.2 mg/dL).
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Figure 1. Distribution of calcium. Calcium
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Extracellular fluid 2.15-2.55 mmol/L (8.6-10.2 mg/dL)
Protein bound 40%
Albumin 80%
The intracellular cytosolic concentration of calcium is only around 0.1 mmol/L, which is less than 1/1000 of that in the ECF4. In plasma, calcium exists in three states: free or ionized calcium, which accounts for approximately 50% of total plasma calcium, protein-bound calcium (40% of total plasma calcium) and calcium complexes with small anions (10% of total plasma calcium)4 (Figure 1). Only ionized calcium is considered physiologically active. Previous studies have suggested that ionized calcium is more sensitive and specific than total or corrected calcium for the diagnosis of hypercalcemia6. Overall, there is a good correlation between ionized calcium and total calcium in determining calcium homeostasis. Thus, plasma total calcium is usually used for determining calcium homeostasis. Of the protein-bound calcium, 80% is bound to albumin and 20% is bound to globulins7,8. Approximately 10% of plasma total calcium forms a complex with small diffusible inorganic and organic anions, including bicarbonate, lactate, phosphate and citrate. Because calcium is bound by serum proteins, total calcium levels are greatly affected by protein concentrations, especially albumin, while the ionized calcium concentration may be normal8. For every 10 g/L decrease in serum albumin below 40 g/L, measured serum calcium decreases by 0.20 mmol/L (0.8 mg/dL). Therefore, to correct for an albumin level of less than 40 g/L, one should add 0.20 mmol/L (0.8 mg/dL) to the measured value of calcium for each 10 g/L decrease in albumin. With this correction, an abnormally low serum calcium level may appear to be normal. The following equation is usually used for total corrected (adjusted) calcium: corrected calcium (mmol/L) ¼ measured total calcium (mmol/L) + 0.02 (40 albumin [g/L]), or corrected calcium (mg/dL) ¼ measured total calcium (mg/dL) + 0.8 (4 albumin [g/dL])4. As Endres indicated, this correction considers only the albumin concentration; other factors that can alter the binding are not included6. Therefore, the utility of this equation is limited. Indeed, the majority of laboratories in the United States and Canada do not report total corrected calcium. Some European laboratories do provide the
Intracellular fluid cytosol 0.1 µmol/L, ionized calcium 50-100 nmol/L
Ionized 50%
Complexed 10%
Globulin 20%
corrected calcium, where it is usually reported at each physician’s discretion6. As calcium binds to negatively charged sites on proteins, its binding is pH-dependent. Acidosis leads to a decrease in binding and an increase in ionized calcium. With each 0.1-unit increase in pH, there will be approximately a 0.05 mmol/L (0.2 mg/dL) increase in ionized calcium. Alkalosis, on the other hand, increases the binding of calcium to proteins and decreases ionized calcium. Changes in calcium ions are usually accompanied by changes in the total calcium in the ECF. Thus, calcium states in plasma can be changed based on the albumin concentration, the number of small anionic molecules and the acid–base status.
Regulation of calcium homeostasis The serum ionized calcium concentration is regulated by PTH, 1,25(OH)2D, calcitonin, and several other calciumregulating hormones. The primary mediator of the intracellular effects of calcium is the calcium-binding regulatory protein, calmodulin. Serum ionized calcium is maintained despite its expansive movements across the gut, bone, kidney and cells. The serum ionized calcium concentration is tightly regulated by PTH and 1,25(OH)2D9. In brief, three major hormones [PTH, 1,25(OH)2D and calcitonin] and three organs (bone, kidneys and intestine) are involved in calcium metabolism (Table 1, Figure 2). PTH and calcium homeostasis PTH is produced and secreted by the parathyroid glands (chief cells). The hormone is synthesized as a 115-amino acid (AA) precursor (pre-pro-PTH) cleaved to pro-PTH (90-AA) and then to the intact PTH (84-AA) in the circulation. The N-terminal region (1-34 AA) of intact PTH possesses the biological activity. The half-life of intact PTH is less than 5 min. PTH is stimulated by ionized calcium that is low by as little as 0.025 mmol/L (0.1 mg/dL) and inhibited by hypercalcemia via the binding of ionized calcium to
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Table 1. Regulation of calcium homeostasis. Hormones
Effect on bones
PTH
"Bone resorption
1,25 (OH)2 D
Weak effect on bone resorption #Bone resorption
Calcitonin
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Effect on intestine
"Ca reabsorption #PO4 reabsorption Weak effect on "Ca2+ reabsorption
"Ca2+ #PO4 "Ca2+, "PO4
No direct effects
#Ca2+, #PO4 reabsorption
#Ca2+, # PO4
↑ PTH ↑ PTH ↑ Bone resorption ↑Renal Ca2+ reabsorption
↑1,25(OH)2D
↑ Ca2+
↑ Ca2+
↑ Ca2+
↑ Serum Ca2+ Figure 2. PTH and calcium homeostasis.
calcium-sensing receptors on the surface of parathyroid cells9. The overall action of PTH is to (1) increase plasma Ca2+ levels and decrease plasma phosphate levels; (2) stimulate bone resorption so as to release calcium and phosphate from bones (PTH can directly activate PTH receptors on surface osteocytes or indirectly act on osteoclasts); (3) increase kidney distal tubule reabsorption of calcium and inhibit tubular reabsorption of phosphate; and (4) stimulate the conversion of 25-hydroxyvitamin D [25(OH)D] to 1,25(OH)2D in kidneys by activating the mitochondrial P450 enzyme 1-a-hydroxylase. Increased production of 1,25(OH)2D enhances intestinal absorption of calcium and phosphate, and to a small extent, bone resorption9.
1,25(OH)2D and calcium homeostasis Vitamin D is acquired from two sources: the skin (via ultraviolet radiation) and the diet. Vitamin D is also not a true ‘‘vitamin’’ because it can be synthesized de novo. On the other hand, vitamin D is not a classic hormone because it is not produced and secreted by an endocrine gland. Indeed, vitamin D is a true hormone that acts on calcium-binding proteins (calbindin) to facilitate calcium uptake by intestinal cells10. 1,25(OH)2D regulates calcium homeostasis in three
2+
Blood level
"1,25(OH)2D Indirectly "Ca2+, "PO4 absorption "Ca2+, "PO4 absorption
↓ Serum Ca2+
↑ GI Ca2+ absorption
Effect on kidneys
ways: (1) it increases calcium and phosphate absorption by the small intestine; (2) it weakly increases the resorption of calcium and phosphate from the skeleton through direct binding and activation of osteoblasts and indirect activation of osteoclasts; and (3) it weakly promotes the reabsorption of calcium by the kidney cells. In addition, 1,25(OH)2D stimulates osteocytic osteolysis and promotes bone calcification and mineralization11.
Calcitonin and calcium homeostasis Calcitonin, a 32-AA peptide, is produced by parafollicular cells of the thyroid gland (C-cells). Overall, the physiological role of calcitonin in normal Ca2+ control is not clearly understood. Calcitonin is secreted in direct response to serum hypercalcemia. The main function of calcitonin is to reduce plasma calcium and phosphate by inhibiting bone resorption, stimulating bone mineralization, reducing the reabsorption of calcium and phosphate in kidneys and antagonizing the effect of PTH on bones4. However, in comparison to PTH and 1,25(OH)2D, the role of calcitonin in the regulation of serum calcium levels in humans is minor. Measurements of serum calcitonin levels are, therefore, not useful in the diagnosis of disorders of calcium homeostasis. Chronic excess of calcitonin does not produce hypocalcemia, and the removal of parafollicular cells of the thyroid gland does not cause hypercalcemia. It is generally accepted that physiological calcium homeostasis is mainly regulated by PTH and 1,25(OH)2D4. Calcitonin is used clinically to treat hypercalcemia and certain bone diseases to suppress bone resorption. It is considered more important in regulating bone remodeling than in Ca2+ homeostasis4. The calcium level is also regulated by PTH-rP, gonadal and adrenal steroids, thyroid hormones, prostaglandins, somatomedins, various growth factors and cytokines. In certain pathological disorders, these mechanisms may become dominant4. Increased expression of fibroblast growth factor 23 has been reported in certain tumors12. Fibroblast growth factor 23 inhibits renal tubular phosphate reabsorption and renal conversion of 25(OH)D to 1,25(OH)2D. This may cause secondary hyperparathyroidism12,13. Circulating calcium is excreted via glomerular filtration and reabsorbed in the proximal tubule. Calcium reabsorption in the proximal tubule is affected by the tubular sodium concentration, whereas PTH induces calcium uptake in the distal tubule and the collecting duct. Excess calcium is excreted in the urine and feces. As PTH increases renal tubular reabsorption of calcium, one would expect patients
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with hyperparathyroidism to be hypocalciuric. However, high urinary calcium/creatinine ratios are found in most patients with primary hyperparathyroidism in the presence of overall highly elevated serum calcium, and less frequently in patients with hypercalcemia resulting from other causes.
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Physiology and pathophysiology Intracellular calcium has key roles in many important physiologic functions, including cell signaling as a second messenger, neural transmission, muscle contraction, enzymatic co-factor, hormone secretion, glycogen metabolism and cell division14. Extracellular calcium provides calcium ions for the maintenance of intracellular calcium, bone mineralization, blood coagulation and plasma membrane potential. Calcium stabilizes the plasma membranes and influences permeability and excitability. A decrease in the serum-free calcium concentration causes increased neuromuscular excitability and tetany while an increased concentration reduces neuromuscular excitability. Hypercalcemia is an abnormally high level of calcium in the blood. The cutoff value for hypercalcemia is not well defined. It is generally defined as a serum calcium concentration of 2.65 mmol/L (10.6 mg/dL) or higher on two occasions15. This disorder is most commonly caused by primary hyperparathyroidism or malignancy. Hypercalcemia of malignancy occurs in approximately 10% of patients with advanced cancers and is the most common life-threatening metabolic disorder during the late stages of cancer. The occurrence of hypercalcemia may rise to as high as 40% in some types of cancer, including breast cancer, lung cancer, multiple myeloma or other tumors with extensive metastasis to the bone. It may also occur in patients with head and neck cancer, cancer of unknown primary origin, lymphoma, leukemia, kidney cancer and gastrointestinal cancer. Individuals with mild or moderate hypercalcemia may have no symptoms or mild non-specific symptoms15–17. The symptoms may vary depending on the organ system(s) affected16,18. The gastrointestinal effects include nausea, vomiting, abdominal pain and constipation. Cardiac effects may include hypertension, bradycardia, shortened QT interval and enhanced sensitivity to digitalis. Renal effects include polyuria, nocturia, polydipsia, dehydration, nephrocalcinosis, renal stones and renal failure. Neurological manifestations include fatigue, lethargy, weakness, confusion, stupor and coma. Bone-related effects include bone pain, arthritis, osteoporosis and osteitis fibrosa cystica. Depending on the preliminary disease and clinical progression, the symptoms can vary but may present as one of ‘‘stones, bones, abdominal moans and psychic groans’’. These symptoms may be due directly to the hypercalcemia, to increased calcium and phosphate excretion, or to skeletal changes. With higher levels of hypercalcemia, patients may have anxiety, depression, personality changes and confusion. Those with more sudden onset and severe hypercalcemia may experience dramatic conditions that include confusion and coma and that may possibly lead quickly to death15,18. Serum calcium levels greater than approximately 3.75 mmol/L (15 mg/dL) are usually considered to be a medical emergency and must be treated aggressively.
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Hypercalcemia due to pre-analytical, analytical and post-analytical factors A number of pre-analytical factors can cause falsely elevated levels of plasma calcium. Patient preparation and specimen collection can significantly affect both free and total calcium measurements. Prolonged tourniquet application and venous occlusion are associated with increased protein-bound calcium and total calcium caused by the efflux of water from the vascular compartment19–22. This can lead to an increase in total calcium of 0.25 mmol/L (1.0 mg/dL)6. Repeated fist clenching or forearm exercise decreases blood pH and thus may increase ionized calcium23. Hyperventilation induces respiratory alkalosis and increases the binding of calcium to albumin and thus reduces the level of ionized calcium20,21,24. In contrast, exercise raises the level of serum ionized calcium because of the change in blood pH20,21,24. Changes in standing posture decrease intra-vascular water and increase protein, and thus increase total calcium concentrations19,25. Prolonged immobilization and bed rest increase both total and free calcium levels as a consequence of increased bone resorption26. Contamination with calcium from corks, glassware, tubes, drywall and water supply pipes can increase measured levels of calcium27. Although false-positive interferences with calcium are rare compared with false-negative interferences, potential analytical interference could occur depending on the methodology28,29. Reference intervals and discrepancies between measured total calcium, corrected total calcium and ionized calcium may affect result interpretations. Clinical causes of hypercalcemia Medical disorders associated with hypercalcemia are listed in Table 2. Of those, primary hyperparathyroidism and cancer account for 90% of cases of hypercalcemia30. In outpatients, approximately 90% of the cases are due to primary hyperparathyroidism, whereas malignancy is the most common cause of hypercalcemia in hospitalized patients6,30. Other common and rare causes are also listed in Table 2 and will be described briefly31–35. Primary hyperparathyroidism The excessive secretion of PTH due to parathyroid adenoma accounts for 80% of cases of primary hyperparathyroidism18,30. Parathyroid gland hyperplasia due to multiple endocrine neoplasia type 1 (MEN 1), MEN 2A, familial hyperparathyroidism, familial benign hypercalcemia (FBH) and parathyroid carcinoma (51%) also lead to increased secretion of PTH and hypercalcemia35–37. Both excess PTH and concomitant excessive production of 1,25(OH)2D are involved in the pathophysiology of primary hyperparathyroidism-induced hypercalcemia38,39. Excess PTH causes hypercalcemia by increasing bone resorption and renal calcium reabsorption. It also indirectly increases intestinal calcium absorption by increasing the synthesis of 1,25(OH)2D16,18. Research has shown that the incidence of hypercalcemia is higher in women than in men, at an approximately 3:1 ratio. Post-menopausal women have five times high incidence compared to the general population, which is about 0.4%40. Primary hyperparathyroidism is diagnosed based on the criteria indicated in Table 3 38,39.
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Table 2. Causes of hypercalcemia. Parathyroid hormone Primary hyperparathyroidism (adenoma, hyperplasia, carcinoma) Sporadic, familial, multiple endocrine neoplasia 1 (MEN 1) or MEN 2A Tertiary hyperparathyroidism Coexisting malignancy and primary hyperparathyroidism Ectopic PTH in malignancy Malignancy Humoral hypercalcemia of malignancy Parathyroid hormone-related protein (PTHrP) Local osteolysis of multiple myeloma Cancer: Breast cancer, lung cancer, hematological malignancy Vitamin D intoxication or related diseases Granulomatous disease: sarcoidosis, tuberculosis, berylliosis, coccidioidomycosis Vitamin D intoxication Vitamin D supplements, vitamin D metabolites or analogues Lymphoma Renal failure Chronic renal failure with treatment with calcium and 1,25(OH)2D or vitamin D analogs Recovery phase of rhabdomyolysis and acute renal failure Renal transplant Other endocrine diseases Hyperthyroidism (thyrotoxicosis) Adrenal insufficiency Acromegaly Pheochromocytoma Medications Thiazide diuretics Lithium-related release of PTH Milk-alkali syndrome (calcium and antacids) Vitamin A intoxication Theophylline Other disorders Immobilization with high bone turnover (e.g. Paget’s disease, bedridden child) Familial hypocalciuric hypercalcemia (FHH) Williams Beuren syndrome Hyperphosphatemia Acute hypomagnesemia
Table 3. Diagnosis of primary hyperparathyroidism. High serum calcium Elevated intact PTH Serum phosphate in the low-normal to mildly decreased range Serum ALP increased Hyperchloremic metabolic acidosis Urine calcium normal to slight increase 24-h urine calcium excretion Used to rule out FHH Values below 2.5 mmol/d (100 mg/24 h) or a calcium creatinine clearance ratio of 50.01 are suggestive of FHH
Hypercalcemia of malignancy Hypercalcemia occurs in 5–30% of patients with cancer41,42. Hypercalcemia associated with malignancy is the second most common cause of hypercalcemia overall and the most common cause of hypercalcemia in hospitalized patients2,31,43. Two types of cancer-associated hypercalcemia have been identified: local osteolytic hypercalcemia and humoral hypercalcemia. Osteolytic hypercalcemia, which accounts for 20% of all malignancy-related hypercalcemia, results from direct bone destruction by a primary or metastatic tumor. Humoral hypercalcemia caused by secretion
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of PTHrP is the most common cause of malignancy-related hypercalcemia. Humoral malignancy-related hypercalcemia accounts for approximately 80% of patients with hypercalcemia-associated malignancy43. The fundamental causes of malignancy-related hypercalcemia are increased bone resorption, bone destruction (osteolysis) and inadequate ability of the kidneys to resolve higher calcium levels. Humoral hypercalcemia occurs commonly in patients with squamous (head and neck, esophageal, lung or cervical), renal, bladder, ovarian and breast carcinomas. PTHrP is considered to be the critical mediator of humoral hypercalcemia of malignancy (HHM). Three forms of PTHrP have been identified, and all are about twice the size of native PTH. PTHrP has marked structural homology with PTH and can bind to the same receptor for PTH on target organs and tissues43–45. Thus, PTHrP reproduces the full spectrum of PTH activities. PTHrP causes biochemical abnormalities including hypercalcemia, hypophosphatemia and increased urinary cyclic AMP excretion, similar to the biological functions of PTH. However, patients with PTHrP-induced hypercalcemia have lower levels of PTH and 1,25(OH)2D, metabolic alkalosis instead of hyperchloremic metabolic acidosis, reduced distal tubular calcium reabsorption and uncoupling of bone formation4. Local osteolysis occurs most commonly in breast cancer, multiple myeloma, lymphoma and leukemia, and is mediated by cytokines and chemokines. Increased synthesis of 1,25(OH)2D by lymphomas also leads to hypercalcemia. Primary hyperparathyroidism can coexist with cancer in some cases46. In such cases, patients can have hypercalcemia and elevated PTH levels. Nearly all individuals with elevated PTH levels have coexisting primary hyperparathyroidism, as PTH is rarely produced ectopically43. Normally, healthy kidneys are able to filter out large amounts of calcium from the blood. However, patients with renal failure are unable to excrete the excess calcium, and this result in hypercalcemia31,47. Patients with chronic renal failure usually have secondary hyperparathyroidism due to reduced inorganic phosphate excretion and consequent phosphate retention. Elevated phosphate levels and hypocalcemia stimulate PTH synthesis and the cellular mass of the parathyroid glands to maintain calcium at low or normal levels. However, hypercalcemia may develop because of treatment with calcium, phosphate binders or vitamin D metabolites and analogues. In chronic renal failure, hypercalcemia occurs mainly due to tertiary hyperparathyroidism18,48, which develops in patients with longstanding secondary hyperparathyroidism; the secondary hyperparathyroidism stimulates parathyroid hyperplasia, which progresses to autonomous hyperparathyroidism. One indication of tertiary hyperparathyroidism is intractable hypercalcemia and/or an inability to control osteomalacia despite vitamin D therapy. The characteristic biochemical changes of primary, secondary and tertiary hyperparathyroidism are listed in Table 4. Because of tertiary hyperparathyroidism and the restoration of 1,25(OH)2D produced by the transplanted kidney, blood calcium levels are elevated following kidney transplantation. Blood calcium levels can also become elevated during the recovery phase of acute renal failure due to rebound increases in levels of PTH and 1,25(OH)2D.
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Table 4. The characteristic biochemical changes of primary, secondary and tertiary hyperparathyroidism. Type Primary HPT Secondary HPT Tertiary HPT
Calcium
Phosphate
PTH
1,25(OH)2D
" #"
# " "
" "" ""
" # #
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MEN Primary hyperparathyroidism is a common feature of the rare MEN syndromes types 1 and 2A49. MEN is a group of syndromes involving two or more tumor types of the endocrine system. These tumors can be either benign or malignant. MEN 1 occurs in 1 in 30 000 persons. Approximately 95% of patients present with hyperparathyroidism, which is the most common and earliest endocrine manifestation. Other endocrine manifestations include gastrinoma (45%), pituitary tumor (25%) or facial angiofibroma (85%). MEN 1 usually involves multiple glands including the pituitary, parathyroid and pancreas. Postoperative hypoparathyroidism may develop due to extensive surgery. On the other hand, 50% of patients who undergo successful subtotal parathyroidectomy have recurrent hyperparathyroidism within 10 years50. MEN 2A generally involves endocrine organs such as the thyroid, parathyroid and adrenal glands. The most common clinical disorders associated with MEN 2A are medullary thyroid carcinoma (MTC; 95%), pheochromocytoma (50%) and primary hyperparathyroidism (20%). MEN 2A is primarily caused by the RET gene mutation (98%). The prevalence of MEN 2A is approximately 1 in 30 000–50 000 people.
Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant condition characterized by lifelong hypercalcemia, hypocalciuria and inappropriately elevated PTH levels. It is caused by loss-of-function mutations in the calcium-sensing receptor gene that needs higher serum calcium to shut off PTH secretion and increase renal tubular calcium reabsorption18,57,58. FHH is a benign condition, and patients with FHH are usually asymptomatic. It is easily misdiagnosed as primary hyperparathyroidism because both entities can manifest as hypercalcemia with an inappropriately normal or elevated level of PTH59,60. FHH must be differentiated from primary hyperparathyroidism, and this differentiation is further discussed below. Multiple myeloma associated hypercalcemia is mainly attributed to widespread tumor-induced bone destruction and osteoclastic bone resorption (osteolysis) mediated through lymphotoxin, interleukin-6, hepatocyte growth factor, receptor activator of nuclear factor-kB ligand, macrophage inflammatory protein-1a and tumor necrosis factors a and b61. Hypercalcemia occurs in most granulomatous disorders, including sarcoidosis, tuberculosis, leprosy, histoplasmosis, berylliosis, eosinophilic granuloma and silicone-induced granuloma. High serum calcium levels are seen in about 10% of patients with sarcoidosis; hypercalciuria is about three times more frequent. Other conditions such as fungal granulomas and lymphomas are also associated with disorders of calcium metabolism. These abnormalities in calcium metabolism are due to increased ectopic production of 1,25-(OH)2D3 (calcitriol) by activated macrophages in pulmonary alveoli and granulomatous inflammation and infection; calcitriol in turn increases intestinal calcium absorption18,32,34,62,63.
Non-parathyroid-related diseases Patients with thyrotoxicosis usually have mild hypercalcemia33. Thyrotoxicosis can occur in patients with primary hyperthyroidism, with hypothyroidism treated with thyroid hormones, and with thyroid cancer treated with thyroid hormones. Excessive osteoclast activity and thyroid hormonemediated bone resorption during thyrotoxicosis can result in hypercalcemia. However, once the thyroid function is corrected, calcium levels return to normal. Patients with pheochromocytoma can present with hypercalcemia; this is mainly attributed to the production of PTHrP and coexisting primary hyperparathyroidism in MEN 2A51. Addison’s disease can be the cause of hypercalcemia. Multiple factors appear to contribute to the hypercalcemia, including increased bone resorption, volume contraction, hemoconcentration, decreased glomerular filtration, increased proximal tubular calcium reabsorption and perhaps increased binding of calcium to serum proteins52,53. Cortisol administration reverses the hypercalcemia within several days. Hypercalcemia has also been reported in patients with secondary adrenal insufficiency due to lymphocytic hypophysitis54. Vasoactive intestinal polypeptide hormone-producing tumors can coexist with primary hyperparathyroidism in MEN 155. The typical features of patients with vasoactive intestinal polypeptide-producing tumors include large-volume diarrhea, hypokalemia and hypercalcemia56. Plasma vasoactive intestinal polypeptide levels are usually markedly elevated in these patients.
Medications Hypercalcemia caused by medications is also seen clinically. Thiazide diuretics can cause hypercalcemia by increasing renal tubular reabsorption of calcium64. Other changes such as metabolic alkalosis, increased intestinal calcium absorption and hemoconcentration that are associated with thiazide diuretics may also contribute to an increase in serum calcium. Hypercalcemia in primary hyperparathyroidism can be exacerbated by thiazide. Increased intake of calcium and calcium-containing antacids can cause hypercalcemia, metabolic alkalosis and renal insufficiency (milk-alkali syndrome)65–67. The metabolic alkalosis augments the hypercalcemia by directly stimulating calcium reabsorption in the distal tubule, thereby diminishing calcium excretion. During the past decade, vitamin D has become a hot topic both publically and medically. There is an increasing concern and debate on inappropriate vitamin D supplementation. High vitamin D levels increase intestinal calcium absorption and bone resorption. Hypercalcemia can be caused by vitamin D intoxication due to excessive exogenous intake or ingestion of vitamin D, its metabolites or vitamin D analogues68–70. Recently, a rare case of hypercalcemia was caused by elevated 1,25(OH)2D due to a mutation in CYP24A1 that failed to metabolize 1,25(OH)2D71. Hypercalcemia can be occasionally caused by taking excessive amounts of vitamin A72–74. Increased intestinal
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DOI: 10.3109/10408363.2014.970266
calcium absorption and increased interleukin-6-mediated bone resorption are potential mechanisms of vitamin A-associated hypercalcemia. Theophylline possesses a beta-adrenergic effect and induces the release of cyclic adenosine monophosphate (cAMP), which can stimulate the release of PTH and cause hyperparathyroidism. Theophylline can also cause dehydration. Together, these effects contribute to high serum calcium levels75. Patients receiving chronic lithium therapy often develop mild hypercalcemia, most likely due to increased secretion of PTH and decreased urinary calcium excretion76–78. Lithium can also unmask previously unrecognized mild hyperparathyroidism. Conversely, lithium can increase serum PTH concentrations without raising serum calcium concentrations. In emergency medicine, patients who have overdosed on calcium channel blockers may be given large amount of calcium to raise their ionized calcium concentrations more than two-fold. In such case, high calcium can be iatrogenic, and in fact helpful. Other rare conditions Several rare conditions are also linked to hypercalcemia. Williams–Beuren syndrome is a genetic disease associated with hypercalcemia79. This syndrome results from the deletion of contiguous genes on chromosome 7; the mechanisms are not known, but increased sensitivity to vitamin D is thought to play a role. Jansen’s metaphyseal chondrodysplasia, a rare form of dwarfism, is associated with asymptomatic but significant hypercalcemia and hypophosphatemia; the primary defect in this condition is a mutation in the PTH-PTH-rP receptor gene, resulting in continuous activation of the receptor at normal or low levels of PTH secretion80. Hypercalcemia has been described in infants with congenital lactase deficiency81; the hypercalcemia may be due to an increase in calcium absorption in the ileum in the presence of non-hydrolyzed lactose, and it resolves rapidly after the institution of a lactose-free diet. Recently, hypercalcemia was reported to be associated with posterior reversible encephalopathy syndrome in a 38-year-old woman who presented with altered mental status82. Factors increasing bone resorption, including immobilization, Paget’s disease and the administration of estrogen, can cause hypercalcemia4,18,34. Hypercalcemia can also develop during the recovery phase of rhabdomyolysis, when calcium that was deposited in the injured muscles is mobilized back to the extracellular space83. Hyperphosphatemia or acute hypomagnesemia stimulates PTH secretion, resulting in hypercalcemia. Hypercalcemia can occur in patients with hyperalbuminemia due to severe dehydration or with multiple myeloma who have a calcium-binding paraprotein. This phenomenon is called pseudohypercalcemia (or factitious hypercalcemia), since the patient has a normal ionized serum calcium concentration4. Hypercalcemia can be classified as mild, moderate or severe based on adjusted serum calcium concentrations15. Mild hypercalcemia is an adjusted serum calcium concentration of 2.65–3.00 mmol/L (10.6–12.0 mg/dL). Moderate hypercalcemia is an adjusted serum calcium concentration of 3.01–3.40 mmol/L (12.04–13.6 mg/dL). Severe hypercalcemia is a serum calcium concentration of greater than
Diagnosis and assessment of hypercalcemia
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3.40 mmol/L (13.6 mg/dL). Hypercalcemic crisis represents a life-threatening emergency84. It is usually defined as a serum calcium concentration of 3.5 mmol/L (14.0 mg/dL) or higher85. The pathogenesis of hypercalcemic crisis is poorly understood. In some cases, the crisis has been ascribed to infarction of a parathyroid adenoma, usually with severely elevated serum calcium levels (43.8 mmol/L or 15.2 mg/dL) and a PTH concentration that is 20 times the upper limit of normal. Most patients experience cardiovascular, neurologic, gastrointestinal and renal manifestations of hypercalcemia. Immediate and effective therapy directed toward the pathophysiology of hypercalcemia is essential.
Laboratory assessment and diagnosis of hypercalcemia Clinically, an investigation into hypercalcemia should start with its underlying causes. Hypercalcemia usually develops in the context of known disease, which is the apparent cause. Sometimes, there may be multiple causes, the details and clinical features of which are described in the preceding sections. Although the symptoms and signs of hypercalcemia are similar, the clinical findings of the associated primary disorders are different and can be the key for differentiating the etiology of hypercalcemia. For instance, chronic hypercalcemia in an asymptomatic, post-menopausal woman may suggest the diagnosis of primary hyperparathyroidism. Patients with malignancy-associated hypercalcemia often have rapidly increasing concentrations of calcium and apparent symptoms. Laboratory testing plays a pivotal role in the assessment of hypercalcemia to confirm the diagnosis of hypercalcemia and to differentiate between primary hyperparathyroidism, FHH and non-PTH mediated hypercalcemia. International societies have issued guidelines and recommendations on the diagnosis and differential diagnosis of primary hyperparathyroidism9,86–91. A laboratory investigation algorithm, based on published evidence, was developed in our laboratory for the assessment of hypercalcemia (Figure 3). Measurement of blood calcium Methods used for quantifying total and ionized calcium levels in the blood are well established and readily available in clinical laboratories4. Ionized calcium is usually measured on blood gas analyzers via calcium ion-selective electrodes. Ionized calcium is the biologically active fraction of blood calcium; it is tightly regulated by PTH and 1,25(OH)2D, and thus is the best indicator of calcium status. Ionized calcium measurements have been recommended because these measurements avoid the consequences of delayed treatment and the cost of working up patients with misleading total calcium results6,92. Various methods have been described for total calcium measurement. At present, only photometry, calcium ion-selective electrodes and, very rarely, atomic absorption spectrophotometry are used in clinical laboratories for measuring serum and urine total calcium4. Current methods for measuring both total and ionized calcium are subject to analytical interference and errors. The analytical accuracy and the degree of analytical interference vary with methodologies. Variations in the patient’s biology, preparation and specimen
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Q. H. Meng & E. A. Wagar
Crit Rev Clin Lab Sci, Early Online: 1–13
Hypercalcemia (Serum calcium >10.2 mg/dL) Clinical evaluation and exclusion of medication (thiazide, lithium etc.)
Hypercalcemia confirmed (Serum calcium, albumin, ionized calcium)
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by McMaster University on 11/03/14 For personal use only.
Intact PTH VitD/vitA excess, Cancer, Milkalkali syndrome Adrenal insufficiency hyperthyroidism
Low
Normal
PTHrP
24-hour urine calcium /creatinine ratio
Low or normal
1,25(OH)2D
Low
Cancer
High
Primary hyperparathyroidism
High
Cancer
24-h urine calcium Ratio0.02 High>250mg
Primary HPT, MEN 1 or 2a, Tertiary HPT
Lymphoma, sarcoidosis
Low
