The immuassay handbook parte75

705© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/B978-0-08-097037-0.00058-0

Bone and Calcium MetabolismWilliam D. Fraser 1 ([email protected])

Kay W. Colston2

John C. Stevenson2

1 This edition.2 Previous editions.

Normal Calcium MetabolismCalcium (Ca) is a divalent cation that is involved in cell pro-liferation, cellular secretion, neural transmission, neuro-muscular function, muscle contraction (including cardiac muscle), the stability and permeability of cell membranes, blood clotting, and the mineralization of bone. Calcium ions (Ca2+) have important intracellular functions, but this chapter will review the regulation of extracellular calcium.

A normal 70 kg adult contains between 700 and 1000 g of calcium with over 99% in the skeleton in the form of hydroxyapatite. Approximately 50% of circulating calcium exists as free ionized calcium. The remainder is bound to albumin or complexed to anions such as citrate, pyruvate, and lactate.

The plasma calcium concentration (total calcium 2.2–2.6 mmol/L, ionized calcium 1.1–1.3 mmol/L) is relatively high compared with the intracellular concentration of ion-ized calcium (usually less than 1 µmol/L). The large mass of calcium in bone is not immediately available, but bone can be resorbed by osteoclasts and, in the long term, can influence the soluble calcium pools.

The calcium concentration in plasma is tightly con-trolled via a classic feedback loop with ionized calcium act-ing on the calcium-sensing receptor (CaSR) located on the chief cells of the parathyroid gland. A decrease in ionized calcium sensed by the CaSR will result in increased synthe-sis and release of parathyroid hormone (PTH), and an increase in ionized calcium will have the opposite effect.

Calcium absorption, excretion, and mobilization from bone are closely regulated. The hormones central to this control include PTH, vitamin D metabolites, and calcito-nin. Of these, PTH and 1,25-dihydroxyvitamin D (1,25(OH)2D) have the greatest effects on plasma-ionized calcium, and their interactions are illustrated in Fig. 1.

1,25(OH)2D acts predominantly to increase calcium absorption from the gut and to increase osteoclast resorp-tion of bone. PTH produces its major regulatory effect on calcium by increasing its reabsorption in the kidney, stim-ulating kidney production of 1,25(OH)2D, and it can cause enhanced bone resorption. Effects of calcitonin on changes in plasma calcium are minimal under normal physiological circumstances. Its secretion is regulated acutely by plasma calcium through the CaSR: an increase in plasma calcium results in a proportional increase in calcitonin, and a decrease elicits a corresponding reduction in calcitonin. Chronic stimulation results in exhaustion of the secretory reserve of the C cells of the thyroid.

For diagnostic purposes, changes in plasma calcium should be interpreted in the context of a thorough medical history and examination. A change in plasma calcium should be adjusted for changes in plasma albumin to better reflect the change in ionized calcium. A general rule of thumb is that for any alteration in albumin from 40 g/L, the plasma calcium should be adjusted in the opposite direction using the equation:

Clinical DisordersHYPERCALCEMIAElevation of plasma calcium above the reference range (>2.6 mmol/L) is often asymptomatic but can cause thirst, polyuria, dehydration, constipation, abdominal pain, renal calculi, nephrocalcinosis, and pancreatitis. With very high levels of calcium (>3.0 mmol/L), diabetes insipidus, hyper-calcemic crisis, and cardiac dysrhythmias may occur; there is impairment of consciousness, coma, and eventual death (see Table 1).

Differentiation between the causes of hypercalcemia is not difficult if the clinical setting is obvious and reliable diagnostic tests are available. If the patient is otherwise well and there is documentation of an elevated plasma calcium concentration several years previously, this suggests primary hyperparathyroidism (1HPT). This diagnosis is particu-larly likely if hypophosphatemia has also been recorded. In hypercalcemia of malignancy (HCM), history, symp-toms, imaging, and biochemical and hematological tests may suggest the diagnosis, which may be confirmed by sub-sequent histology. Iatrogenic causes of hypercalcemia have become very common, and vitamin D or its metabolites

C H A P T E R

9.4

FIGURE 1 Effects of PTH and 1,25(OH)2D on Ca2+.

706 The Immunoassay Handbook

plus calcium administration are often the causes of hyper-calcemia. Steroid suppression tests are unreliable for distin-guishing between 1HPT and other causes of hypercalcemia. Most patients with hypercalcemia have either 1HPT, Vita-min D/Ca excess, or HCM. Other causes are rarer but may need to be considered and excluded.

The intact PTH assay is used to distinguish patients with 1HPT from those with HCM (Fig. 2). The term humoral hypercalcemia of malignancy (HHM) has been used to refer to those patients with HCM, who display biochemical features similar to those seen in 1HPT.

Parathyroid hormone-related protein (PTHrP) is thought to be the main humoral factor responsible for hypercalcemia, associated with cancer. It is mainly secreted into the circulation by solid malignancies of the breast, lung, and genitourinary system but can also have significant local paracrine effects in many malignancies. The amino-terminal portion of PTHrP possesses PTH-like activity that causes hypercalcemia because of increased bone resorption by osteoclasts and decreased urinary calcium excretion. PTHrP also promotes renal phosphate excretion and increased nephrogenous cyclic AMP. Whereas a mild hyperchloremic acidosis is often a feature in 1HPT, hypokalemic alkalosis is frequently found in HHM.

PARATHYROID DISORDERS1HPT is a common disorder occurring in 1 in 500–1000 of the general population, but the incidence is highest

among postmenopausal women. The condition is most commonly associated (80–85%) with the presence of a solitary parathyroid adenoma autonomously secreting PTH. 1HPT may also develop when certain undefined pathological processes lead to hyperplasia of the parathyroid glands. This may occur sporadically or form part of one of the syndromes of multiple endocrine neoplasia (MEN). Classically, the first presentation of MEN1 is 1HPT, and since the gene is highly penetrant, 88–97% of carriers will develop 1HPT by the age 50.

Patients with 1HPT are often asymptomatic but may present with nonspecific gastrointestinal symptoms, poly-uria, polydypsia, and occasionally psychiatric disorders, renal stones, or nephrocalcinosis. In elderly patients, the presentation may be that of acute hypercalcemic crisis characterized by confusion and dehydration. 1HPT is often associated with reduced bone mineral density (BMD), but overt evidence of bone disease is seen in about 10% of patients. Frequently, hypercalcemia is an incidental finding during biochemical testing for the investigation of an unrelated condition. Plasma calcium is usually but not invariably raised; plasma phosphate con-centration is low or below normal; and plasma chloride is usually in the upper reference range or clearly elevated with reverse changes in plasma bicarbonate. Plasma alkaline phosphatase (ALP) activity is usually normal but may be raised when there is bone disease. Circulating concentrations of PTH are raised or inappropriate for the prevailing plasma calcium. The development of assays measuring the intact PTH has improved the diagnostic utility of this test (Fig. 2).

TABLE 1 Causes of Hypercalcemia

Common1. Primary hyperparathyroidism2. Hypercalcemia of malignancy Metastases in bone e.g., carcinoma of the breast

Humoral hypercalcemia e.g., squamous cell carcinoma of the lung

Hematological malignancies e.g., multiple myeloma3. Vitamin D intoxication IatrogenicLess Common4. Renal disease Iatrogenic (vitamin D metabolites)

Diuretic phase of acute necrosis5. Sarcoidosis and other granulomatous disease6. Thyrotoxicosis7. Familial benign hypocalcuric hypercalcemia8. Milk alkali syndromeVery Rare9. Thiazide diuretics (often transient)10. Immobilization11. Infantile hypercalcemia 24-hydroxylase deficiency

(possible genetic cause)12. Lithium treatment13. Vitamin A toxicity14. Addison’s disease

707CHAPTER 9.4 Bone and Calcium Metabolism

HYPOPARATHYROIDHypoparathyroidism is diagnosed from the combination of hypocalcemia and hyperphosphatemia with low or undetectable PTH concentrations. Hypocalcemia and hyperphosphatemia may occur in chronic renal failure and pseudohypoparathyroidism, but circulating PTH concen-trations are usually high in these conditions.

Decreased secretion of PTH mainly occurs because of surgical damage to the parathyroid gland or because of spontaneous failure of parathyroid gland function, usually due to autoimmune factors. The clinical features result from acute or chronic effects of hypocalcemia.

HYPOCALCEMIAA decrease in the concentration of extracellular calcium ions causes hyperexcitability of the nervous system. The main clinical manifestations are numbness, tingling (perioral or peripheral), tetany, either latent or overt, proximal weakness, and ECG abnormalities. Effects of long-standing hypocalcemia include cataracts, cutane-ous candidiasis, calcification of the basal ganglion, and psychiatric disorders, particularly mental depression. (See Table 2).

VITAMIN D DISORDERSVitamin D intoxication can present clinically as hyper-calcemia with its attendant features.

Vitamin D deficiency manifests itself predominantly as skeletal defects resulting from impaired mineraliza-tion of bone. It had been decreasing in the UK, but is

increasingly seen in growing children, the dark-skinned immigrant or indigenous population, and in the elderly, particularly those who are housebound. It is more common in Asian communities, mainly because of tradi-tional habits in diet and dress. In growing children, the disease may manifest as bone pain, tenderness and defor-mities with expanded epiphyses, muscle weakness, and even symptomatic hypocalcemia; this is the clinical pic-ture of rickets. In adults, the condition is known as osteomalacia, which may be manifested as bone pains and tenderness, fractures, proximal myopathy, and symp-tomatic hypocalcemia.

History and examinationNo Obvious Drug Causes

E.g., Lithium,Thiazides

Plasma ACa >2.65 mmol/L on TWO occasions

Normal Renal Function

PTH >3.0 pmol/L*

Yes No

Non Parathyroid CauseHematology malignancy,

MyelomaVit D Excess,Sarcoid,

Toxicosis, Immobilization

PTHrP measurementIf >1.8 pmol/L

Malignancy Very Likely

HyperparathyroidPrimary (majority)

TertiaryMalignancy Not Excluded

Urine CaCl/CrCL<0.01+/-Family History

Familial hypocalciuric hypercalcemia

Dual PathologyPossible

* Intact PTH (1-84) measured using ECLIA assay

FIGURE 2 Initial biochemical investigation of hypercalcemia. (The color version of this figure may be viewed at www.immunoassayhandbook.com).

TABLE 2 Main Causes of Hypocalcemia

ProtractedPrivational vitamin D deficiencyHypoparathyroidismPseudohypoparathyroidismChronic renal diseaseMalabsorption syndromeVitamin D-dependent rickets

TransientPostoperative:

ThyroidParathyroidOther neck surgery

Acute pancreatitisHypomagnesemia


Biochemical ChangesPlasma calcium may be subnormal in advanced cases, but initially the tendency to hypocalcemia is corrected by increased parathyroid activity, resulting in increased PTH concentration (secondary hyperparathyroidism) and main-tenance of 1,25(OH)2D within the reference range so that a normal plasma calcium concentration is not uncommon. Plasma phosphate concentration is usually in the low normal or subnormal range. There is generally an increase in the activity of the bone isoenzyme of ALP because of increased osteoblast activity. Some patients do not have increased plasma total ALP so that a normal result does not exclude osteomalacia. Urinary calcium excretion is usually low as a result of decreased glomerular filtration and increased PTH secretion stimulating calcium reabsorption.

The development of vitamin D insufficiency, due in part to defective 1α-hydroxylation of 25OHD, occurs in chronic renal failure (CRF). A wide spectrum of bio-chemical abnormalities is present because of the combined effects of secondary hyperparathyroidism, osteomalacia, and renal failure. Serum 25OHD concentrations may be normal or low, while 1,25(OH)2D concentrations are often decreased. PTH measurement is complicated by metabolism in CRF with the retention of C-terminal fragments that can interfere in the intact PTH assays. Development of the “whole-molecule” PTH assays has not completely resolved these problems. Standardization of PTH assays is increasingly recognized as problematic and contributing to the variability in treatment of CRF-associated metabolic bone disease (MBD).

Patients on long-term anticonvulsant therapy have an increased prevalence of osteomalacia. Such patients have low-plasma 25OHD concentrations, thought to result from induction of hepatic microsomal enzymes, leading to increased catabolism and a decreased half-life of vitamin D and its metabolites. Antituberculous drugs may similarly cause osteomalacia.

Both hereditary and acquired forms of rickets and osteomalacia occur due to defective 1,25(OH)2D synthe-sis. Vitamin D-dependent rickets type I is a rare disorder inherited as an autosomal recessive trait. The disorder is thought to be a hereditary defect in the renal 1α-hydroxylase enzyme responsible for the production of 1,25(OH)2D. Severe rickets usually manifest before the age of 6 months. Plasma biochemistry shows the typical changes of vitamin D deficiency: hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and increased ALP. The rickets can be healed by treatment with small doses of 1,25(OH)2D (calcitriol) or very large doses of cholecalciferol (1000–3000 µg/day).

Vitamin D-dependent rickets type II is a disorder characterized by rickets or osteomalacia that is present despite marked increases in circulating 1,25(OH)2D. The disorder appears to be one of target organ resistance to the hormonal form of vitamin D. It is likely that structural abnormalities in the receptor account for the defective response of target organs to 1,25(OH)2D.

Because the renal activation of vitamin D is regulated by the vitamin D status, high doses of vitamin D are needed to produce hypercalcemia. Vitamin D intoxication may occur as a complication of therapy in hypoparathyroidism,

especially when using active vitamin D metabolites, but occasionally vitamin D ingestion is surreptitious. Hypercalcemia due to intoxication with vitamin D itself is often protracted, lasting weeks or occasionally months, unless treated with glucocorticoids. Intoxication with 1,25(OH)2D3 (calcitriol) also produces hypercalcemia, but the duration is much shorter (days), because of its shorter biological half-life. Assay of 25OHD (vitamin D intoxica-tion) or 1,25(OH)2D will reveal the diagnosis if this is not clinically obvious.

About 10% of patients with sarcoidosis exhibit hypercalcemia, and a much higher proportion have hyper-calciuria (about 50%). The hypercalcemia is associated with increased absorption of calcium in the intestine, and it is due to extra-renal production of 1,25(OH)2D by sar-coid tissue, producing inappropriately increased circulat-ing 1,25(OH)2D concentration. Other granulomatous diseases such as berylliosis and tuberculosis can also be associated with this mechanism for hypercalcemia.

In lymphoma and in acute and chronic leukemia, hyper-calcemia is sometimes associated with inappropriately high 1,25(OH)2D concentrations, suggesting that the tumor cells may be the site of unregulated hydroxylation of 25OHD.

OSTEOPOROSISOsteoporosis is associated with aging, and with increasing longevity, a greater percentage of the population will become susceptible to osteoporosis and its sequelae. BMD decreases from a peak achieved by the age of 20–30 in men and women, and the rate of bone loss is accelerated in women after loss of estrogen secretion at the menopause. The progressive loss of bone that takes place with aging is a result of uncoupling of bone turnover over a prolonged period of time, with a rela-tive increase of bone resorption or decrease in bone forma-tion. This leads to an increased risk of fracture.

PAGET’S DISEASE OF BONEPaget’s disease of bone is a disorder characterized by areas of accelerated bone turnover initiated by increased osteoclast-mediated bone resorption. Osteoclasts in Paget’s disease are large, numerous, and multinucleate (up to 100 nuclei); their activity is coupled to increased osteoblast number and activity. Affected bones become expanded and deformed and may be more liable to fracture. A number of other complications can occur. A common biochemical abnormality is increased ALP, which is indicative of increased osteoblast activity. Increased collagen breakdown by osteoclasts results in a high plasma and urine concentra-tion of hydroxyproline, pyridinolines (deoxypyridinoline (DPD) and pyridinoline (PYD)), and telopeptides (C-terminal telopeptide (CTX) and N-terminal telopeptide (NTX)). The bisphosphonates, particularly zoledronic acid, have significant anti-osteoclastic activity and are the drugs of first choice for treating Paget’s disease.

MEDULLARY CARCINOMA OF THE THYROIDMedullary carcinoma of the thyroid, which may present as a lump in the neck, is associated with hypersecretion of


calcitonin. Families in whom this condition is an inherited trait may require screening for excessive calcitonin secre-tion. This carcinoma may form a component of MEN type 2A (MEN2A) and is often the first presentation of this disease.

AnalytesIn all immunoassays, the following are important.

� The correct sample type and appropriate sampling procedures (see Stokes et al., 2011).

� Any initial extraction or displacement procedures should be efficient and reproducible.

� The tracer should be stable and of sufficiently high specific activity.

� The solid phase employed for the capture antibody should have a high capacity, giving a linear relationship between signal and analyte concentrations.

� An efficient separation of bound from free should be achieved.

� Internal quality assurance samples should be included in every assay; these should have concentrations covering the assay range.

� If available, external quality assurance (proficiency testing) samples should be assayed regularly (2–3 times monthly).

VITAMIN D METABOLITES: 25-HYDROXYVITAMIN D AND 1,25-DIHYDROXYVITAMIN DVitamin D metabolites consist of a steroid nucleus with the important structural variations of the different metab-olites being related to the number and position of hydroxyl groups.

The synthesis of vitamin D and its metabolites involves metabolic pathways in a number of different tissues. Vita-min D3 (cholecalciferol) is produced in the skin by the action of sunlight on the precursor molecule 7-dehydro-cholesterol. Ultraviolet irradiation leads to fission of the ring between carbons 9 and 10 to produce vitamin D3. Chronic lack of sunlight may result in a deficiency of this vitamin. Vitamin D is converted in the liver to 25OHD. A second hydroxylation step takes place in the kidney to form 1,25(OH)2D, the biologically active metabolite. Dis-turbed tissue function at any of these sites has the potential to alter vitamin D concentrations and thus calcium metab-olism. An alternative hydroxylation in the 24-position pro-duces 24,25-dihydroxyvitamin D (24,25(OH)2D), a relatively inert metabolite (see Table 3).

Typically, 80–90% of circulating vitamin D is produced as a result of sunlight exposure; dietary sources of vitamin D contribute 10–20%. Plasma transport of vitamin D metabolites is on a specific carrier protein, vitamin D-binding protein (VDBP). A second form of vitamin D, vitamin D2 (ergocalciferol), can be produced by ultraviolet irradiation of the sterol, ergosterol. Its side-chain structure differs slightly from that of vitamin D3, and there is a debate regarding whether vitamin D2 and D3 have equiva-lent biological potencies in humans.

MetabolismInterest in the catabolism of 1,25(OH)2D has led to the elucidation of a pathway involving C24 oxidation and side-chain cleavage with ultimate formation of calcitroic acid. Metabolites containing 24 hydroxylations have very little biological activity, although they may form part of the controlling mechanism for the regulation of production of metabolites. Loss of function of the 24-hydroxylase may result in significant clinical hypercalcemia.

FunctionThe major target organ for 1,25(OH)2D is the small intes-tine, where it acts to increase active calcium absorption. This action involves vitamin D-induced changes in the cal-cium transport system, of which an increase in the concen-tration of a specific calcium-binding protein, calbindin, is an important part. Vitamin D metabolites also act on bone to increase calcium resorption by an indirect effect on osteoclasts, and act directly on osteoblasts to stimulate their activity, resulting in increased ALP in plasma. However, the predominant role of vitamin D in bone metabolism is to promote mineralization and thus retention of calcium in bone by raising extracellular calcium concentrations.

Although in the past, vitamin D has been considered solely as a calcium-regulatory hormone, there are now suggestions that it may have more of a general role in cel-lular metabolism via modulation of intracellular calcium transport. It is now clear that vitamin D has many different effects in various tissues, and its receptors are widely dis-tributed throughout the body. Vitamin D has suggested roles in both classical (calcium and bone metabolism, neu-romuscular function) and nonclassical diseases, e.g., arthri-tis, cardiovascular, cancer, diabetes, multiple sclerosis, and psychiatric illness.

Significant debate has centered on the optimal circulat-ing 25OHD concentration. The Institute of Medicine (IOM) has recently produced a classification based on optimal concentrations for bone health.

TABLE 3 Source of Major Metabolites of Vitamin D

DietVitamin D3 (cholecalciferol)Vitamin D2 (ergocalciferol)

SkinUltraviolet light7-DehydrocholesterolVitamin D3

Liver25-Hydroxyvitamin D3 (25OHD3)25-Hydroxyvitamin D2

Kidney1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) or24,25-Dihydroxyvitamin D3 (24,25(OH)2D3)1,25-Dihydroxyvitamin D2 (1,25(OH)2D2) or24,25-Dihydroxyvitamin D2


Clinical ApplicationsThe measurement of vitamin D metabolites is useful in the investigation of hypo- and hypercalcemia. 25OHD con-centrations are elevated in vitamin D intoxication and reduced in vitamin D deficiency. Measurement of vitamin D metabolites can be useful in the investigation of patients with hyper- and hypocalcemia. Cutaneous production of vitamin D is related to intensity of sunlight exposure and decreases with advancing age. In elderly subjects living in Northern Europe, vitamin D supplementation has been recommended during the winter months to maintain adequate 25OHD concentrations. Plasma concentrations of 25OHD show a seasonal variation with the highest in the late summer months and a nadir in late winter/early spring.

In chronic renal failure, plasma 25OHD concentrations may be normal or low while 1,25(OH)2D concentrations are decreased. Patients on long-term anticonvulsant therapy or taking antituberculous drugs have an increased prevalence of osteomalacia, with low-plasma 25OHD concentrations.

Vitamin D-dependent rickets type I, also known as pseudo-vitamin D-deficiency rickets (PDDR), is an inborn error in conversion of 25OHD to 1,25(OH)2D3 due to deficiency of the 1α-hydroxylase. Plasma 1,25(OH)2D3 concentrations are low or undetectable, and patients with this condition can be successfully treated with physiological doses of the metabolite. Vitamin D-dependent rickets type II (hereditary vitamin D-resistant rickets, HVDRR) is a disorder characterized by early onset rickets with a marked increase in circulating 1,25(OH)2D, which is attributable to end-organ resistance to the hormone due to a receptor defect.

In lymphoma and in acute and chronic leukemia, hypercalcemia is sometimes associated with inappropri-ately high 1,25(OH)2D concentrations. In patients with sarcoidosis, hypercalcemia may be due to synthesis of 1,25(OH)2D3 by sarcoid tissue. Concentrations of 1,25(OH)2D3 may also be elevated in a subgroup of stone-forming patients who have absorptive hypercalciuria and normal parathyroid function (Bataille et al., 1987) (see Tables 4 and 5).

Assay TechnologyAntirachitic potency is used to be assessed by the “line test”. Nutritional rickets was induced in test rats and the degree of linear calcification in the radial epiphysis in response to vitamin D or an unknown sample quantified. More sensitive assays for metabolites of vitamin D were subsequently developed that took advantage of the high affinity and selectivity of target tissue receptors and serum transport proteins (radio receptor assays [RRA]). These have now been superseded by immunoassays or liquid chromatography mass spectrometry (LCMS).

25-Hydroxyvitamin D25OHD is the major circulating form of vitamin D in plasma, accounting for more than 80–90% of total vitamin D metabolites. Measurement of plasma 25OHD concen-tration is a useful index of overall body stores of vitamin D. A competitive protein-binding assay for 25OHD was

developed in the early 1970s, which gained widespread use. Initially, VDBP present in rat serum or kidney cytosol was utilized as a specific binding agent (Haddad and Chyu, 1971). The procedure involved organic extraction of the serum sample with ethanol, acetonitrile, or other solvents, followed by sample pre-purification using Sephadex or silica columns, or Sep-Pak cartridges. These methods used [3H]25OHD3 as tracer and required individual sample recovery estimation to correct for endogenous losses of 25OHD during extraction and chromatographic steps. This method measured both 25OHD3 and 25OHD2. The first valid direct UV-quantitative HPLC assay was introduced in 1977 (Eisman et al., 1976). HPLC detection provided the advantage of being able to separately quanti-tate 25OHD3 and 25OHD2. The disadvantages of this assay included the requirement for expensive equipment, a large sample size, and the considerable expertise required. These approaches to determine circulating 25OHD concentrations have been superseded by radioimmunoas-says (RIAs), which eliminate the requirement for sample chromatography, although a solvent extraction or protein displacement step is required. Immunoassay methods reported in the 1980s described an RIA that initially utilized a 3H-labeled 25OHD tracer and subsequently developed into a 125I-labeled 25OHD assay with higher throughput and improved performance (Hollis et al., 1993). This assay formed the basis for a subsequent chemiluminescent detection-based system that has been fully automated (Ersfeld et al., 2004). A commercial two-step extraction RIA was produced subsequently that developed into an enzyme immunoassay (EIA) without

TABLE 4 25-Hydroxyvitamin D Concentrations in Various Conditions Associated with Disturbances of Calcium Metabolism

Low Normal High

Privational osteomalacia (diet, sunlight)

Sarcoidosis Vitamin D intoxicationVitamin D-dependent

ricketsCirrhosisAnticonvulsant therapyRickets/osteomalacia

TABLE 5 Plasma 1,25(OH)2D Concentrations

Causes of Increased 1,25-Dihydroxyvitamin D Levels

1. Physiological: growth, pregnancy, and lactation2. Hyperparathyroidism3. Sarcoidosis4. Acromegaly5. Hypothyroidism6. Vitamin D-dependent rickets type IICauses of Decreased 1,25-Dihydroxyvitamin D Levels

1. Renal failure2. Vitamin D deficiency3. Hypoparathyroidism4. Vitamin D-dependent rickets type I5. Hyperthyroidism


extraction that could also be automated (Hypponen et al., 2007). Commercial 25OHD immunoassays have been produced where solvent extraction and chromatographic separation have been replaced by various blocking agents that displace 25OHD from VDBP, with varying success. This approach facilitates automation of these assays but data suggest that some of these assays are affected by variations in VDBP concentrations, resulting in differ-ences in results between assay formats.

An area of concern in relation to immunoassays is the variability in the detection of 25OHD2. Some assays claim 100% cross-reactivity with exogenously added 25(OH)D2 and 25OHD3 and are therefore equipotent for the two metabolites. Other assay manufacturers admit to lower cross-reactivity with exogenous 25OHD2 (e.g., 75%, 52%), while other assays were specifically designed to measure only 25OHD3. Reports have confirmed the vari-ability of commercial immunoassays to detect 25OHD2 (Binkley et al., 2010). Some assays have problems detecting ingested vitamin D2 when converted in vivo to 25OHD2 with a significant underestimation of 25OHD2 by three commercial immunoassays in some samples in a vitamin D2 (ergocalciferol) supplementation study (Glendenning et al., 2006). There appears to be a change in the 25OHD2 “synthesized” in vivo following oral consumption of vitamin D2 in some individuals that alters recognition by the antibodies in several immunoassays.

The first direct UV detection-based HPLC assays for 25OHD were published in 1977 (Eisman et al., 1977). A cumbersome chloroform–methanol extraction was fol-lowed by chromatography on Sephadex/silica gel columns followed by HPLC with UV detection. Improvements in HPLC methods centered around the introduction of reversed-phase HPLC mainly using C18 columns, improved internal standard material, and improved sample extraction using chloroform–methanol, methanol–hexane, and then extraction of samples using semiautomated technology employing acetonitrile postsample precipitation.

The techniques of sample extraction allied to chroma-tography with MS detection methods offer increased spec-ificity for the molecule of interest, and so the combination of HPLC and MS in tandem, LC–MS/MS, has become a commonly used technique. Early methods employed fast atom bombardment with Cookson-type reagents, result-ing in derivatization of 25OHD, which improved detec-tion of 25OHD. Isotope dilution- electrospray LC–MS/MS methods performed on “benchtop” analyzers became popular in the mid 2000s with protein precipitation of the sample, liquid–liquid extraction, short run times, com-puter programming, and processing of chromatograms contributing to higher throughput and ease of use (Maunsell et al., 2005). The use of deuterated 25OHD2 and D3 internal standard material improves accuracy, with better extraction processes helping to remove phospholip-ids, which reduces the problem of ion suppression. Recent developments have centered around reducing the manual component of sample preparation prior to chromatogra-phy, and elimination of liquid–liquid extraction using either separate automated solid-phase extraction (SPE), online SPE, or online turbulent flow extraction. A step change in technology has been published that has increased

sample throughput multiplexing samples by differential mass tagging in LC–MS/MS (Netzel et al., 2011).

Isotope dilution LC–MS/MS is currently considered the gold standard method for 25OHD measurement, simulta-neously quantitating 25OHD2 and 25OHD3. In 2011, a reference method (Tai et al., 2010) was recognized by the Joint Committee for Traceability in Laboratory Medicine.

1,25-Dihydroxyvitamin DIn 1978, the first RIA for 1,25(OH)2D was published (Clemens et al., 1978). The assay required sample purifica-tion due to poor specificity of the rabbit antibody and a tritiated tracer was employed, but overall it was insensitive compared with an RRA (detection limit 20 ng/L). Improved antibody technology resulted in RIAs with better perfor-mance and detection limits, but antibody cross-reactivity with 25OHD and 24,25(OH)2D meant that extensive purification of 1,25(OH)2D was required. By manipulation of extraction and chromatography or use of particular anti-bodies, assays specific for 1,25(OH)2D2 and 1,25(OH)2D3 were produced. A major advance was the development and commercialization of an RIA for 1,25(OH)2D that required minimal sample pre-purification, did not need internal standardization, utilized calibrators in an equivalent matrix to samples, and utilized an 125I-labeled tracer. This assay involved acetonitrile extraction, treatment of the extract with sodium periodate (converting the 24,25(OH)2 D3 and 25,26(OH)2 D3 to aldehyde and ketone forms, reducing cross-reactivity), extraction and purification of 1,25(OH)2D by solid-phase chromatography (C18-OH and silica car-tridges), followed by quantification by RIA. A detection limit of 2.4 ng/L was quoted, recovery of 1,25(OH)2D2 was 64–71% and 1,25(OH)2D3 90–101% (Hollis et al., 1996). Further analytical and clinical validation of the commer-cial assay was published in 2002 (Clive et al., 2002). A novel approach to sample purification and extraction was adopted in a commercial assay, utilizing immunoextraction separat-ing 1,25(OH)2D from delipidated samples using an antibody bound to a mini immunocapsule. Elution of the sample from the capsule, evaporation, and then reconstitu-tion prior to RIA resulted in a rapid assay (Fraser et al., 1997). This RIA has been modified to a non-isotopic for-mat. Both the RIA and EIA show excellent correlation with an RRA assay (Seiden-Long & Veith, 2007). Concerns have been raised about possible contribution to 1,25(OH)2D measurement from other 1α-hydroxylated metabolites, and cross-reactivity for 1,25 (OH)2 D3, 26,23-lactone, 1,24,25 (OH)3D3, and 1,25,26(OH)3D3 has been demonstrated in commercial assays.

Immunoassays for 1,25 (OH)2D may under-recover exogenous 1,25 (OH)2D2, probably due to the antibodies having poor cross-reactivity with 1,25(OH)2D2. A recent study has confirmed a similar problem to that discussed previously for 25OHD immunoassays, where endoge-nous vitamin D2 given as a single bolus of 300,000 IU by intramuscular injection was not fully quantified by a commercial immunoassay as 1,25(OH)2D2 after in vivo metabolism, in comparison with an LC–MS/MS technique.

Measurement of 1,25(OH)2D by direct detection UV methods is not possible due to the low concentration in blood (pmol/L). 1,25(OH)2D has few ionizable polar


groups and so techniques to increase the ionization efficiency (e.g., derivatization) have been incorporated in all of the published tandem MS methods.

An isotope dilution–mass fragmentography assay for 1,25(OH)2D was first published in 1979 (Bjorkhem et al., 1979). Extraction of 20 mL of serum was performed using chloroform/methanol after addition of [26-2H3]-1,25(OH)2D3 and purification by liquid chromatography. The purified material was converted into the trimethylsilyl ether and analyzed by gas chromatography-mass spec-trometry (GCMS). The LLOQ was 5 ng/L with a CV of 5%, but the large sample volume limited the general appli-cability of the assay. An LC/thermospray (TSP) MS tech-nique using positive- and negative-ion detections after online post-column Diels–Alder derivitization obtained an LLOQ of 1 nmol/L. Derivatization using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a Cookson-type reagent, of a solid-phase extracted sample and measurement using ultra-performance liquid chromatography (UPLC) elec-trospray tandem MS allowed simultaneous quantification of a profile of vitamin D metabolites (1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D3, 25OHD2, and 25OHD3) with an LLOQ of 25 pg/mL and CV of 5–16% for 1,25(OH)2D3 (Aronov et al., 2008). PTAD was also used in a method able to quantitate the same four metabolites with significantly improved sensitivity, 5 ng/L (12 pmol/L) for 1,25(OH)2D3, that employed selective SPE and microflow LC MS/MS. Lithium acetate has been used to produce ionizable adducts in a method that uses a complex online sample-processing procedure with the use of a perfusion column, followed by a chain of two monolithic columns to clean and enrich the sample prior to quantification on a highly sensitive LC–MS/MS (Casetta et al., 2010). Both 1,25(OH)2D2 and 1,25(OH)2D3 can be measured with an LLOQ for 1,25(OH)2D3 of 15 ng/L (36 pmol/L), a CV of 5–15% across physiological concentrations. A commercial immunoaffinity column and reagents were incorporated into a sample preparation procedure following protein precipitation and SPE. Lithium acetate was used to pro-duce adducts prior to LC–MS/MS analysis. This method removed isobaric interferences and matrix effects resulting in significantly reduced ion suppression with the resultant LLOQs of 3.9 ng/L (9.1 pmol/L) for 1,25(OH)2D2 and 3.4 ng/L (8.2 pmol/L) for 1,25(OH)2D3 with inter-assay CVs of 2.5–7.0% (Yuan et al., 2011). A very similar approach using the commercial columns for immunoex-traction but derivitizing using PTAD prior to UPLC MS/MS resulted in improved sensitivity. All of the LC–MS/MS methods described are labor intensive with manual workflows and limited throughput.

Type of SamplePlasma or serum, store frozen.

Limitations

� Low 25OHD concentrations indicate vitamin D defi-ciency. Diagnosis of osteomalacia requires histomorphometry.

� Immunoassays have variable cross-reactivity with 25OHD2, and so care is required interpreting results in

patients receiving ergocalciferol supplementation (vitamin D2).

� Major differences in 25OHD concentrations in differ-ent assays obtained on the same samples highlight the problems of standardization of assays as well as VDBP effects.

� Almost all 25OHD immunoassays show a high cross-reactivity with 24,25(OH)2D that increases in concentration in blood with increasing sun exposure and as 25OHD increases.

� Monitoring vitamin D replacement therapy by measurement of 1,25(OH)2D levels is only useful in patients treated with alfacalcidol (1α hydroxyvitamin D) or calcitriol (1,25(OH)2D3).

Frequency of Use25OHDHigh. Primary care and secondary care physician requests are increasing due to recognition of the high prevalence of vitamin D deficiency and association of low 25OHD with many conditions.

1,25(OH)2DLow. Specialized bone and endocrine units and renal physicians.

Reference Intervals25OHD

� Vitamin D deficiency may be defined as a serum 25OHD <30 nmol/L.

� A serum 25OHD of 30–50 nmol/L may be inadequate for bone health in some people.

� A serum 25OHD >50 nmol/L is sufficient for bone health for almost the whole population.

(commercially available RIA method, IOM recommendations)

1,25(OH)2D40–120 pmol/L (adults) (RIA; Hollis, 1997).

PARATHYROID HORMONEPTH, parathyrin, is a single-chain polypeptide composed of 84 amino acids, produced by two discrete pairs of para-thyroid glands located at the upper and lower poles of the thyroid gland in the neck. Like many peptide hormones, PTH is synthesized as a larger precursor molecule, preproparathyroid hormone (a 114 amino acid form), and this peptide is cleaved to form proparathyroid hormone. The pro sequence of six amino acids is cleaved to form the 84 amino acid active peptide (PTH[1–84]), which is stored in secretory granules of the parathyroid cells. The fusion of the secretory granules to the chief cell membrane is regulated by magnesium (Mg).

FunctionIn the circulation, PTH (1–84) has a short half-life (less than 5 min) compared with the biologically inactive middle


and carboxy-terminal fragments (20–30 min). The main actions of PTH are on bone, where it stimulates bone turnover, and the kidneys, where it acts directly to pro-mote calcium reabsorption (and promote phosphate excre-tion), and indirectly via its ability to increase the activity of the 1α-hydroxylase enzyme controlling 1,25(OH)2D syn-thesis. The major factor regulating the release of PTH is the extracellular calcium concentration. Secretion is mark-edly increased by a decrease in plasma-ionized calcium, sensed at the CaSR on the chief cells. Other factors have also been shown to influence PTH release, including phosphate, which increases PTH (1–84) synthesis and secretion, and 1,25(OH)2D, which is reported to suppress the PTH gene expression. PTH (1–84) has a marked cir-cadian rhythm with a significant rise to a peak around 0300–0400 and a nadir at 0800–0900.

Clinical ApplicationsIn 1HPT, circulating PTH (1–84) concentrations are raised or inappropriate for the prevailing plasma calcium. The development of assays that measure PTH (1–84) has improved the diagnostic utility of this test.

Decreased secretion of PTH (1–84) occurs because of surgical damage to the parathyroid gland, spontaneous failure of parathyroid gland function (usually due to auto-immune factors), or suppression by ingested calcium/vita-min D (Manning & Fraser, 1993). Hypoparathyroidism is diagnosed from the combination of hypocalcemia and hyperphosphatemia with low or undetectable PTH (1–84) concentrations. Hypocalcemia and hyperphosphatemia also occur in chronic renal failure and pseudohypopara-thyroidism, but circulating PTH (1–84) concentrations are high in these conditions.

Assay TechnologyImmunometric assays are the commonest methods used for routine measurement of circulating PTH (1–84). A serious problem is the variety of immunological species of the hormone present in plasma. The major immunoreac-tive species is the biologically inactive C-terminal fragment. Intact PTH (1–84) and its fragments are cleared from the circulation both by the kidneys and the liver. The clearance of C-terminal fragments is slower than that of PTH (1–84). It is also more dependent upon renal mechanisms, and consequently, renal impairment leads to greater accumula-tion of C-terminal fragments. Most of the early RIAs for PTH utilized antibodies directed against the C-terminal region. Although these assays showed raised values in the majority of patients subsequently shown to have 1HPT, a variable proportion had normal values, and patients with renal impairment had raised concentrations regardless of parathyroid secretion rate because of impaired clearance of the C-terminal fragments. The early assays were also based on antisera, calibrators and tracer from nonhuman species, which contributed to the insensitivity of the assay and non-parallel dilution characteristics.

Development of the two-site immunoassays measuring the “intact” hormone PTH (1–84) has improved the sensi-tivity and reproducibility of the measurement of PTH, and they are the commonest methods in use today. One early commercial intact PTH immunoradiometric assay

(IRMA) that was developed (Nussbaum et al., 1987) used two different polyclonal antibodies. One, directed against amino acids 39–84, was bound to a solid phase. The sec-ond antibody, which recognized the first 34 amino acids, was labeled with 125Iodine. Samples were incubated simul-taneously with both antibodies followed by a washing pro-cedure to remove any unbound labeled antibody. This methodology helped eliminate interference by the major C-terminal and mid-region fragments. Several intact PTH immunometric assays have been developed using radioac-tive, enzyme, and electrochemiluminescence labels, and have been incorporated on automated analyzers. Rapid format PTH (1–84) assays with short incubation times, for near-patient testing, allowing intra-operative PTH measurement to help guide parathyroid surgery, are avail-able. In the late 1990s, it was demonstrated that the sec-ond-generation IRMAs or intact PTH assays cross-reacted with some C-terminal fragments, subsequently shown to include PTH (7–84) (D’Amour et al., 2005), and the per-centage cross-reactivity varied depending on the antibod-ies and assay technology employed. Third-generation assays highly specific for PTH (1–84), initially termed “whole” PTH assays (Gao et al., 2001), lacking cross-reactivity with PTH (7–84), have become available although they are not yet widely used in clinical practice. The advantage of such assays is the lack of detection of most C-terminal fragments that accumulate in renal fail-ure. The expansion in numbers and the type of PTH assay available has resulted in reports of significant variability in results and a lack of comparability between assays, especially in patients with renal failure. Establishing method-related reference ranges in local populations is essential, and better standardization of assays is required in future.

Types of SampleEach assay manufacturer has established the optimal sam-ple type for their own assay, and the manufacturer’s pack insert should be consulted regarding the best collection tubes. Some patients with particular diseases (e.g., pancreatitis) can rapidly metabolize PTH (1–84) and so collection onto ice, separation without delay (<30 min), with rapid analysis, may be required to obtain an accurate result.

Limitations

� Standardization of PTH (1–84) is a current problem and the introduction/use of WHO IS 95/646 may help reduce assay variability in the future.

� C-terminal fragments produced in renal failure have variable cross-reactivity in different commercial intact PTH (1–84) assays.

� Optimal sample type (EDTA plasma, heparin plasma, or serum) may be different for each available commer-cial assay for PTH (1–84).

Frequency of UseHigh. Guidelines for the treatment of CRF and mineral bone disorders (MBD) incorporate PTH (1–84) concentrations.


Reference Interval1.1–6.9 pmol/L.(Commercial immunometric assay for intact PTH)

PTH-RELATED PROTEINA humoral factor responsible for hypercalcemia and asso-ciated with cancer has been isolated from several solid tumors. The gene for this factor has been cloned leading to the recognition of the protein now known as PTHrP. On the basis of analysis of messenger RNA (mRNA) from tumor tissue, at least three polypeptides of different lengths have been predicted. The amino-terminal portion of PTHrP has close sequence homology with PTH.

Mechanism of ActionThe mechanism by which PTHrP induces hypercalcemia is by interaction with PTH receptors. As with excess secre-tion of PTH, PTHrP production by tumors results in hypercalcemia because of increased bone resorption and reduced urinary calcium excretion. The protein also pro-motes renal phosphate excretion and increases nephroge-nous cyclic AMP. However, whereas a mild hyperchloremic acidosis is often a feature in 1HPT, hypokalemic alkalosis is frequently found in HHM.

Assay TechnologyInitial immunoassays for PTHrP were of limited utility because of poor sensitivity and the requirement in early assays for sample extraction. Ratcliffe et al. (1991) reported the development and validation of an IRMA for PTHrP in unextracted plasma. The assay involves a polyclonal anti-body to amino acids 1–34 of PTHrP coupled to cellulose particles as the capture antibody and a rabbit anti-PTHrP (37–67) as radiolabeled antibody. Ratcliffe et al. reported increased PTHrP concentrations in 95% of patients stud-ied with HCM. Plasma samples from normal subjects and patients with 1HPT had undetectable levels (detection limit of assay 0.23 pmol/L). Pandian et al. (1992) reported the development of a modified IRMA for PTHrP that uses affinity-purified polyclonal antibody. Antibodies recogniz-ing PTHrP 37–74 were immobilized onto polystyrene beads, and antibodies to epitopes within the 1–36 amino acid region of PTHrP were labeled with 125I. The detec-tion limit of this assay was reported to be 0.1 pmol/L, and low but detectable concentrations of PTHrP were reported in some normal individuals. In the study, 91% of patients with hypercalcemia associated with nonhematological malignancies had increased concentrations of PTHrP. A commercial IRMA that was a development of this assay was described (Fraser et al., 1993), which proved to be a reliable and robust measurement system commonly detect-ing elevated PTHrP in patients with HCM and breast, lung, kidney, and genitourinary malignancy. Different antibodies and assay technology have resulted in several commercial IRMAs in current use.

Type of SampleEDTA plasma with protease inhibitors is the preferred sam-ple type. Separate within 30 min and store frozen at −20 °C.

Limitations

� Secreted in concentrations high enough to be detect-able in circulation using current assays late in the course of malignancy.

� Requires specific sample collection procedure and sam-ple must remain frozen during transport to laboratory-performing analysis.

Frequency of UseRare. Specialized reference laboratory-supplied assay.

Reference Interval>1.8 pmol/L strongly indicative of PTHrP production by tumor.

1.0–1.7 pmol/L. Detectable PTHrP likely tumor source. Repeat estimation to confirm.

(Commercial PTHrP IRMA assay)

CALCITONINCalcitonin is a 32 amino acid peptide hormone produced by the C cells located predominantly in the thyroid but also present in the parathyroid, thymus, and lung. As with many other peptide hormones, calcitonin is derived from a larger precursor molecule with posttranslational modification cleaving both N- and C-terminal segments. As well as exist-ing in plasma as both mono- and dimeric forms, alternative splicing of mRNA results in heterogeneous circulating pep-tides with both immunological and biological activity.

FunctionThe major action of calcitonin is on bone, where it inhibits osteoclastic resorption. This is achieved by inhibition of osteoclast activity and, in the longer term, a reduction in the number of osteoclasts. Acute intravenous administra-tion decreases plasma calcium, but physiologically calcito-nin is not thought to play a major role in the control of plasma calcium concentrations. A role for calcitonin in minimizing postprandial increases in serum calcium has been suggested. Elevated concentrations are seen in preg-nancy suggesting that a physiological role may be that of skeletal protection.

Clinical ApplicationsPlasma calcitonin is considered important clinically in situations where there is hypersecretion. The classic example is medullary carcinoma of the thyroid. Families in whom this condition is an inherited trait may require screening for excessive calcitonin secretion although genetic testing is a routine procedure in this disease and in MEN2A. Increased calcitonin levels following pro-vocative tests of intravenous calcium, pentagastrin or oral alcohol can be used to confirm the diagnosis, although occasional false negatives occur. Plasma calci-tonin may be useful as a tumor marker in a variety of different conditions.

It is not clear whether calcitonin deficiency per se results in any clinical deficit, although the development of osteo-porosis may be enhanced.


Assay TechnologyEarly RIAs required extraction and concentration tech-niques to detect the low circulating levels of calcitonin. The assay of Hillyard et al. (1977) involved an initial extraction using sepharose beads and elution of calcitonin with acetone. A rabbit antibody was used with 131I calcito-nin as tracer and, after a 24 h incubation, separation was achieved using charcoal. The sensitivity was 4–8 ng/L. Inter- and intra-assay variations were <10% and <14%, respectively. Although the sensitivity can be improved to 2 pg per tube with a 7 day incubation, this is of little use clinically, because the main indication for calcitonin mea-surement is as a tumor marker, where high levels are pres-ent. For the same reason, in the assay of Body and Heath (1983), which used a silica extraction method and a goat antibody, long pre-incubation and incubation periods were involved, and sensitivity was <1 ng/L.

Other techniques, including a bioassay and competitive RIA binding to cell membranes, are largely of historical interest.

Current two-site immunometric assays have satisfactory precision and sensitivity for all clinical uses.

Limitations

� Lack of specificity; most assays use polyclonal antibodies.

� Nonspecific interference with binding from other serum constituents.

� To achieve the desired level of sensitivity, extraction procedures or long incubations are necessary.

Type of SamplePlasma samples should be placed on ice immediately and stored frozen at −20 °C.

Reference IntervalsThere is a marked sex difference in calcitonin concentra-tions with males having higher values than females.

Males: <120 ng/L.Females: <60 ng/L.Pregnancy: <120 ng/L.(RIA)

Biochemical Markers of Bone TurnoverThe activity of cells that regulate bone remodeling, the osteoblasts and osteoclasts, is reflected in the serum concentration of the products of cellular activity. There has been extensive development of biochemical assays that measure markers of both bone formation and resorption. During these phases of bone activity, osteo-blastic and osteoclastic enzymes and other proteins are secreted together with release of components of the organic extracellular matrix. Measurement of these markers of bone turnover in conjunction with the cal-ciotropic hormones provides an important adjunct to

imaging procedures for clinical assessment of the skeleton.

MARKERS OF BONE FORMATION

Alkaline PhosphataseBone-specific ALP (EC3.1.3.1) is a marker of bone forma-tion, since this enzyme is present in the osteoblast mem-brane and appears to play a role in phosphate acquisition in the formation of the hydroxyapatite complex. ALP occurs in the body as four isoenzymes: placental, intestinal, germ cell, and liver/bone/kidney—the latter being the predomi-nant form in serum. The isoforms from bone, the liver and the kidney are encoded by the same gene but posttransla-tional modification gives rise to differences that can be detected electrophoretically. Because of lack of tissue spec-ificity, total ALP activity is of limited value. However, in diseases where there is significant skeletal involvement, such as Paget’s disease of bone, ALP remains a clinically useful test. In patients with less dramatic biochemical changes, such as in osteoporosis, any changes in bone ALP are obscured by the small contribution that they make to the circulating pool of the enzyme. Many assay procedures have been developed to improve identification of bone ALP in plasma. These have relied mainly on electropho-retic characteristics; however, resolution has been improved by heat inactivation, lecithin precipitation, and more recently by immunoassay. Immunoassays are not 100% specific for bone ALP and a 10–15% cross-reactivity with liver ALP is observed. Commercial immunoassays for bone ALP estimate either enzyme activity or mass and concentration, and cross-reactivity differs between these assays.

Type of sample

Total ALPHeparin plasma or serum.

Bone ALPSerum or plasma. Consult manufacturer’s guidelines.

Reference intervals

Total ALPAdult men and women 20–60 years: 20–125 U/L.(Commercial automated platform)

Bone ALPAdult men 20–60 years: 10–40 U/L.

Adult premenopausal women 20–50 years: 10–26 U/L.Adult postmenopausal women 50–90 years: 14–50 U/L

(Commercial enzyme-linked immunosorbent assay (ELISA))

OsteocalcinOsteocalcin, also known as bone γ-carboxy glutamic acid protein (bone gla protein), is the most abundant non-collagenous bone matrix protein, comprising 1–2% of total bone protein. Initially synthesized by osteoblasts and odontoblasts as pro-osteocalcin, a 75 amino acid peptide,


the secreted osteocalcin peptide consists of 49 amino acids and is unique in having three glutamic acid residues in the central region of the molecule, which are carboxylated by a vitamin K-dependent process (Eriksen et al., 1995). Syn-thesis of osteocalcin is dependent on the actions of 1,25(OH)2D3, which promotes transcriptional activation of the osteocalcin gene. Osteocalcin detected in plasma derives almost exclusively from synthesis by osteoblasts since very little is released during the bone resorption. In addition to the intact molecule, a large N-terminal “mid fragment” of 43 amino acids, as well as smaller fragments, has been identified in plasma.

Osteocalcin is cleared by the kidneys, and consequently circulating concentrations are affected by impaired renal function. The plasma half-life is 15–70 min, and there is a pronounced circadian variation with levels peaking during the night and a nadir in the afternoon. Plasma concentra-tions of osteocalcin may be measured by RIA or by ELISA or electrochemiluminescent immunoassay (ECLIA). Variability between assays can be attributed to differing antibody specificities to fragments and the intact mole-cule. In postmenopausal women, plasma osteocalcin is 10–30% higher compared with the concentration in pre-menopausal women. In osteoporosis, the concentration may be normal or slightly raised above the expected post-menopausal range (Price and Thompson, 1995). This reflects the variable bone turnover states observed in this condition and the fact that patients may have high or low osteoblastic activity. However, bone formation is invari-ably reduced relative to levels of resorption. Plasma osteo-calcin concentration is increased in most conditions associated with bone mineralization, but the concentra-tions do not always parallel those seen with bone ALP. Dis-eases characterized by increased concentrations of circulating osteocalcin include Paget’s disease (although this is variable), hyperparathyroidism, hyperthyroidism, osteomalacia, renal osteodystrophy, and acromegaly. Decreased osteocalcin levels have been reported in hypo-thyroidism, hypoparathyroidism, growth hormone defi-ciency, and early pregnancy. Steroid treatment markedly decreases osteocalcin concentrations. There is a need for consensus regarding standardization and collection proce-dures. Clinical studies correlating circulating osteocalcin with other biochemical or bone histomorphometric mea-surements of bone turnover have shown that osteocalcin can be a useful marker of bone formation.

Type of sampleDepends on assay used. Consult manufacturer’s guidelines.

Reference intervalsPremenopausal women: 3.0–7.4 ng/mL.

Men: 2.3–5.4 ng/mL.(Commercially available immunoradiometric assay kit

that detects 1–49 intact osteocalcin (carboxylated and uncarboxylated) and the 1–43 peptide fragment).

Procollagen I Extension PeptidesCollagen is the major bone protein, and over 90% of bone collagen is type I. It is synthesized by osteoblasts as a

precursor molecule, procollagen, which has a central triple helix domain comprising two α1 chains and one α2 chain flanked by carboxy- and amino-terminal extension pep-tides. These extension peptides are cleaved before collagen becomes incorporated into the bone matrix. Measurement of circulating levels of these cleaved peptides by immuno-assay can provide an indication of the rate of collagen type I synthesis.

The procollagen type I carboxy-terminal peptide (PICP) has a molecular weight of approximately 100 kDa and therefore is not subject to excretion by glomerular filtra-tion. It can be detected in the circulation by RIA. The majority of studies of this marker of bone formation have been undertaken with an RIA utilizing an antibody raised against the carboxy-terminus of the propeptide. Increases in PICP in the serum are seen in conditions associated with cancellous bone formation that correlate with other indices such as bone histomorphology and whole-body calcium kinetics where there is coexisting matrix forma-tion and mineralization (Eriksen et al., 1993). However, when these are uncoupled, the correlation is not apparent (Price and Thompson, 1995).

Procollagen type 1 amino terminal peptide (P1NP) circulates in trimeric (~100 kDa) and monomeric (27 kDa) forms with thermal transition taking place of the labile trimeric form at 37 °C. Immunoassays (ELISA, ECLIA, and RIA), some fully automated, have been developed utilizing antibodies directed against the α1 chain of P1NP that measure only the trimeric form or both tri-meric and monomeric forms of P1NP (Orum et al., 1996; Melkko et al., 1996; Garnero et al., 2008). The RIA mea-sures only the trimeric form, and the ELISA/ECLIA measures both high and low molecular weight forms. The differences in the measurement of the two forms of P1NP are thought to reflect degradation of pN-collagen rather than denaturation of the propeptide. Pre-analytical advantages of PINP include a low diurnal and intra-individual variability, and stability at room temper-ature. Circulating P1NP concentrations are directly proportional to the amount of new collagen matrix formed and subsequently newly mineralized bone. P1NP is significantly increased in diseases with increased bone formation/turnover such as Paget’s disease, hyperpara-thyroidism, malignancy involving bone, thyrotoxicosis, and acromegaly. In patients treated with anti-osteoclast therapies such as bisphosphonates, a significant decrease in circulating concentration of P1NP is observed. P1NP concentration increases significantly and rapidly, follow-ing treatment with anabolic agents (Glover et al., 2009), and change in P1NP is a good predictor of BMD response at the end of treatment. It has been suggested that measurement of P1NP be incorporated into an assess-ment algorithm when using injectable daily PTH therapy for osteoporosis (Eastell et al., 2006).

Type of samplePlasma or serum.

Reference intervals

P1CP50–170 µg/L (from Eriksen et al., 1993).


P1NPAdult men, 19–65 years: 20–76 µg/L.Premenopausal women, 19–50 years: 19–69 µg/L.(ECLIA ref. range)

MARKERS OF BONE RESORPTION

Collagen Cross-link Molecules (Pyridinoline and Deoxypyridinoline)The extracellular matrix is stabilized by the formation of covalent cross-links between adjacent collagen chains. There are two major cross-link molecules: hydroxylysyl pyridinoline (PYD) and lysyl pyridinoline (DPD). These molecules form small nonreducible cross-links that stabi-lize the collagen fibrils. PYD is mainly present in cartilage with a small amount in bone. DPD is less abundant than PYD but is almost exclusively found in bone. The two pyridinoline compounds are not degraded by osteoclast resorption of bone or metabolized in vivo and are excreted in urine as free (40%) or peptide-bound (60%) forms. The fact that the cross-link molecules are only found in mature collagen means that the excretion in urine only reflects degradation of mature collagen and does not include collagen, which has been synthesized but not incorporated into collagen fibrils. As the great majority of cross-links in urine are bone derived, there is good correlation between cross-link excretion and bone resorp-tion (Delmas and Garnero, 1998). There is a pronounced circadian variation with the lowest urinary excretion of DPD observed in the early afternoon. Cross-link excretion has been shown to decrease by approximately 30% between 0800 and 1100, and thus standardization of sampling time is of critical importance for serial measure-ments. They are released into the circulation and excreted into the urine when collagen is catabolized, reflecting collagen degradation.

Initial assays of PYD and DPD were performed by HPLC with fluorescent detection, following hydrolytic sample derivatization. A variety of commercial ELISAs mainly measuring free DPD have become available with some incorporated onto automated immunoassay platforms (Eriksen et al., 1995). In order to measure total PYD and DPD (free plus peptide-bound cross-links), acid hydrolysis (4N HCl) of the sample is required.

PYD and DPD are elevated in diseases associated with high bone resorption including Paget’s disease, osteomala-cia/rickets, HCM, bone metastases, myeloma, thyrotoxi-cosis, and immobilization hypercalcemia. They are significantly decreased as a result of anti-osteoclast treat-ment such as bisphosphonate therapy. Due to the large variability in urine samples and the requirement for correction of results to creatinine, their use has largely been superseded by plasma markers of resorption.

Type of sampleFor measurement of urinary-free DPD and PYD, a preser-vative-free random urine sample (first or second morning void collection) or a 24 h urine sample is required. Samples may be stored at −20 °C. Exposure to ultraviolet light should be avoided.

DPD excretion is corrected for urinary concentration by calculating the urine DPD or PYD:creatinine ratio.

Reference intervals

PYDAdult men, 20–80 years: 5.0–21.8 nmol/mmol creatinine.Premenopausal women, 20–50 years: 7.8–21.2 nmol/mmol creatinine.

DPDAdult men 20–80 years: 0.4–6.4 nmol/mmol creatinine.Premenopausal women 20–50 years: 1.8–6.7 nmol/mmol creatinine.

(In-house HPLC assay.)

Pyridinium Cross-Linked Carboxy-Terminal TelopeptidesDuring collagen type I degradation by osteoclasts under the action of cathepsin K, the carboxy-terminal cross-linked telopeptide region is liberated into the circulation as an immunologically intact fragment that resists further degradation. This trimeric antigen has been isolated, and the β-isomerized octapeptide (EKAH(β)DGGR) on the nonhelical carboxy-terminal telopeptide of the type I collagen molecule has been used as an antigen to raise antibodies for immunoassay (Delmas and Garnero, 1998). The CTX synthetic peptide sequence containing the cross-link site can be measured in serum or urine [C-terminal cross-linked telopeptide of type I collagen (CTX)]. There are four isomers of CTX, according to the isomerization of the aspartate (native α- and transformed β-CTX) and to its racemization (L or D). Both racemiza-tion and isomerization increase with tissue age; thus measurement of the different forms could give an insight into the mean age of bone tissue (with α/β higher if bone turnover is increased). There are two commercial EIA assays, measuring the α-CTX and β-CTX in urine, and β-CTX can be measured in plasma/serum with ECLIA and ELISA. Plasma and urine β-CTX values are highly correlated. β-CTX has a circadian rhythm with an increase to peak around 0600–0900 and a nadir at 1500–1700. Food intake significantly decreases plasma βCTX, so sample collection needs to be standardized for optimal results. βCTX concentrations in plasma show a significant correla-tion with the rate of bone resorption as indicated by histomorphometry (Eriksen et al., 1993).

Measurement of the cross-linked N-telopeptide (NTX) of type I collagen in urine and serum has been reported to be a sensitive and specific marker of bone resorption. The antibody in the NTX assays recognizes an epitope of the N-terminal telopeptide of the α-2 chain of collagen I. This telopeptide is liberated from type 1 collagen by osteoclas-tic hydrolysis with cathepsin K. Commercial ELISAs exist in both plate and automated platform formats (Hanson et al., 1992; Clemens et al., 1997). NTX demonstrates a significant circadian rhythm similar to CTX. A near-patient testing device has been developed for urine NTX.

CTX and NTX are elevated in diseases associated with high bone resorption including Paget’s disease, osteomalacia/rickets, HCM, bone metastases, myeloma, thyrotoxicosis,


and immobilization hypercalcemia. They are significantly decreased as a result of anti-osteoclast treatment such as bisphosphonate therapy. Urine NTX has been the bone marker used in the majority of trials and routine clinical assessment of osteoporosis therapies, but the use of plasma/serum-based markers has increased due to ease of collec-tion, improved technical performance, and reduced bio-logical variability.

Type of sampleFor measurement of urinary α/β-CTX or NTX, a preser-vative-free random urine sample (first- or second-morning void collection) or a 24 h urine sample is required. Samples may be stored at −20 °C. Exposure to ultraviolet light should be avoided.

The α/β-CTX or NTX excretion is corrected for urinary concentration by calculating the urine α/β-CTX or NTX creatinine ratio.

Reference intervals

Urine NTXAdult men, 20–80 years: 15–40 BCE/mmol creatinine.Premenopausal women, 20–50 years: 12–38 BCE/mmol creatinine.(Commercial automated immunoassay)

Plasma β-CTXAdult men and premenopausal women: 0.1–0.5 µg/L.(Commercial ECLIA)

Serum Tartrate-Resistant Acid PhosphataseOsteoclasts contain an isoenzyme of acid phosphatase that can be distinguished from prostatic acid phosphatase because it is tartrate resistant (type 5 tartrate-resistant acid phosphatase [TRAP5]). The measurement of total TRAP activity in serum has been used as an index of osteoclast

activity. Total TRAP is influenced by enzymes originating from the erythrocytes and platelets, and its measurement can be hampered by circulating inhibitors. RIAs were originally developed to measure total TRAP but have been replaced by ELISAs performing either mass measurement or enzyme activity of the captured protein. Commercial ELISAs measuring type 5b TRAP, a desialylated isoenzyme present only in osteoclasts and alveolar macrophages, are available. One assay that does not cross-react with other TRAP5 molecules or metabolized fragments is described as a fragment-absorbed immunocapture enzyme assay (FAICEA) system (Ohashi et al., 2007). TRAP5b is secreted by active osteoclasts even when not resorbing bone. Increased TRAP5b concentrations have been described in diseases, characterized by increased bone resorption, such as primary or secondary hyperparathy-roidism, Paget’s disease or metastatic bone disease (Halleen et al., 2001). There are a number of studies on TRAP5b in osteoporosis treatment indicating a significant decrease following anti-osteoclast therapy. TRAP5b correlates with the number of osteoclasts present in bone and metabolism in liver, and subsequent clearance is not affected by renal function. It is relatively unstable on storage and requires rapid analysis or short-term storage at −80 °C.

Type of sampleSerum.

Reference intervalsAdult men, 25–82 years (Japan): 1.7–5.8 U/L.Premenopausal women, 25–55 years (Japan): 1.2–4.4 U/L.(Commercial FAICEA)

LIMITATIONS OF BONE MARKERS

� Bone markers are not diagnostic for a specific disease. They reflect bone formation and resorption.

TABLE 6 Summary of Changes in Ions, Hormones, and Bone Markers in Conditions Associated with Abnormal Calcium and Bone Metabolism

Ca2+ PO42− PTH 25OHD 1,25D ALP β-CTX P1NP

Hypercalcemia1HPT +/++ n/− n/++ n/− n/+ n/+ n/++ n/+HCM +/+++ n/− −/u n/− n/− n/+++ n/+++ n/++Vitamin D intoxication +/+++ n/+ −/u +/+++ n/+++ n/+ n/+ nSarcoidosis +/++ n/+ n/u n/− n/++ n/+ n/+ nHypocalcemiaHypoparathyroid −/−−− +/++ u n n/− n n n2HPT (vitamin D deficient) −/−− −/−− +/+++ −/−− n/−− n/+++ n/++ n/++Pseudohypopara −/−−− +/++ +/+++ n n/− n n nCRF −−/++ +/+++ n/+++ n/− n/−−− n/++ n/+++ n/++Bone diseaseOsteoporosis n n n/+ n/−− n n/+ n/++ n/+Paget’s disease n n n n/− n n/+++ n/+++ n/+++Bone metastases n/+++ +/− n/u n/− n/−− n/+++ n/+++ n/++

1HPT: primary hyperparathyroid; HCM: hypercalcemia associated with malignancy; 2HPT: secondary hyperparathyroid; Pseudohypopara: pseudohypoparathyroid; CRF: chronic renal failure.The range of possible concentrations measured in the clinical scenario is given for each analyte.u: undetectable; −: low; −−−: very low concentration; n: within the reference range; +: elevated; +++: markedly elevated concentration.


� The production of bone markers depends not only on the rate of bone turnover but also on the skeletal size, reflecting mainly trabecular bone turnover, which is four to five times more active than cortical bone.

� A localized bone disease, bed rest, and fracture healing can significantly contribute to bone marker measurements.

� Biological variability for some markers is high, and sampling procedures need to be consistent to ensure optimal clinical utility.

� Urine measurements need to be corrected for creati-nine, and in very dilute samples bone markers can be difficult to quantify accurately.

Summary of Changes in Hormones and Bone Markers in Conditions Associated with Abnormal Calcium and Bone MetabolismPlease see summary of changes in ions, hormones and bone markers in conditions associated with abnormal calcium and bone metabolism in Table 6.

References and Further ReadingAronov, P.A., Hall, L.M., Dettmer, K., Stephensen, C.B. and Hammock, B.D.

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Health & Medicine

calcium absorption

plasmaionized calcium

calcium administration

circulating calcium

g of calcium

plasma calcium results

free ionized calcium

calcium metabolism william