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
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.
1HPT 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
1. Primary hyperparathyroidism
2. Hypercalcemia of malignancy Metastases in bone e.g., carcinoma of the breast
Humoral hypercalcemia e.g., squamous cell carcinoma of the
Hematological malignancies e.g., multiple myeloma
3. Vitamin D intoxication Iatrogenic
4. Renal disease Iatrogenic (vitamin D metabolites)
Diuretic phase of acute necrosis
5. Sarcoidosis and other
7. Familial benign hypocalcuric
8. Milk alkali syndrome
9. Thiazide diuretics (often transient)
11. Infantile hypercalcemia 24-hydroxylase deﬁciency
(possible genetic cause)
12. Lithium treatment
13. Vitamin A toxicity
14. Addison’s disease
707CHAPTER 9.4 Bone and Calcium Metabolism
Hypoparathyroidism 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.
A 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 DISORDERS
Vitamin 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-
History and examination
No Obvious Drug Causes
Plasma ACa >2.65 mmol/L
on TWO occasions
Normal Renal Function
PTH >3.0 pmol/L*
Non Parathyroid Cause
Vit D Excess,Sarcoid,
If >1.8 pmol/L
Malignancy Very Likely
Malignancy Not Excluded
Familial hypocalciuric hypercalcemia
* 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
Privational vitamin D deﬁciency
Chronic renal disease
Vitamin D-dependent rickets
Other neck surgery
708 The Immunoassay Handbook
Plasma 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
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
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
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
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.
Osteoporosis 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 BONE
Paget’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.
OF THE THYROID
Medullary carcinoma of the thyroid, which may present
as a lump in the neck, is associated with hypersecretion of
709CHAPTER 9.4 Bone and Calcium Metabolism
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
In all immunoassays, the following are important.
G The correct sample type and appropriate sampling
procedures (see Stokes et al., 2011).
G Any initial extraction or displacement procedures
should be efﬁcient and reproducible.
G The tracer should be stable and of sufﬁciently high
G The solid phase employed for the capture antibody
should have a high capacity, giving a linear relationship
between signal and analyte concentrations.
G An efﬁcient separation of bound from free should be
G Internal quality assurance samples should be included
in every assay; these should have concentrations
covering the assay range.
G If available, external quality assurance (proﬁciency
testing) samples should be assayed regularly (2–3 times
VITAMIN D METABOLITES:
25-HYDROXYVITAMIN D AND
Vitamin 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
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.
Interest 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
The 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
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
Vitamin D3 (cholecalciferol)
Vitamin D2 (ergocalciferol)
25-Hydroxyvitamin D3 (25OHD3)
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) or
24,25-Dihydroxyvitamin D3 (24,25(OH)2D3)
1,25-Dihydroxyvitamin D2 (1,25(OH)2D2) or
710 The Immunoassay Handbook
The 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
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
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
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).
Antirachitic 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).
25OHD 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
Sarcoidosis Vitamin D
TABLE 5 Plasma 1,25(OH)2D Concentrations
Causes of Increased 1,25-Dihydroxyvitamin D Levels
1. Physiological: growth, pregnancy, and lactation
6. Vitamin D-dependent rickets type II
Causes of Decreased 1,25-Dihydroxyvitamin D Levels
1. Renal failure
2. Vitamin D deﬁciency
4. Vitamin D-dependent rickets type I
711CHAPTER 9.4 Bone and Calcium Metabolism
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
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.
In 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 20ng/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
by solid-phase chromatography (C18-OH and silica car-
tridges), followed by quantiﬁcation by RIA. A detection
limit of 2.4ng/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
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
712 The Immunoassay Handbook
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 20mL 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 5ng/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 1nmol/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 25pg/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, 5ng/L (12pmol/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 15ng/L (36pmol/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 3.9ng/L (9.1pmol/L) for 1,25(OH)2D2 and
3.4ng/L (8.2pmol/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 Sample
Plasma or serum, store frozen.
G Low 25OHD concentrations indicate vitamin D deﬁ-
ciency. Diagnosis of osteomalacia requires
G Immunoassays have variable cross-reactivity with
25OHD2, and so care is required interpreting results in
patients receiving ergocalciferol supplementation
G 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
G 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.
G 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 Use
High. 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
Low. Specialized bone and endocrine units and renal
G Vitamin D deﬁciency may be deﬁned as a serum
G A serum 25OHD of 30–50nmol/L may be inadequate
for bone health in some people.
G A serum 25OHD >50nmol/L is sufﬁcient for bone
health for almost the whole population.
(commercially available RIA method, IOM
40–120pmol/L (adults) (RIA; Hollis, 1997).
PTH, 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).
In the circulation, PTH (1–84) has a short half-life (less
than 5min) compared with the biologically inactive middle
713CHAPTER 9.4 Bone and Calcium Metabolism
and carboxy-terminal fragments (20–30min). 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.
In 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.
Immunometric 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
Types of Sample
Each 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 (<30min),
with rapid analysis, may be required to obtain an accurate
G 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.
G C-terminal fragments produced in renal failure have
variable cross-reactivity in different commercial intact
PTH (1–84) assays.
G Optimal sample type (EDTA plasma, heparin plasma,
or serum) may be different for each available commer-
cial assay for PTH (1–84).
Frequency of Use
High. Guidelines for the treatment of CRF and mineral bone
disorders (MBD) incorporate PTH (1–84) concentrations.
714 The Immunoassay Handbook
(Commercial immunometric assay for intact PTH)
A 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 Action
The 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.
Initial 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 0.23pmol/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 0.1pmol/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 Sample
EDTA plasma with protease inhibitors is the preferred sam-
ple type. Separate within 30min and store frozen at −20°C.
G Secreted in concentrations high enough to be detect-
able in circulation using current assays late in the course
G Requires speciﬁc sample collection procedure and sam-
ple must remain frozen during transport to laboratory-
Frequency of Use
Rare. Specialized reference laboratory-supplied assay.
>1.8pmol/L strongly indicative of PTHrP production by
1.0–1.7pmol/L. Detectable PTHrP likely tumor source.
Repeat estimation to conﬁrm.
(Commercial PTHrP IRMA assay)
Calcitonin 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.
The 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
Plasma 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
It is not clear whether calcitonin deﬁciency per se results
in any clinical deﬁcit, although the development of osteo-
porosis may be enhanced.
715CHAPTER 9.4 Bone and Calcium Metabolism
Early 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 24h incubation, separation was
achieved using charcoal. The sensitivity was 4–8ng/L.
Inter- and intra-assay variations were <10% and <14%,
respectively. Although the sensitivity can be improved to
2pg 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 <1ng/L.
Other techniques, including a bioassay and competitive
RIA binding to cell membranes, are largely of historical
Current two-site immunometric assays have satisfactory
precision and sensitivity for all clinical uses.
G Lack of speciﬁcity; most assays use polyclonal
G Nonspeciﬁc interference with binding from other
G To achieve the desired level of sensitivity, extraction
procedures or long incubations are necessary.
Type of Sample
Plasma samples should be placed on ice immediately and
stored frozen at −20°C.
There is a marked sex difference in calcitonin concentra-
tions with males having higher values than females.
of Bone Turnover
The 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
MARKERS OF BONE FORMATION
Bone-speciﬁc ALP (EC188.8.131.52) 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
Type of sample
Heparin plasma or serum.
Serum or plasma. Consult manufacturer’s guidelines.
Adult men and women 20–60 years: 20–125U/L.
(Commercial automated platform)
Adult men 20–60 years: 10–40U/L.
Adult premenopausal women 20–50 years: 10–26U/L.
Adult postmenopausal women 50–90 years: 14–50U/L
(Commercial enzyme-linked immunosorbent assay
Osteocalcin, 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,
716 The Immunoassay Handbook
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 15–70min, 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 sample
Depends on assay used. Consult manufacturer’s
Premenopausal women: 3.0–7.4ng/mL.
(Commercially available immunoradiometric assay kit
that detects 1–49 intact osteocalcin (carboxylated and
uncarboxylated) and the 1–43 peptide fragment).
Procollagen I Extension Peptides
Collagen 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
The procollagen type I carboxy-terminal peptide (PICP)
has a molecular weight of approximately 100kDa 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 (~100kDa) and monomeric (27kDa)
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 sample
Plasma or serum.
50–170µg/L (from Eriksen et al., 1993).
717CHAPTER 9.4 Bone and Calcium Metabolism
Adult men, 19–65 years: 20–76µg/L.
Premenopausal women, 19–50 years: 19–69µg/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
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 sample
For measurement of urinary-free DPD and PYD, a preser-
vative-free random urine sample (ﬁrst or second morning
void collection) or a 24h 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.
Adult men, 20–80 years: 5.0–21.8nmol/mmol creatinine.
Premenopausal women, 20–50 years: 7.8–21.2nmol/mmol
Adult men 20–80 years: 0.4–6.4nmol/mmol creatinine.
Premenopausal women 20–50 years: 1.8–6.7nmol/mmol
(In-house HPLC assay.)
Pyridinium Cross-Linked Carboxy-Terminal
During 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,
718 The Immunoassay Handbook
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-
Type of sample
For measurement of urinary α/β-CTX or NTX, a preser-
vative-free random urine sample (ﬁrst- or second-morning
void collection) or a 24h 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.
Adult men, 20–80 years: 15–40BCE/mmol creatinine.
Premenopausal women, 20–50 years: 12–38BCE/mmol
(Commercial automated immunoassay)
Adult men and premenopausal women: 0.1–0.5µg/L.
Serum Tartrate-Resistant Acid
Osteoclasts 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 sample
Adult men, 25–82 years (Japan): 1.7–5.8U/L.
Premenopausal women, 25–55 years (Japan): 1.2–4.4U/L.
LIMITATIONS OF BONE MARKERS
G 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
2− PTH 25OHD 1,25D ALP β-CTX P1NP
1HPT +/++ n/− n/++ n/− n/+ n/+ n/++ n/+
HCM +/+++ n/− −/u n/− n/− n/+++ n/+++ n/++
Vitamin D intoxication +/+++ n/+ −/u +/+++ n/+++ n/+ n/+ n
Sarcoidosis +/++ n/+ n/u n/− n/++ n/+ n/+ n
Hypoparathyroid −/−−− +/++ u n n/− n n n
2HPT (vitamin D deﬁcient) −/−− −/−− +/+++ −/−− n/−− n/+++ n/++ n/++
Pseudohypopara −/−−− +/++ +/+++ n n/− n n n
CRF −−/++ +/+++ n/+++ n/− n/−−− n/++ n/+++ n/++
Osteoporosis 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
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.
719CHAPTER 9.4 Bone and Calcium Metabolism
G 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.
G A localized bone disease, bed rest, and fracture healing can
signiﬁcantly contribute to bone marker measurements.
G Biological variability for some markers is high, and
sampling procedures need to be consistent to ensure
optimal clinical utility.
G 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
Please see summary of changes in ions, hormones and
bone markers in conditions associated with abnormal
calcium and bone metabolism in Table 6.
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