Misurare vitamina d

1. Introduction
25-hydroxyvitamin D (25OHD) determination is of diagnostic
importance for the investigation of vitamin D deficiency and
much more rarely, intoxication. Despite the name, vitamin D is
apre-hormone,beingendogenouslysynthesisedprovidedthere
is adequate sunlight. Its biological function, exerted through
the active form 1,25 dihydroxyvitamin D3
(1,25(OH)2
D) is
to maintain calcium and phosphate levels in the blood.1
In
addition, it has important roles in immune regulation.2
Demand for 25OHD and 1,25(OH)2
D assays has increased
substantially worldwide since the introduction of commercial
kit assays, but performance is relatively poor with only just
over half of laboratories achieving acceptable performance.3
A recently emerging problem is that some immunoassays
underestimate 25-hydroxyvitamin D2
metabolites due to
differences in affinity between the antibodies or D-binding
proteins employed.4
It is likely that achievement of reliable, clinically appropriate
vitamin D results will require a combination of careful use of
commercial systems by skilled operators, validated reference
ranges, good quality control schemes and the availability of
reference methods of analysis.
Vitamin D Metabolism
VitaminD3
(cholecalciferol)isformedintheskinbyphotolysis
of 7-dehydrocholesterol by ultraviolet radiation from sunlight.
Cholecalciferol is transported in the circulation bound to
vitamin D binding protein and is hydroxylated in the liver to
25OHD.Afurther hydroxylation reaction occurs in the kidney
to form the active hormone 1,25(OH)2
D. This reaction is
tightly regulated by induction of the CYP27B1 enzyme which
is stimulated by PTH and inhibited by hyperphosphataemia
and 1,25(OH)2
D.5
Ergocalciferol (vitamin D2
) is produced commercially by
ultraviolet irradiation of a provitamin D sterol (ergosterol)
that occurs in plants.6
It differs from vitamin D3
in having an
additional methyl group at C24 and a double bond at C22-23. It
undergoes the same hydroxylation reactions as cholecalciferol
to form 25OHD2
and 1,25(OH)2
D2
. Ergocalciferol is the only
prescriptionpharmaceuticalavailableinAustralia7
andtheonly
high dose preparation available in the USA8
so D2
metabolites
can represent a significant fraction of the circulating hormone
in patients treated with these preparations (see later).
Nomenclature
Notwithstanding the International Union of Pure and Applied
Chemistry (IUPAC) recommendation9
for the use of the trivial
names calcidiol (25-hydroxyvitamin D, 25OHD) and calcitriol
(1,25-dihydroxyvitamin D, 1,25(OH)2
D), these two terms
have not come into widespread use. As a consequence the
abbreviations 25OHD and 1,25(OH)2
D are used in this review.
Vitamin D Status
Although cholecalciferol and 1,25(OH)2
D can be measured
in the circulation, the best estimates of vitamin D status are
provided by measurement of 25OHD.
10,11
This is due to its
long serum half-life (approximately 3 weeks) and because
the 25-hydroxylation step is unregulated, thus reflecting
substrate availability. A number of commercial kit assays are
available for the clinical laboratory and will be discussed
further.
In contrast, cholecalciferol has a short half-life (approximately
24 h) so that serum levels depend on recent sunlight exposure
and vitamin D ingestion. The assay is difficult due to the
lipophilic nature of the molecule and no commercial versions
are available.12
Since production of 1,25(OH)2
D is tightly regulated and
serum half life is 4-6 h, circulating levels provide limited
information about nutritional vitamin D status. Although
commercial radioimmunoassays and ELISAs are now
available, measurement of 1,25(OH)2
D is principally of
Clin Biochem Rev Vol 26 February 2005 I 33
Analytical Commentary
Improving the Measurement of 25-hydroxyvitamin D
Andrew M Wootton
Division of Laboratory Medicine, RMIT University, Bundoora, VIC 3083, Australia
For correspondence: Dr Andrew Wootton e-mail: andrew.wootton@rmit.edu.au

2. interest only in renal disease and remains the preserve of
specialist laboratories.
Measurement of 25OHD
Approximately 85% of 25OHD is bound to D binding
protein (DBP), 15% to albumin, and 0.03% free.13
Thus
chromatographic separation requires an extraction step to
release 25OHD from its binding proteins and this can be
subject to variable co-precipitation.14
On the other hand,
non-extracted immunoassay methods may be susceptible
to matrix effects, particularly due to the lipophilic nature of
25OHD.15
Taken together with the low (nanomolar) levels
of vitamin D metabolites in serum, these factors have made
the routine measurement of 25OHD an analytical challenge.
Clinical assessment of vitamin D status in patients receiving
ergocalciferol requires measurement that is equally reactive
to both the 25OHD3
and the 25OHD2
metabolites. It has
recently become clear that not all of the currently available
commercial immunoassays will detect 25OHD2
with adequate
sensitivity.15
Although D2
is used in the USA and Australia, D3
is the form given in Europe and so these problems may be
confined to Australia and the USA. However, these analytical
issues may be exacerbated by the less efficient conversion of
D2
to 25OHD2
compared to the D3
forms.16
Furthermore, the
half life of 25OHD2
is shorter and clinical potency is less than
one third.8
Together, this means that the clinical picture in an
ergocalciferol-treated patient may be highly uncertain.
Reference Methods
Definitive methods employ GC coupled with mass
spectrometry detection.17,18
Recently a candidate reference
method using LC-tandem mass spectrometry was published.14
Whilst these reference methods are suitable for validating the
recovery and accuracy of routine methods, their complexity
and derivatisation requirements mitigate against regular use.
HPLC
Alarge number of HPLC methods for vitamin D determination
have been published. Chromatographic separation is suitable
as a reference method, since it is capable of resolving D2
and
D3
forms as well as the 25OH, 1,25(OH)2
and 24,25(OH)2
metabolites. Early assays utilised normal phase separation19,20
and these have been followed by reverse phase separation.21
Many adaptations and variations have been published
(reviewed15,22
). The most widely adopted methods use liquid-
liquid or liquid-solid pre-sample cleanup with UV detection
after column separation. A C18 reverse phase column with
isocratic or gradient elution using acetonitrile/water is now
the standard procedure and diode array detection following
hexane extraction gives results of sufficient clinical
sensitivity.23
Thus determination of 25OHD by HPLC with
UV detection can be considered the gold standard method.15
However, it is accepted that these methods are unsuitable for
routine, clinical laboratory use.
Immunoassay
The earliest immunoassay method used DBP and 3
H-25OHD
tracer.24
An RIA using antibody that is cospecific to both
25OHD2
and 25OHD3
was developed by Hollis25
and was
improved with the use of 125
I-labelled tracer.26
More recently,
chemiluminescent assays have utilised both DBP27
and
antibody-based binding.28
A range of immunoassays is now
commercially available (in Australia, the DiaSorin, IDS, and
Nichols Institute assays comprise 90% of the market29
).
Diasorin RIA
The Hollis assay25,26
was released commercially with
FDA approval and has become widely used. Acetonitrile
extraction is followed by competitive radioimmunoassay
using 125
I-labelled 25OHD and antibody to 25OHD. A second
antibody is used as precipitating agent. Although Hollis found
that the primary antibody recognises 25OHD2
and 25OHD3
equally,4
other authors have published data indicating that the
assay under-recognises 25OHD2
.30
IDS Gamma-B
The sample obtained after acetonitrile extraction is incubated
with antibody to 25OHD in competition with 125
I-labelled
25OHD. A second antibody coupled to cellulose is used for
separation of bound radioactivity. The manufacturers state
that there is 75% cross reactivity of the antibody with 25OHD2
compared to 100% for the 25OHD3
form.31
Interestingly, data
from the DEQAS indicate that the EIA version of this IDS
assay performed better than the RIA, despite using the same
antibody.32
Nichols Advantage
This assay first separates 25OHD from DBPusing a denaturing
agent. Competition is then established in the same sample well
between the sample 25OHD and 25OHD on magnetic particles
for human DBP. Separation utilises the magnetic particles and
detection is by chemiluminescence using acridinium-ester.27
Despite the Nichols Advantage manufacturers earlier claims
for 100% reactivity with 25OHD2
, it has become apparent that
the assay is unable to measure samples containing substantial
amounts of 25OHD2
reliably.15
Recently, data from the UK
quality assurance program has shown that the assay displays
a positive bias (~31%) with 25OHD3
and a negative one with
25OHD2
.32
This under-recovery of 25OHD2
in some patient
samples is now acknowledged in the information supplied
with the kit.
Diasorin Liaison
This chemiluminescent assay has recently become available.
34 I Clin Biochem Rev Vol 26 February 2005
Wootton A

3. Serum is incubated with antivitamin-D coated microparticles
and isoluminol derivative-conjugated 25OHD before
measurement of the chemiluminescent signal.28
The antibody
is said to be same as that used in the DiaSorin RIA33
and the
results correlate well with the radioimmunoassay.34
However,
this assay was found to recognise 25OHD2
more than
25OHD3
,35
an anomaly that remains to be explained.
Comparability of Assays
Standardization of results between methods and laboratories
remains a significant problem.30,36,37
This may be due to
variability with temperature of antigen-antibody or protein-
binding protein interactions as well as to differences in
recognition of the 25OHD2
and 25OHD3
forms. Furthermore,
the Diasorin RIA showed good correlation with HPLC in
experienced hands but compared poorly in a laboratory that
had not validated the method,37
emphasising the need for
good quality assurance. The Nichols Advantage showed poor
comparison with HPLC and overestimated basal 25OHD
levels whilst underestimating exogenous 25OHD3
. Recently
this kit underwent a re-standardisation exercise due to the
results having drifted downwards in the Australian QAP. This
resulted in values increasing by 17% in an unselected patient
population (Martin P, 2004 personal communication).
Aspects of Quality Control
An international quality assurance scheme (DEQAS, UK)
has been in operation since 1989.3
At present there are 16
Australian laboratories participating (DEQAS, personal
communication). Additionally in Australia, the RCPA-
AACB QAP has 36 laboratories participating in the Vitamin
D Endocrine program.29
Results from the DEQAS scheme
have highlighted the 25OHD2
detection problem32
as well
as confirming that only half the participants are achieving
acceptable performance (80% of results within 30% of All
Laboratories Trimmed Mean).
Sample
Serum is the preferred specimen although plasma (EDTA and
Li-heparin) samples are satisfactory.34
Vitamin D analytes
have been shown to be stable for up to 2 weeks at 30°C3
and
to be unaffected by up to 4 freeze-thaw cycles.38
Storage of
frozen serum samples at –20°C for up to one year has also
been reported to cause no loss in vitamin D metabolites.12
Conclusions
Measurement of 25OHD by immunoassay will remain
the method of choice for reasons of convenience, speed,
turnaround and cost. Although the current generation of
commercial assays have shortcomings in defining the vitamin
D status of patients being treated with ergocalciferol, now
that this problem has been recognised, performance will
undoubtedly improve with the development of better assays.
Given the problems caused by ergocalciferol outlined here, it
may also be reasonable to recommend a change to using D3
as
the pharmaceutical of choice.
Continuing efforts to improve laboratory performance and
vigilance with quality assurance programs are required.
Laboratories offering vitamin D assays are urged to participate
in the local and international programs.
The establishment and maintenance of reference assays for
25OHD are required and have been supported by the AACB.
An HPLC assay is being established at RMIT to provide
estimationofboth25OHD2
and25OHD3
metabolitesinplasma
samples so that the performance of routine immunoassays
on quality assurance samples containing mixtures of these
metabolites can be monitored.
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