227

LABORATORY INSIGHTS
Proceedings of Singapore Healthcare Volume 22 Number 3 2013
Vitamin D Measurements Facts and Fancies
Tar Choon Aw, FRCP (Edin), FRCPA, Clementine Yap, MSc, DLM(ASCP)
Department of Laboratory Medicine, Changi General Hospital, Singapore
ABSTRACT
Clinical laboratories have seen an increased interest in vitamin D measurements as new knowledge about the
vitamins pathobiology escalates. Clinicians now recognise that besides bone health, vitamin D insufficiency
may be associated with potential health risks such as coronary artery disease, diabetes, depression, epilepsy,
polycystic ovaries, musculoskeletal pain, autoimmune disease, multiple sclerosis, and cancers. While vitamin D
testing and supplementation has been touted for these patients, benefit is evident only for the prevention of falls
and fractures. The main form of vitamin D measured is 25-hydroxy-vitamin D (25OHD). While 25OHD assays have
improved, they remain a work in progress.
Keywords: 1,25-dihydroxyvitamin D3 (Calcitriol), Automated immunoassays, Liquid chromatography-tandem
mass spectrometry (LC-MS/MS), Vitamin D2 (Ergocalciferol), Vitamin D3 (cholecalciferol)
INTRODUCTION
There has been over 25,000 scientific articles on
vitamin D since 2000
1
. A scholarly monograph
on vitamin D with over 2000 pages is into its
third edition
2
. An international gathering of
experts on vitamin D met in March 2012 and their
deliberations are available
3
. Over the last decade
there has been great interest in the measurement of
vitamin D given the availability of rapid automated
assays for 25-hydroxy-vitamin D (25OHD). New
understanding on vitamin D pathophysiology
has emerged from its extra-skeletal roles. Arising
from the unbridled measurements of 25OHD
worldwide is the concern over the possibility
of widespread inadequate vitamin D status in
the population. Consequently, many guidelines
and recommendations have emerged over
intake of vitamin D, use of supplements, and
exposure to sunlight. To understand the intense
fascination with vitamin D we briefly review its
new pathobiology and highlight the vagaries of
25OHD analysis.
VITAMIN D PATHOBIOLOGY
Metabolism
The metabolism of vitamin D is increasingly
complex
48
, but some elaboration is necessary
to understand its newer actions and the vagaries
of its measurement. Vitamin D exists in two
forms vitamin D2 (ergocalciferol) and vitamin
D3 (cholecalciferol). Vitamin D2 is derived from
ergosterol, a yeast sterol found in mushrooms.
Vitamin D3 is synthesised in the skin from photo-
degradation of 7-dehydrocholesterol present in the
suprabasal layers of the epidermis
7
. Commercially,
vitamin D2 is manufactured through the ultraviolet
irradiation of ergosterol, and vitamin D3 through
the ultraviolet irradiation of 7-dehydrocholesterol
from lanolin
9
. Both forms of vitamin D are used in
fortified foods and vitamin supplements but the
type of vitamin D present may differ for different
products and in different countries; prescription
vitamin D is often ergocalciferol. The major source
of vitamin D in children and adults is dermal
synthesis of vitamin D3
10,11
. Both vitamins D2 and
D3 are believed to be bioequivalent
8
, but their
relative efficacy has been debated
1214
.
Ingested vitamin D is incorporated into
chylomicrons, absorbed into the lymphatic system,
and enters the blood stream. Vitamin D from the
skin (D3) and gut (D2 and D3) is transported to the
liver within hours by proteins principally vitamin
D binding protein (DBP), and albumin; 99% of the
228
Laboratory Insights
Proceedings of Singapore Healthcare Volume 22 Number 3 2013
D is protein bound. Vitamin D is biologically inert
and requires hepatic hydroxylation to 25-hydroxy-
vitamin D or calcidiol (25OHD) by the vitamin
D-25-hydroxylase, a microsomal P450 cytochrome
CYP2R1. Other minor 25-hydroxylases include
CYP27A1 and CYP3A4. While calcidiol formation
is not regulated and depends largely on vitamin
D supply, its long half-life of several weeks renders
25OHD a good index of vitamin D supply and hence
reserve in the body.
Calcidiol requires a further hydroxylation by the
renal-1--hydroxylase, a mitochondrial P450
cytochrome CYP27B1. The end-product is the
biologically active form of vitamin D, calcitriol or
1,25-dihydroxy-vitamin D [1,25(OH)2D]. Calcitriol
binds to a nuclear receptor, the vitamin D receptor
(VDR), present in the kidneys, small intestine, and
bone. VDR, a member of the nuclear receptor
superfamily, heterodimerizes with the retinoid
X receptor. Together with coregulatory proteins,
VDR binds to vitamin D response elements
resulting in the induction or suppression of
vitamin D target genes. In the kidney, 1,25(OH)2D
stimulates proximal tubule calcium reabsorption.
In the intestine, 1,25(OH)2D stimulates calcium
and phosphate absorption. In bone, 1,25(OH)2D
stimulates the expression of receptor activator of
nuclear factor -B (RANK) ligand or RANK-L. RANK-L
then interacts with RANK to induce maturation of
osteoclasts from immature monocytes to mobilise
calcium and phosphate from the skeleton in the
process of bone resorption. Calcitriol is vital for
calcium regulation and bone health
15
because
of its counter-regulatory effects on parathyroid
hormone (PTH), a promoter of bone loss. Calcitriol
interacts with VDR in the parathyroid gland to
suppress PTH synthesis.
The calcium endocrine regulatory network
is expanding
1618
. Renal systems ensure that
1,25(OH)2D synthesis is responsive to the bodys
calcium needs via the stimulatory effects of PTH on
CYP27B1. Production of 1,25(OH)2D also depends
on the bodys phosphate requirements. Phosphate
homeostasis is controlled by fibroblast growth
factor 23 (FGF23). FGF23 acts on the parathyroid and
kidney. When serum phosphate is elevated, FGF23
inhibits CYP27B1 and thus 1,25(OH)2D synthesis.
1,25(OH)2D in turn, induces the release of FGF23
from bone and Klotho (another cytokine) from
the kidney. Klotho is a co-receptor for FGF23. The
phosphatemic actions of vitamin D are opposed by
the phosphaturic effects of FGF23. PTH is repressed
by 1,25(OH)2D, FGF23, and calcium, whereas FGF23
is induced by 1,25(OH)2D, PTH, and phosphate.
Calcitriol also exerts control over its own synthesis
by direct feedback inhibition on CYP27B1 activity.
In addition, 1,25(OH)2D further modulates its own
activity through enhancing the expression of the
24-hydroxylase, CYP24A1, which metabolises
25OHD, and 1,25(OH)2D into less active water-
soluble forms. These metabolites, also bound by
DBP, can circulate in substantial concentrations
(ng/mL) and interfere with some of the 25OHD
assays. In the failing kidney these complex renal-
vitamin D metabolic processes become even
more complex
19,20
.
A simplified scheme of vitamin D metabolism is
given in Figure 1 below.
Fig. 1. Current understanding of vitamin D metabolism.
229
Vitamin D Measurements Facts and Fancies
Proceedings of Singapore Healthcare Volume 22 Number 3 2013
Extraskeletal Effects
VDR as well as CYP27B1 is present in most tissues
and cells in the body including skin, colon, brain,
pancreas, and breast as well as activated T and B
lymphocytes, monocytes, and macrophages
57
.
Thus local production of 1,25(OH)2D in these
tissues from 25OHD in an autocrine or paracrine
fashion may then be responsible for modulating
the 200 genes
21
that govern many of the pleiotropic
effects ascribed to vitamin D (e.g. cancer,
cardiac, cerebrovascular, immune, pancreatic,
vascular)
8,10,2225
. Although observational studies
support a strong case for an association between
vitamin D and its extra-skeletal effects, a true
causal link between poor vitamin D status and
these diseases has not been proven
26,27
. Meanwhile,
interest in this area is rapidly mounting. A recent
comprehensive review
26
contains notable take-
home messages:
serum levels of 24 ng/mL of 25OHD appear
beneficial for musculoskeletal health; vitamin D
may have immune function as it may be involved
in the pathogenesis of infections and autoimmune
diseases; vitamin D deficient individuals are at
increased risk of cardiovascular disorders; risk
for breast and colorectal cancer decreases as
serum 25OHD level increases to 3040 ng/mL
(75100 nmol/L); all-cause mortality in the general
population appears lowest at 25OHD levels ranging
from 3045 ng/mL (75112.5 nmol/L).
An interesting report
28
has suggested that the
extraneous effects of vitamin D may be related
to the influence of its binding protein (DBP)
concentration on serum 25OHD levels.
VITAMIN D ASSAYS
Tests Commonly Included
Calcium, Phosphate, Alkaline Phosphatase, Parathyroid
hormone.
Specimen
Serum or plasma (ethylenediaminetetraacetic acid
and heparin) are acceptable specimens for analysis.
Methodology
2932
Serum concentration 25OHD is the best indicator
of vitamin D status as it reflects both cutaneous
vitamin D3 production and dietary vitamin D (D2
and D3). In addition, it has a fairly long circulating
half-life of three to four weeks. In contrast,
1,25(OH)2D has a short circulating half-life of
only four hours and is a thousand-fold lower in
concentration than 25OHD. Moreover, the decline
in 1,25(OH)2D in vitamin D deficiency occurs late. In
addition its serum levels are affected by hormones
and several drugs (bisphosphonates, heparin,
ketoconazole, and steroids).
Calcidiol (25-OHD) is challenging to measure
accurately
33,34
. Analytical difficulties include its
hydrophobic and lipophilic nature, strong affinity
to its binding protein DBP, binding to serum
albumin, wide range of 25OHD concentrations
in clinical samples, cross-reacting D metabolites
(e.g. 24,25-dihydroxy-vitamin D, C3-epimer of 25-
OHD), and assay interference from heterophilic
antibodies. Heterophilic antibodies are
encountered at rates of less than 1 in 1000 clinical
samples. In the original assay, 25OHD in serum is
extracted from its binding proteins with an organic
solvent (e.g. acetonitrile or ethanol) followed by
liquid chromatography to separate 25OHD from
other interfering D metabolites before entering
a protein-binding assay. This procedure is time-
consuming and labour-intensive.
Several types of assays are now available for
measurement of serum 25OHD, but the two most
common platforms are immunoassays
35
and mass
spectrometry-based
36
methods. The first automated
25OHD immunoassay (Nichols Advantage)
employed DBP in a binding protein assay. This
assay, beset with many problems (under- and over-
estimation, non-linearity, and imprecision), has
been discontinued. DiaSorin and Roche introduced
assays that employed specific antibodies to capture
25OHD. The DiaSorin LIAISON polyclonal antibody
assay cross-reacted completely with both 25OHD3
and 25OHD2, but the Roche assay could not detect
25OHD2. While this drawback was acceptable in
Europe where vitamin D2 containing supplements
were rare, it was not suitable in the United States
of America and the United Kingdom where vitamin
D2 containing supplements were common. The
Abbott ARCHITECT and Siemens Centaur have a
competitive format but different modes for signal
generation. The Abbott assay underestimates
25OHD2 while the SIEMENS method overestimates.
Roche has launched a new total 25OHD assay,
which uses recombinant DBP instead of an antibody
for analyte capture. The method is standardised
against liquid-chromatography tandem mass
spectrometry (LC-MS/MS) with traceability to the
National Institute of Standards and Technology
230
Laboratory Insights
Proceedings of Singapore Healthcare Volume 22 Number 3 2013
(NIST) standard reference material (SRM) 972
available since 2009. However, the new assay cross-
reacts with the 25-OHDepi metabolite. There is
still concern about the performance of all 25OHD
assays today
3743
. Clinical laboratories offering
vitamin D as a routine service usually measure
25OHD on automated immunoassay platforms
from Abbott, Roche, or Siemens that they already
use for other analytes.
Some laboratories have migrated to LC-MS/MS due
to local arrangements and higher reimbursement.
While LC-MS/MS is generally considered the gold
standard and a reference method is available, many
laboratories do not harmonise their procedures to
this standard
43
. Many variables can affect LCMS/
MS methods, including assay standardisation/
calibration, chromatography, and MS conditions.
MS/MS alone cannot distinguish metabolites such
as 3-epi-25OHD3. Interference can also occur
if non-standard conditions are employed. The
introduction of reference material NIST SRM 972 has
improved the comparability of LC-MS/MS methods,
but proficiency testing programmes still show
substantial variation between them. Inspection
of the 2013 College of American Pathologists
(CAP) proficiency testing program data for 25OHD
reveals the marked variation between methods
including LC-MS/MS and even within the same
vendor (Tables 1 and 2, please see overleaf ). For
a sample with low 25OHD (BGS-01) Diasorin, IDS
and Siemens methods recorded much higher
values compared to LC-MS/MS. For a sample with
higher 25OHD (BGS-03) Roche instruments gave
very low values (1/3 of LC-MS/MS) while Diasorin
and IDS were 3-fold and Siemens 5-fold LC-MS/
MS concentrations. Such a broad spread of values
reported for the same sample tested on different
analytical platforms is clearly unsatisfactory.
Limitations
Clinicians should always know their assay and be
cognizant of their limitations. Repeat testing using
the same or different method should be considered
if the 25OHD result is inconsistent with the clinical
impression. They should be particularly aware
of discrepant results in haemodialysis patients,
probably due to possible matrix effects from urea
38

as well as cross-reacting metabolites.
A change in 25OHD assay can have a significant
impact on results, patient classification, and
treatment recommendations especially when
applying vitamin D treatment guidelines. Two assays
(IDS-RIA and Diasorin Liason) agreed in only 41
68% of subjects, depending on the criteria used to
define agreement and on the desired target 25OHD
level 20 ng/mL versus 30 ng/mL
45
. It underscores
the urgent need for assay standardisation, akin to
the National Glycohemoglobin Standardization
Program for HbA1c. A start has been made with
the United States (US) National Institutes of
Health Office of Dietary Supplements Vitamin
D Standardization Program in 2010 and the
recent US Center for Disease Control Vitamin D
Standardization-Certification Program
46
.
Melanin in the epidermis reduces synthesis of
provitamin D, with consequent lower substrate
availability for 25OHD formation. Thus subjects
with darker skin have lower serum 25OHD levels
47
.
The Endocrine Society
5
feels that such subjects
are at risk for vitamin D deficiency and should be
screened for 25OHD while the Institute of Medicine
disagrees. It is noteworthy that in Singapore
25OHD levels decline in proportion to skin
pigmentation and gender: Chinese males > Malay
males > Chinese females > Malay females > Indian
males > Indian females
48
. However, the role of skin
pigmentation and vitamin D is complex and needs
further investigation
49
.
Obesity is also associated with low 25OHD levels,
mostly because vitamin D is sequestered in
adipose tissue
50
. With the growing popularity of
probiotics in the modern diet, increased circulating
25OHD has been reported following oral intake
of probiotics
51
.
VITAMIN D TESTING
Reference Values
The desired level of serum 25OHD remains
unclear, as does the dose of vitamin D needed for
replacement and maintenance of optimum health.
Most authorities agree that vitamin D deficiency
can be defined as a 25OHD level of < 20 ng/mL
9
.
When serum 25OHD levels fall below 30 ng/mL
there is an associated increase in PTH signifying
a relative insufficiency of vitamin D. Thus serum
25OHD > 30 ng/mL is believed to indicate vitamin
D sufficiency
9
. The Endocrine Society echoed this
view in its clinical practice guidelines
5
. The Institute
of Medicine published dietary reference intakes
for vitamin D
52
and disputed this decision limit
of 30 ng/mL
5355
prompting a firm rebuttal from
the endocrinologists
56
. The two different views of
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Vitamin D Measurements Facts and Fancies
Proceedings of Singapore Healthcare Volume 22 Number 3 2013
Table 1. 25OHD Results from the CAP Proficiency Testing 2013 Survey Set BGS-01.
Method Participating laboratories (n) Mean (ng/mL) Standard deviation C.V. (%)
^
Abbott Architect i 45 0.94 1.41 151.1
Abbott Architect i 135 < 13.0*
Diasorin, Liaison 206 15.83 4.17 26.4
Diasorin, Liaison 7 < 7.0*
IDS EIA 31 14.28 4.8 33.6
IDS iSYS 38 13.02 4.15 31.9
LC-MS/MS 15 4.74 1.63 34.3
LC-MS/MS 13 < 5.0*
Mass Spectrometry 10 5.32 1.54 29.1
Siemens Advia Centaur/XP 245 22.53 6.48 28.7
Siemens New Reagent 150 22.84 6.29 27.5
Roche Cobas e411/Elecsys 23 < 5.0*
Roche Cobas e6000/E170 53 <5.0*
^
Co-efficient variation (C.V. %): ;* Laboratories reporting their results as below the lower detection limit of their assay
Table 2. 25OHD Results from the CAP Proficiency Testing 2013 Survey Set BGS-03.
Method Participating laboratories (n) Mean (ng/mL) Standard deviation C.V. (%)
^
Abbott Architect i 162 15.05 1.19 7.9
Abbott Architect i 19 < 13.0*
Diasorin, Liaison 192 51.85 5.77 11.1
Diasorin, RIA 11 18.86 2.72 14.4
IDS EIA 31 47.64 7.64 16.0
IDS iSYS 40 38.05 10.85 28.5
LC-MS/MS 29 16.99 2.00 11.8
Mass Spectrometry 13 16.08 1.34 8.3
Roche Cobas e411/Elecsys 15 5.59 1.15 20.5
Roche Cobas e411/Elecsys 8 < 5.0*
Roche Cobas e600/E170 35 5.44 0.82 15.1
Roche Cobas e600/E170 25 < 5.0*
Siemens Advia Centaur/XP 231 75.33 10.01 13.3
Siemens New Reagent 143 75.82 10.19 13.4
* Laboratories reporting their results as below the lower detection limit of their assay
Table 3. Recommendations for Vitamin D Insufficiency.
Institute of Medicine Endocrine Society
Vitamin D status 25OHD level (ng/mL) Vitamin D status 25OHD level (ng/mL)
Increased risk deficiency < 12 D deficient < 20
Increased risk of inadequacy < 16 D insufficient 2129
Adequate > 20 D sufficient 30-100
Increased risk of excess > 50
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Laboratory Insights
Proceedings of Singapore Healthcare Volume 22 Number 3 2013
vitamin D insufficiency are summarised in Table 3
(please see overleaf ).
Indications for Testing
57
Over the past few years, requests for serum
vitamin D levels, especially in the paediatric and
geriatric populations, have greatly increased due
in part to the availability of rapid and reasonably
reliable 25OHD methods. It behooves the
clinician to consider the patients clinical history,
presentation, and functional outcomes when
prescribing and monitoring a patient on vitamin
D supplementation. In children, 25OHD levels
are determined and followed for the diagnosis
and treatment of vitamin D-dependent rickets
while the elderly are monitored during vitamin D
treatment for such diseases as osteoporosis and
osteomalacia. There is no evidence of benefit for
vitamin D deficiency screening at a population
level. Screening for vitamin D deficiency is
recommended for individuals at risk for vitamin
D deficiency. The Endocrine Society guidelines
5

include 25OHD testing for the following
fourteen conditions:
Rickets, Osteomalacia, Osteoporosis, chronic
kidney disease, malabsorption syndromes,
hyperparathyroidism, medications (acquired
immune deficiency syndrome, seizures, antifungals,
steroids, cholestyramine), granulomatous
disorders, some lymphomas, older adults with a
history of fractures, older adults with a history of
non-traumatic fractures, obesity (children and
adults with BMI > 30), pregnancy and lactating
women, African Americans and Hispanics.
However, the Institute of Medicine is against
25OHD screening for the last four conditions on
the Endocrine Society list
55
. Widespread testing
of Vitamin D status in asymptomatic people
is unhelpful
58
.
Despite the many diseases purported to be
associated with vitamin D deficiency, randomised
clinical trial data demonstrating the benefit of
vitamin D supplementation only exists for the
prevention of falls and fractures
5961
. The US
Preventive Services Task Force (USPSTF) has
concluded that there is insufficient evidence of
benefits or harm to take more than 400 IU of
vitamin D3 for primary prevention of fractures in
postmenopausal women. USPSTF also recommends
against daily supplementation with less than
400 IU of vitamin D3
62
. Consequently, 25OHD
measurement should be restricted to subjects at
high risk of falls or fractures. Controversy regarding
the desired therapeutic levels of 25OHD remains.
Until resolved, widespread adoption of screening
programmes and measurement of 25OHD even in
at-risk patients may seem premature. Conclusions
about causality extrapolated from observational
data are tenuous
63
. While concomitant vitamin D
and calcium supplements (in appropriate doses)
might reduce the risk of fractures in elderly
osteoporotics (diagnosed on the basis of dual
energy x-ray imaging scans), routine measurement
of 25OHD in these people is questionable since
treatment is likely to include vitamin D supplements
irrespective of the result
58
.
There is a critical need for large-scale randomised
clinical trials (RCTs) to determine the role, if any, of
vitamin D supplementation in extra-skeletal health
outcomes. At least one such RCT is currently under
way in cardiovascular disease (CVD). The Vitamin
D and Omega-3 Trial is currently randomising
25,000 healthy older men and women throughout
the US to receive either 2000 IU of vitamin D3 per
day or placebo, as well as 1 g of omega-3 fatty
acids (fish oil) per day or placebo, for five years.
The primary outcomes of cancer, heart disease,
and stroke will be self-reported and confirmed by
medical record review
1
; first results are expected
by 2017. In the mean time more reports on vitamin
D and its cardiac effects accumulate, including
association of race
64
and mortality during acute
coronary events
65
. A large recent meta-analysis of
12,000 deaths showed strong and independent
associations of 25OHD with vascular and non-
vascular mortality, thus arguing against its causal
relationship to CVD
66
.
CONCLUSION
Over the past 1015 years, vitamin D deficiency
has been associated with many adverse health
outcomes
67
beyond merely bone health
68
,including
skeletal muscle
69
, pregnancy
70
, reproduction
71
, cancer
72
,
immune function
73
, allergic diseases
74
, infections
75
,
type 2 diabetes
76
, non-alcoholic steato-hepatitis
77
,
cardiovascular disease
78
, multiple sclerosis
79
, cognitive
decline
80
, and all-cause mortality
81
. Increasing media
coverage of these findings has compounded the
increase in vitamin D testing and expenditure on
vitamin D supplements. In light of the complexities
of vitamin D pathobiology as well as the vagaries of
vitamin D testing any associations between vitamin
233
Vitamin D Measurements Facts and Fancies
Proceedings of Singapore Healthcare Volume 22 Number 3 2013
D and disease states must remain tentative. Equally,
the use of vitamin D tests must be judicious as they
remain a work in progress.
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