Elecsys Vitamin D

3
(25-OH)
More than a classical bone marker
25-hydroxyvitamin D
Vitamin D seems to be implicated in the development of several
diseases. Insuffcient levels of vitamin D are correlated with an
increased risk of developing non-skeletal pathologies, e.g. car-
diovascular diseases, cancer, autoimmune diseases, diabetes and
pregnancy risks.
1,2

Worldwide it is estimated that 1 billion people have vitamin D
defciency or insuffciency.
2
A vitamin D serum level of at least 30 ng/mL or 75 nmol/L is
desirable.
3

The importance of vitamin D
Vitamin D plays a central role in calcium and
phosphorus metabolism and skeletal health
The natural form of vitamin D is cholecalciferol, vitamin D
3
.
Vitamin D
2
(ergocalciferol) can be found in some plants and
fungi and can also be synthesized by irradiation of plant sterols.
Vitamin D
3
is converted in the liver to 25-hydroxyvitamin D
3

and is further converted in the kidney into the active hormone
1,25-dihydroxyvitamin D
3
.
Until very recently, vitamin D
2
and D
3
were considered to be
interchangeable and equivalent. It has become clear that vita-
min D
2
is substantially less potent than vitamin D
3
.
4,5
Vitamin
D
2
and D
3
seem to be absorbed from the intestine and to be
25-hydroxylated in the liver with equal efficiency, but vitamin
D
2
leads to an increased metabolic degradation of endogenous
vitamin D
3
. Although it is certainly possible to treat patients
satisfactorily with vitamin D
2

6
it seems to have no advantage
over vitamin D
3
.
25-hydroxyvitamin D
3
is the best indicator
to assess the vitamin D status of a patient
Synthesis of vitamin D
Cholecalciterol D
3
7-dehydrocholesterol pre-Vit.D Vit. D
temp. dependent
Isomerization
Increases
Ca
2+
absorption (small intestine)
Urinary calcium re-absorption (kidney)
Bone mineralisation
(25-OH) Vit. D
low Ca
2+
, PO
4
3-
PTH
24,25 (OH)
2
D
1,24,25 (OH)
3
D
+
several other metabolites
UVB radiation
(290 - 315 nm)
Liver
Bloodvessel
Skin
Kidney
Lipoproteins Vitamin D
binding
protein with
bound vitamin D
Albumin
1,25 (OH)
2
Vit. D
active hormone
Synthesis of vitamin D
3
in human skin
The sunlight is an essential part
to convert the proform of vitamin D
to its biologically active form
Fortifies bone and muscle strength
and eliminates rickets
Low bone density is common in adults. Currently over 200 million
people worldwide suffer from low bone density and osteoporosis,
80 % of them are women.
7

Vitamin D supplementation in combination with calcium can
help to decrease the incidence of low bone density, and in turn,
fractures and osteoporosis especially in the elderly.
Vitamin D also plays an essential role in muscle growth and
development
8
and in regulating muscle contractility.
9
The predominant cause for rickets is vitamin defciency. Rickets,
a softening of the bones in children which can lead to fractures
and deformity, is among the most frequent childhood diseases in
less developed countries.
10

The classical role of vitamin D
Cytokine
regulation
Immunoglobin
synthesis
Activated T lymphocyte
Activated B lymphocyte
1-OHase
1-OHase
1-OHase
Decreased
renin Decreased
parathyroid
hormone
Parathyroid hormone regulation Blood pressure regulation
VDR-RXR
VDR-RXR
VDR-RXR
TLR-2/1
24-OHase
25 (OH)D
Calcitroic Acid
1,25 (OH)
2
D
1,25 (OH)
2
D
1,25 (OH)
2
D
1,25 (OH)
2
D
Immunomodulation
Breast, colon, prostate, etc.
Parathyroid glands
Innate immunity
Macrophage/monocyte
25 (OH)D
> 30 ng/mL
Blood
Kidneys
Pancreas
Increased insulin
Increased
cathelicidin
Tuber-
culosis
tubercle
Lipopolysaccharide
or tuberculosis
tubercle
Blood sugar control
Enhances p21 and p27
Inhibits angiogenesis
Induces apoptosis
Increased VDR
Increased 1-OHase
The classical role of vitamin D
Adapted from reference 2
Biologically active 1,25-dihydroxyvitamin D
binds to the vitamin D receptor and targets
more than 200 genes
The importance of vitamin D and potential
health consequences of vitamin D deciency
Increasing evidence from clinical studies links
vitamin D deficiency with a higher risk
of developing several diseases
Vitamin D seems to lower cancer risks
A large population-based casecontrol study assessing the rela-
tionship of serum 25(OH)D concentrations and breast cancer
risk in post-menopausal women shows that the concentration of
serum 25(OH)D was inversely associated with the risk of post-
menopausal breast cancer.
11
An analysis of two studies with more than 800 cases and 800
controls showed that serum 25(OH)D level of 50 ng/mL are asso-
ciated with 50% lower incidence of breast cancer, compared to a
baseline of <10 ng/mL.
12
Results of a randomized trial with more than 1000 healthy women
show that an improved vitamin D nutritional status reduced the
relative risk for all cancers in postmenopausal women by 77%.
13
Vitamin D deciency is associated with several autoimmune
diseases
The net effect of the vitamin D system on the immune response
is an enhancement of innate immunity coupled with multifaceted
regulation of adaptive immunity.
14
In a prospective, nested case-control study (148 cases, 296
controls) among whites, the risk of multiple sclerosis signif-
cantly decreased with increasing levels of 25-hydroxyvitamin D
to > 40 ng/mL. The inverse relation with multiple sclerosis risk
was particularly strong for 25-hydroxyvitamin D levels measured
before the age of 20.
15
In women for every 10 nmol/L increase of serum 25(OH)D level
the odds of MS was reduced by 19%, suggesting a protective
effect of higher 25(OH)D serum levels.
16
Vitamin D deciency is a risk factor for developing
cardiovascular diseases
Vitamin D is important for blood pressure regulation. Low vitamin D
levels can cause cardiovascular diseases including hypertension
17

and hardening of artery walls.
18
Low levels of 25(OH)D ( < 15 ng/mL) are associated with higher
risk of myocardial infarction in a graded manner. This was shown
in a study of > 400 men originally free of diagnosed cardiovascular
disease and who developed myocardial infarction or coronary heart
disease during a 10 year follow-up.
19
Low 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels
are independently associated with all-cause and cardiovascular
mortality. Appropriate serum levels of 25-hydroxvitamin D are
associated with a decrease in mortality. This data have been
obtained in a seven year follow-up study of > 3000 patients
referred for angiography.
20
Vitamin D deciency increases the risk for type 1 diabetes
Meta-analysis of data from four case-control studies showed that
the risk of type 1 diabetes was signifcantly reduced in infants
who were supplemented with vitamin D compared to those who
were not supplemented.
21
In children who received vitamin D supplementation regularly
(daily dose of 2000 IU), the risk for type 1 diabetes was reduced
by about 80% compared with those receiving less.
22
Vitamin D and calcium insuffciency may negatively infuence
glycemia, whereas combined supplementation with both nutri-
ents may be benefcial in optimizing glucose metabolism.
23
10 years follow up in the prospective population based Ely-study
showed an inverse association of baseline serum vitamin D con-
centration with future glucose levels and insulin resistance in 524
non-diabetic adults.
24
Vitamin D deciency seems to increase pre-eclampsia risks
and may inhibit fetal growth
A nested case-control study of pregnant women followed
from less than 16 week gestation to delivery showed a relation
between serum 25(OH)D concentrations at less than 22 week
and risk of preeclampsia.
25
In a longitudinal study involving 198 children, it was found that
children born to mothers with defcient levels of vitamin D (< 11
ng/mL) had signifcantly lower whole-body and lumbar spine bone
mineral content at age 9 years than those whose mothers were
vitamin D replete ( > 20 ng/mL).
26

Vitamin D supplementation of such mothers, especially when the
last trimester of pregnancy occurs during the winter months, could
lead to an enhanced peak bone-mineral accrual and a reduced
risk of fragility fracture in offspring during later life.
Everybody with insufcient sun exposure
2,10

Due to lifestyle reasons and spending most of the time indoors,
many people with insuffcient sun exposure are at risk for vitamin D
defciency.
The elderly
27
The natural process of aging can reduce vitamin D levels as the
mechanisms for vitamin D synthesis become less effcient over time.
Pregnant women
28
Vitamin D plays a vital role in the regulation of cell growth,
immunity and cell metabolism. It is important for the normal
growth of the fetus. Defciencies have been prevalent even in
studies where > 90% of the women took prenatal vitamins.
Who is at risk for vitamin D deciency?
Standardization to the reference method LC-MS/MS
and good comparability with HPLC delivers
a realistic situation on vitamin D levels
Reference method LCTMS [ng/mL]
0
20
40
60
80
100
0 20 40 60 80 100
Passing/Bablok
16-18
Linear regression
y = 0.27 + 1.00x
y = 0.31 + 0.90x
= 0.719
r = 0.903
E
l
e
c
s
y
s

V
i
t
a
m
i
n

D
3

(
2
5
-
O
H
)

a
s
s
a
y

[
n
g
/
m
L
]
HPLC Vitamin D 25 OH [ng/mL]
0
20
40
60
80
100
0 20 40 60 80 100
Passing/Bablok
16-18
Linear regression
y = 2.69 + 1.03x
y = 4.73 + 0.96x
SD (md 95) = 8.14
Sy.x = 5.85
= 0.771
r = 0.936
E
l
e
c
s
y
s

V
i
t
a
m
i
n

D
3

(
2
5
-
O
H
)

[
n
g
/
m
L
]
Reference standardization vitamin D
3
(25-OH) pooled data from
4 external labs show the method comparison Elecsys Vitamin D
3

(25-OH) vs reference method LC-MS/MS.
Method comparison between the Elecsys Vitamin D
3
(25-OH) assay
(y) and the HPLC method (x) on the MODULAR ANALYTICS E170
analyzer in 291 patient samples.
0 10 40 30 20 50 60
25-OH vitamin D [ng/mL]
70 80 90 100
40
35
30
25
20
15
10
5
0
C
V

[
%
]
Roche Competitor
Roche specification
Interassay precision
Interassay precision: Data combined from 3 studies, 3 Roche
controls, 3 human serum sample pools and one additional low
concentrated native human serum in different concentration
ranges (measured at 15 sites on the Elecsys 2010 and MODULAR
ANALYTICS E170 analyzer and four competitor systems as single
determination on 10 days).


+
+
+
1
9 min.
Ru
Ab<VitD>S-lgG

Ru
Bi
Bi-vit.D (25-OH)
3
Detection
2
9 min.
Sample [35L]
Vit D binding protein is
inactivated during the incubation
Sandwich-complex
between ruthenylated antibody and
biotinylated vit. D (25-OH) bound to a SA-microparticle
Ab<Vit.D>S-IgG-Ru = ruthenylated antibody against vit.D
3
(25-OH)
Bi-vit.D (25-OH) = biotinylated vit.D (25-OH)
SA = Streptavidin
SA-Microparticle
Bi
Ru
The Elecsys Vitamin D
3
(25-OH) assay principle
Inactivation of the vitamin D binding protein
is the key for correct analyte recovery
Total assay duration 18 minutes
Assay principle Competitive principle
Sample volume 35 L
Analytical sensitivity 4 ng/ml (10 nmol/L)
Measuring range 4 100 ng/mL (10 250 nmol/L)
Traceability Candidate Reference Method LC-MS-MS
Total precision E2010: CV% 6.6 9.9
E170: CV% 3.7 7.8
Sample material Serum, K3-EDTA plasma, Li-heparin
Reagent stability 8 weeks after first opening
Onboard stability 1 week on E2010, 2 weeks on E170
Dilution Manual 1:2 dilution of samples above the measuring range
Expected values




5 95% percentile: 11 43 ng/mL (27.7 107 nmol/L)
Health based reference values are recommended to replace population
based reference values
Consensus expert opinion: minimal vitamin D
3
(25-OH) level for bone
health: 20 - 32 ng/mL (50 - 80 nmol/L)
more recent consensus of experts: > 30 ng/mL ( > 75 nmol/L) is desirable
3
Key performance overview
31
Why does the Elecsys test specifically measure vitamin D
3

(25-OH) and not vitamin D
2
(25-OH)?
Vitamin D
3
(25-OH) is the main metabolite in human serum or plasma
for assessing the overall vitamin D status
> 95 % of vitamin D (25-OH) in serum is metabolized to vitamin D
3
(25-
OH), not as vitamin D
2
(25-OH)
Is a vitamin D
3
measurement equivalent to or better than a total
vitamin D assay?
The vitamin D
3
assay is not subject to interference from the non-physio-
logic and less potent vitamin D
2
that may also be present in a specimen.
Therefore, a vitamin D
3
assay is preferred to a total vitamin D assay.
2
How should the results of the vitamin D
3
assay be used
by physicians?
The results should be interpreted in an identical manner to the interpre-
tation and use of a total vitamin D assay.

Vitamin D
2
is not endogenously found in humans. In almost all patients,
the results will be similar, except in very rare patients whose major
source of vitamin D is supplementation by vitamin D
2
.
4,5
What is the therapeutic relevance of vitamin D
3
vs. D
2
? According to its molecular structure, vitamin D
2
has a lower half-life
than vitamin D
3
, which reduces its biological activity. This is due to a
formation of vitamin D
2
(24-OH) which has a lower affinity for the
vitamin D receptor.

Vitamin D
2
hampers the hydroxylation of vitamin D
3
, which leads to a
depletion of vitamin D
3
(25-OH).
4,5
Vitamin D
3
and its metabolites have a higher affinity for the vitamin D
receptor and the vitamin D binding protein than vitamin D
2
.
Therefore supplementation with vitamin D
3
is more desirable, since some
years the majority of patients are supplemented with vitamin D
3
.
Why is it possible that different assays may deliver different test
results?
Due to the lack of an accepted international reference preparation for
a vitamin D test every manufacturer uses different ways of traceability
and standardization.
The Elecsys Vitamin D
3
(25-OH) assay is standardized against liquid
chromatography tandem mass spectrometry (LC-MS/MS) which nowa-
days is an accepted reference method in clinical chemistry.
29
Our reference method uses a specific mass transition for vitamin D
3

(25-OH) in combination with two dimensional HPLC which leads to an
accurate and precise determination of vitamin D
3
(25-OH).
What is an optimal vitamin D status? Currently there is no standard definition of the optimal vitamin D status.
It is widely accepted that especially in winter times but also during
summer a high number of normal, apparently healthy individuals have
suboptimal levels of vitamin D.
It should be taken into consideration that differences in vitamin D
3

levels may exist with respect to gender, age, season, geographical lati-
tude and ethnic groups, but these population based reference ranges
should not be taken as clinical cutoff to recommend or dissuade from
vitamin D supplementation. Guidance for supplementation should be
taken from recent literature.
30

Health-based reference values are recommended to replace population
based reference values. There is a consensus opinion that the minimal
vitamin D
3
(25-OH) level for bone health is between 20-32 ng/mL (50-80
nmol/L). A more recent consensus of experts leads to the conclusion
that for general health a desirable concentration of vitamin D
3
(25-OH)
is > 30 ng/mL ( > 75 nmol/L).
The frequently asked questions
of vitamin D testing
COBAS, LIFE NEEDS ANSWERS and ELECSYS
are trademarks of Roche.
2010 Roche
Roche Diagnostics Ltd.
CH-6343 Rotkreuz
Switzerland
www.roche.com
References
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+
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+
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See package insert of Roche Elecsys Vitamin D3 (25-OH) assay. 31.

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