Methylmalonic acid: A biomarker for vitamin B12 deficiency

DIALOG
Methylmalonic acid:
A biomarker for
vitamin B12 deficiency
C hromSystemS
2014/1
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Prof Dr Rima Obeid, Department of Clinical Chemistry and Laboratory Medicine,
University Hospital of the Saarland, Homburg/Germany
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Page 1–3
Methylmalonic acid: A biomarker for
vitamin B12 deficiency
Prof Dr Rima Obeid, University Hospital of the
Saarland, Homburg/Germany
Vitamin B12 deficiency
Subclinical vitamin B12 (cobalamin) deficiency frequently
occurs and over the years numerous clinical symptoms may
become apparent [1, 2]. These include neurological and
psychological disorders, anaemia, pregnancy complications and miscarriages. However, through early diagnosis
and vitamin B12 supplementation, irreversible neurological
damage and other health problems can be prevented.
Recent studies have shown that additional intake of vitamin B12 along with B6 and folic acid in the form of dietary
supplements may protect against age-related diseases [3].
In contrast to bacteria, some of which can themselves synthesise vitamin B12, humans have to intake the vitamin through
the consumption of foods of animal origin. So in vegans
(83 %) and vegetarians (65 % of the total cohort) increased
levels of methylmalonic acid (MMA) in plasma/serum are
found indicating a disturbed vitamin B12 metabolism. For
vegetarians, the need for vitamin B12 cannot be met through
consumption of eggs, milk and milk products, therefore, the
vitamin should be supplied through dietary supplements [4].
In general, in the western population absorption of vitamin B12 from food is situated over the recommended daily
dose for adults of 2.4 µg per day. In the German population,
regardless of gender, this value is about twice as high (NVS II,
National Nutrition Survey 2012) [5]. This situation offers
no adequate explanation for the widespread vitamin B12
deficiency found in some population groups.
Especially in elderly people (> 60 years) and in people who,
due to a malfunction, are unable to reabsorb the ingested
vitamin B12, it can lead to a clinically significant vitamin B12
deficiency, even though enough vitamin B12 may be taken
from food [6]. In addition, certain conditions have a strong
influence over the vitamin B12 absorption [7]. Thus, gastrointestinal disturbances are associated with a vitamin B12
deficiency including achlorhydria (a missing or inadequate
acid production in the stomach), the presence of antibodies
against the intrinsic factor, the intake of H2 blocker, or an
infection with Helicobacter pylori.
Physiology
In order to understand how a vitamin B12 deficiency arises, the
physiology of the vitamin must first be considered. Between
70 and 80 % of the vitamin B12 in plasma are bound to the
®
transport protein haptocorrin. This percentage is inactive
and cannot be absorbed by the cells (Fig. 1). Transcobalamin is another blood serum transport protein that binds
the remaining 20–30 % of the vitamin B12 and thus is also
called holotranscobalamin (holoTC) or the “active B12”. Only
holoTC can bind a specific receptor on the cell and ensure
the supply of vitamin B12. In the cell, vitamin B12 serves as a
cofactor of two important enzyme reactions. After binding to
the cytosolic methionine synthase, homocysteine (HCY) can
be converted into methionine. In mitochondria, it is required
for the conversion of methylmalonyl-CoA into succinyl-CoA.
An excess of methylmalonyl-CoA is converted into MMA.
Lack of vitamin B12 results in an increased concentration of
HCY and MMA in plasma [8] as well as in an increase in
the amount of MMA in urine [9].
In the case of vitamin B12 deficiency due to inadequate
intake or due to impaired absorption, the holoTC level
decreases first in the plasma. Next, the gradual increases
in the concentration of MMA and HCY can be detected
in the plasma. Clinical symptoms, which often have nonspecific manifestations, occur only at a relatively advanced
stage of undersupply. A reliable diagnosis of a vitamin B12
deficiency is therefore only possible through the combined
determination of holoTC and MMA in serum. In clinical
studies, in which holoTC or total B12 concentrations were
compared with MMA concentrations, it was found that
holoTC is a better indicator than total B12 of an inadequate
level of vitamin B12 supply [10]. Therefore, with an elevated
MMA level a decreased holoTC value can be anticipated
[10]. Thus, the MMA concentration can be used as a marker
for the vitamin B12 status [11, 12].
The holoTC level in plasma is a direct indicator for the amount
of “active B12” that is available to the cells. There are two
possibilities, if a low holoTC concentration, generally from
< 35 pmol/l, is identified: 1. the individual affected has
either an existing condition of early negative balance (B12
depletion) or 2. an advanced deficiency caused by a B12
related cell reaction disorder, resulting in a high MMA-level
in plasma or urine. The increased plasma concentration of
MMA occurs mainly in people with a holoTC < 35 pmol/l,
but it has also been detected at concentrations of up to
75 pmol/l [10]. It is therefore strongly recommended that
MMA levels should be measured at least in samples with a
holoTC value of less than 75 pmol/l. In particular patients
who exceed this limit, and who moreover have unexplained
neurological symptoms, gastrointestinal disorders, diabetes
Page 3–4
LC-MS/MS analysis of MMA in plasma/
serum and urine
Katrin Bernhardt, Dr habil. Richard Lukačin,
Chromsystems GmbH
Page 5–7
Nutritional importance of vitamin D
Prof Dr Jutta Dierkes, Dr Frank Hirche,
University of Bergen, Norway,
Martin Luther University Halle-Wittenberg,
Germany
Page 8–9
Special vitamin D diagnosis in infants:
meaning of the C3-epimers
Dr Inga Unterieser, Dr habil. Richard Lukačin,
Chromsystems GmbH
Page 10–11
Vitamin B1 and B6 analysis in veterinary
medicine – comparison of HPLC and
UHPLC
Dr Anja Müller, Sebastian Raich,
Samuel Bauder, Vet Med Labor GmbH,
Ludwigsburg/Germany
Page 12
News/Calendar/Quiz/Imprint
DIALOG 2014/1
Page 2
mellitus or kidney diseases should be investigated for a lack
of vitamin B12. The intake of certain medications, a vegetarian
diet as well as a higher age also enhance the probability
of an insufficient provision of vitamin B12.
Laboratory diagnosis of MMA
The reference method for the analysis of MMA in plasma
is GC-MS, a time-consuming method that can only be
performed in a few laboratories. However, an LC-MS/MS
method (see next article) has recently been developed, which
enables a fast, reliable and reproducible determination of
this biomarker. To assess vitamin B12 status, the MMA concentration in plasma/serum or urine should be determined:
1. The MMA concentration specifically increases in blood
and urine of individuals with vitamin B12 deficiency. The
blood HCY concentration is also increased with vitamin B12
deficiency, but also with deficiency in folate and therefore
cannot be used as a specific marker for the assessment of
a potential vitamin B12 undersupply.
2. In combination with a holoTC laboratory test the determination of MMA concentration is an important criterion
for distinguishing vitamin B12 depletion from an intracellular
deficiency of this cofactor. A low holoTC plasma level in
conjunction with a normal MMA concentration suggests that
the vitamin B12 concentration is too low in the plasma only. In
contrast, a low holoTC level and a high MMA concentration
of > 270 nmol/l in plasma indicates that there is insufficient
supply of vitamin B12 for B12-specific intracellular processes.
3. MMA is a valuable marker to identify and optimise the
effect of vitamin B12 supplementation and for monitoring the
success of the B12-treatment. MMA concentration decreases
approximately 1–2 weeks after initiation of vitamin B12
supplementation, depending on the dosage and the initial
MMA concentration.
4. In patients with renal diseases, where both MMA and
holoTC levels are high, a significant reduction of MMA
concentration (by > 270 nmol/l) as a result of a vitamin B12
treatment is a simple and reliable way to identify a vitamin B12 deficiency that existed in any of these patients before
treatment. The MMA diagnosis should be performed before
and 2–4 weeks after vitamin B12 supplementation [13].
5. The determination of MMA in serum/plasma enables
a reliable diagnosis of vitamin B12 deficiency in patients
that have been treated with metformin (a drug for the oral
treatment of diabetes mellitus). Plasma levels of vitamin B12
(holoTC and total vitamin B12) are low in patients treated with
metformin [14]. It has therefore been assumed for some time
that metformin may cause a vitamin B12 deficiency. However,
the decrease in vitamin B12 and/or holoTC-concentration
in the plasma of patients treated with metformin cannot be
associated with a change in MMA plasma concentration
so far [15,16].
6. For vitamin B12 status determination or respectively for
nutritional assessment of the population’s vitamin B12 supply,
the determination of total vitamin B12 is insufficient (Austrian
Nutrition Report 2012). It is preferable that the holoTC level is
determined first, followed by analysis of MMA concentration.
Critical reflections on MMA
There are some restrictions on the use of MMA as a biomarker for vitamin B12 status:
1. MMA plasma levels may be non-specifically increased in
patients with renal insufficiency [12, 17, 18] . This false-positive
result, however, can be countered by measuring MMA levels
before and after supplementation with vitamin B12.
2. MMA plasma levels may be significantly elevated in
people with bacterial overgrowth, as intestinal bacteria,
among others, produce propionic acid, which is subsequently
converted into MMA [19]. In this case, the holoTC level is
in the normal range, the MMA concentration is increased
and does not substantially decrease following treatment
with vitamin B12.
There is a broad consensus that subclinical vitamin B12 deficiencies occur frequently. However, to date it is not clear whether patients with low holoTC and elevated MMA levels actually already have a clinically significant vitamin B12 deficiency.
In most cases the vitamin B12 status is established by determination of the plasma levels for the whole vitamin bound
to the transport proteins, although it is well known that
only the active portion bound to transcobalamin should
be considered. It can therefore be concluded that this test
is inadequate in the majority of cases. For example, from
analysing 1,034 samples a lower vitamin B12 level was
only detected in 27 [12]. However, after determination
of the holoTC value in 254 samples and after measurement of the MMA concentration in plasma in 184 samples, a critically low vitamin B12 status was revealed [12].
Currently, MMA determination is more expensive than tests
for determination of total vitamin B12 content. However,
considering the number of cases with actual vitamin B12
deficiency and relating that to the high cost of clinical complications due to false-negative test results, it appears that
the slightly higher cost could be amortised by an earlier
administration of vitamin B12 supplements.
Conclusion
MMA determination provides valuable information on vitamin B12 status and enables the early and specific diagnosis
of vitamin B12 deficiency. In addition, MMA is a good marker
for monitoring the success of the treatment and is of great
benefit for selecting the vitamin supplement‘s administration
method, its dosage, form and duration.
References
3. MMA plasma levels can also be increased in smokers.
Compared to non-smokers, a slower decrease of MMA concentration in vitamin B12 supplementation can be observed
[9]. The reasons for this are not yet known.
4. Standard values for MMA concentration in plasma are
below 271 nmol/l. Various studies point out higher limits
and, according to our estimation, even an increased MMA
plasma level of > 450 nmol/l is probably clinically relevant,
while values of 271–450 nmol/l are borderline, even so
should be reduced by supplementation.
[1] Obeid R, Schorr H, Eckert R, Herrmann W. (2004) Vitamin B12 status in the elderly as
judged by available biochemical markers. Clin Chem 50(1): 238–41.
[2] Herrmann W, Obeid R, Schorr H, Geisel J. (2005) The usefulness of holotranscobal
amin in predicting vitamin B12 status in different clinical settings.
Curr Drug Metab 6(1): 47–53.
[3] Douaud G, Refsum H, de Jager CA, Jacoby R, Nichols TE, Smith SM, Smith AD. (2013)
Preventing Alzheimer‘s disease-related gray matter atrophy by B-vitamin treatment.
PNAS 110(23): 9523–8.
[4] Herrmann W, Schorr H, Obeid R, Geisel J. (2003) Vitamin B12 status, particularly
holotranscobalamin II and methylmalonic acid concentrations, and hyperhomocystein
emia in vegetarians. Am J Clin Nutr 78(1): 131–6.
[5] Deutsche Gesellschaft für Ernährung e. V. Bonn. (2012) 12. Ernährungsbericht.
DGE 40–85.
[6] Henoun LN, Noel E, Ben AM, Locatelli F, Blickle JF, Andres E. (2005) Cobalamin
deficiency due to non-immune atrophic gastritis in elderly patients. A report of 25
cases. J Nutr Health Aging 9(6): 462.
[7] Howard JM, Azen C, Jacobsen DW, Green R, Carmel R. (1998) Dietary intake of
cobalamin in elderly people who have abnormal serum cobalamin, methylmalonic
acid and homocysteine levels. Eur J Clin Nutr 52(8): 582–7.
[8] Hvas AM, Lous J, Ellegaard J, Nexo E. (2002) Use of plasma methylmalonic acid in
diagnosing vitamin B12 deficiency in general practice. Scand J Prim Health Care
20(1): 57–9.
[9]
Hill MH, Flatley JE, Barker ME, Garner CM, Manning NJ, Olpin SE, Moat SJ, Russell J,
Powers HJ. (2013) A vitamin B12 supplement of 500 µg/d for eight weeks does
not normalize urinary methylmalonic acid or other biomarkers of vitamin B12 status
in elderly people with moderately poor vitamin B12 status. J Nutr 143(2): 142–7.
[10]Herrmann W, Obeid R. (2013) Utility and limitations of biochemical markers of
vitamin B12 deficiency. Eur J Clin Invest 43(3): 231–7.
[11] Bor MV, Nexo E, Hvas AM. (2004) Holo-transcobalamin concentration and transco
balamin saturation reflect recent vitamin B12 absorption better than does serum
vitamin B12. Clin Chem 50(6): 1043–9.
[12] Clarke R, Sherliker P, Hin H, Nexo E, Hvas AM, Schneede J, Birks J, Ueland PM,
Emmens K, Scott JM, Molloy AM, Evans JG. (2007) Detection of vitamin B12
deficiency in older people by measuring vitamin B12 or the active fraction of vitamin
B12, holotranscobalamin. Clin Chem 53(5): 963–70.


[13] Obeid R, Kuhlmann MK, Kohler H, Herrmann W. (2005) Response of homocys
teine, cystathionine, and methylmalonic acid to vitamin treatment in dialysis patients.
Clin Chem 51(1): 196–201.
[14] de Jager J, Kooy A, Lehert P, Wulffelé MG, van der Kolk J, Bets D, Verburg J, Donker AJ,
Stehouwer CD. (2010) Long term treatment with metformin in patients with type 2
diabetes and risk of vitamin B12 deficiency: randomised placebo controlled trial.
BMJ 340: c2181.
[15]Obeid R, Jung J, Falk J, Hermann W, Geisel J, Friesenhahn-Ochs B, Lammert F,
Fassbender K, Kostopoulus P. (2013) Serum vitamin not reflecting vitamin B12 sta
tus in patients with type 2 diabetes. Biochimie 95(5): 1056–61.
Figure 1: Source of a vitamin B12 deficiency.
A vitamin B12 deficiency occurs either through insufficient intake or insufficient absorption. In both cases, first the holoTC in plasma decreases, then
the concentrations of biochemical markers (MMA and HCY) increase. Only holoTC can enter the cell to deliver B12. An elevated MMA level
suggests that the cells do not receive enough vitamin B12 for an optimal activity of the mitochondrial enzyme (methylmalonyl-CoA mutase).
[16] Greibe E, Trolle B, Bor MV, Lauszus FF, Nexo E. (2013) Metformin lowers serum
cobalamin without changing other markers of cobalamin status: A study on women
with polycystic ovary syndrome. Nutrients 5(6): 2475–82.
[17]Lindgren A. (2002) Elevated serum methylmalonic acid. How much comes from
cobalamin deficiency and how much comes from the kidneys? Scand J Clin Lab
Invest 62(1): 15-9.
[18] Hvas AM, Juul S, Gerdes LU, Nexo E. (2000) The marker of cobalamin deficien
cy, plasma methylmalonic acid, correlates to plasma creatinine.
J Intern Med 247(4): 507–12.
[19] Lindenbaum J, Savage DG, Stabler SP, Allen RH. (1990) Diagnosis of cobalamin
deficiency: II. Relative sensitivities of serum cobalamin, methylmalonic acid, and
total homocysteine concentrations. Am J Hematol 34(2): 99–107.
DIALOG 2014/1
Page 3
Central atom
Vitamin B12
The coenzyme B12 is the active form of vitamin B12 and an important representative of cobalamins that harbours the trace
element cobalt as the central atom. It is a cofactor of two enzymes
which are among others involved in the synthesis of amino acids.
The demand of the human body can be met almost entirely of
products of animal origin such as fish, meat, eggs and milk.
LC-MS/MS analysis of MMA in plasma/serum
and urine
Katrin Bernhardt, Dr habil. Richard Lukačin, Chromsystems GmbH
Vitamin B12 deficiency is more common in the population
than would be expected; an inadequate supply can cause
various hematologic, gastrointestinal and neurological diseases [1]. Disorders occurring in the nervous system due to
vitamin B12 deficiency appear non-specific, but left untreated
cause permanent damage. The affected vulnerable groups
mainly include the elderly, vegetarians, pregnant women,
smokers and also patients with autoimmune, kidney and
various intestinal diseases [2, 3]. An appropriate diagnosis
for the early detection of vitamin B12 deficiency is therefore
absolutely essential.
Vitamin B12 content is usually determined by the serum analysis
of holo-transcobalamin (holoTC), the metabolically active
form of vitamin B12. Due to its insufficient specificity and
sensitivity, this laboratory parameter is of limited relevance
as, in the worst case, it can lead to an incorrect assessment
of the physical supply status. In any event, 10 % of patients
with normal holoTC levels in serum show a deficiency in the
essential cofactor of amino acid metabolism [4]. A possible
misjudgement can be prevented by the additional supply
of a far more sensitive, selective and also endogenously
existing biomarker, methylmalonic acid (MMA), which is
Propionyl-CoA
biotin-dependent
Propionyl-CoA carboxylase
D-methylmalonyl-CoA
D,L-methylmalonyl-CoA racemase
O
O
HO
OH
CH3
L-methylmalonyl-CoA
L-methylmalonyl-CoA mutase
Methylmalonic acid
vitamin B12-dependent
Succinyl-CoA
Figure 1: Conversion of propionyl-coenzyme A into succinyl-coenzyme A
DIALOG 2014/1
Page 4
a dicarboxylic acid. Vitamin B12 is, among other things, a
cofactor in methionine metabolism and is also needed in the
conversion of methylmalonyl-coenzyme A (methylmalonylCoA) into succinyl-CoA (Fig. 1). Vitamin B12 deficiency
leads to a strong increase in the concentration of MMA in
plasma, serum and urine [1] at a very early stage, when the
decrease in vitamin B12 concentration in serum is not necessarily detectable [5]. Also, compared to the photosensitive
vitamin B12 molecule, MMA molecules have significantly
greater stability in patient samples, which has brought the
dicarboxylic acid into the focus of laboratory medicine.
Chromsystems GmbH has now taken this into account and
developed a reagent kit for the specific determination of
MMA in plasma, serum and urine using LC-MS/MS. Key
aspects of this innovative methodology are the use of cleanup tubes, so that patient samples are “gently purified“.
Furthermore, the visible chromatographic separation of the
MMA-isobaric succinic acid avoids false-positive test results
(Fig. 2 and 3). Finally, the use of a specific internal standard
minimises matrix effects, thereby ensuring the precision and
robustness of the method (Fig. 3A and 3B). In addition,
for calibration of the data system matrix-specific 3PLUS1®
multilevel calibrators are provided for plasma/serum as well
as for urine. Quality controls are also available for plasma/
serum and urine.
Conclusion
O
MMA is being increasingly used as a laboratory diagnostic parameter for early detection of vitamin B12 deficiency. The analysis of MMA has the advantage that
this sensitive and specific biomarker can reveal shortages much sooner than the classical analysis of vitamin B12, and thus make an early diagnosis possible.
Chromsystems offers a complete solution for LC-MS/MS
that is characterised by fast sample preparation, minimised
matrix effects, high precision and robustness. The new
MassChrom® reagent kit for the determination of MMA in
serum/plasma and urine ensures the safe, reliable and rapid
determination of vitamin B12 status, which allows an early
start for treatment in the event of deficiency.
HO
O
O
O
HO
OH
Molecular formula:
C4H6O4
OH
Succinic acid
CH 3
Molecular formula:
C4H6O4
Methylmalonic acid
Figure 2: Chemical structures of the isobaric compounds methylmalonic acid and succinic acid
(A) Plasma sample
100 %
Succinic acid
90 %
80 %
70 %
60 %
50 %
40 %
(B) Urine sample
30 %
Methylmalonic acid
20 %
10 %
100 %
Succinic acid
90 %
0%
0.0
80 %
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
2.0
2.2
2.4
2.6
2.8
Time, min
70 %
60 %
50 %
Methylmalonic acid
40 %
30 %
100 %
20 %
Internal Standard
90 %
10 %
80 %
0%
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
Time, min
70 %
60 %
50 %
40 %
30 %
20 %
100 %
10 %
Internal Standard
90 %
0%
0.0
80 %
0.2
0.4
0.6
0.8
1.0
70 %
1.2
1.4
1.6
1.8
Time, min
60 %
50 %
Figure 3: Chromatograms of
(A) plasma sample and (B) urine
sample with physiologically
normal methylmalonic acid
concentration
40 %
30 %
20 %
10 %
0%
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
Time, min
References
[1] Erdogan E, Nelson GJ, Rockwood AL, Frank EL. (2010) Evaluation of reference
intervals for methylmalonic acid in plasma/serum and urine.
Clin Chim Acta 411(21–22): 1827-9.
[2] Herrmann W, Obeid R. (2008) Ursachen und frühzeitige Diagnostik von Vitamin B12 Mangel. Dtsch Ärztebl 105(40): 680–5.
Product Information
MMA in Plasma
Kit for
400 analysis
[3] Müller MJ. (2007) Ernährungsmedizinische Praxis: Methoden-Prävention-Behand
lung, Springer-Verlag.
Specifications
Sample Preparation
Linearity: 2–600 μg/l
Limit of quantification: 3.5 μg/l
Recovery: 104 %
Intraassay: CV = 2.3–2.5 %
Interassay: CV = 3.2–3.6 %
Analysis time: 3.5 min
→ Pipette 100 μl sample/
calibrator/control in
Clean-Up Tubes.
→ Add 50 μl Internal Standard
and mix (vortex) briefly.
→ Centrifuge 10 min at
14 000 x g.
→ Transfer filtrate into an auto sampler vial.
→ Injection volume: 10–20 µl
Linearity: 86–9500 μg/l
Limit of quantification: 86 μg/l
Recovery: 98 %
Intraassay: CV = 1.8–2.2 %
Interassay: CV = 2.4–2.9 %
Analysis time: 3.5 min
→ Pipette 200 μl sample/
calibrator/control in transpa rent reaction vials.
→ Add 25 μl Internal Standard and mix 10 s (vortex).
→ Centrifuge 10 min at
14 000 x g.
→ Dilute sample with Dilution Buffer in an autosampler vial.
→ Injection volume: 10–20 µl
[4] Koch CA. (2005) Diskussion zu dem Beitrag Gesundheitliche Bedeutung der Folsäurezufuhr. Dtsch Ärztebl 102(4): A215.
[5]
Rasmussen K, Nathan E. (1990) The clinical evaluation of cobalamin deficiency by
determination of methylmalonic acid in serum or urine is not invalidated by the
presence of heterozygous methylmalonic-acidaemia.
J Clin Chem Clin Biochem 28(6): 419–21.
MMA in Urine
Urine set for
200 analysis
DIALOG 2014/1
Page 5
Nutritional importance of vitamin D
Prof Dr Jutta Dierkes, Dr Frank Hirche, Institute of Medicine, Section of Clinical Nutrition, University of Bergen, Norway;
Institute of Agricultural and Nutritional Sciences, Martin Luther University Halle-Wittenberg, Germany
Figure 1: Number of publications with the keyword “vitamin D“ in the
database of the National Institute of Health (www.pubmed.org) up to
end 2012.
An important reason for the increasing interest in vitamin D
is the realisation that this cholesterol-derived vitamin clearly
has important functions in metabolism besides calcium,
phosphate and bone metabolism, the significance and
mechanisms of which have only recently been elucidated
or are still not fully understood. Epidemiological studies
support these findings and suggest a link between a vitamin D undersupply and the occurrence of several chronic
diseases, such as diabetes mellitus, hypertension, cancer,
as well as cardiovascular and autoimmune diseases [1, 2].
In this context it should be noted that studies on vitamin D
have been classified into a series of different hypotheses on
vitamins that, over the last 30 years, have led to a veritable
“vitamin hype”. Ultimately, however, there is a lack of a
1.Radioimmunoassay (IDS, Frankfurt/Deutschland)
2.LC-MS/MS (MassChrom® reagent kit Chromsystems,
Munich/Germany).
Table 1: Overview of the different hypotheses that increased vitamin intake could turn out to be a chronic disease prevention or treatment.
Years
Vitamins
Assumed effects
1980s
Antioxidants
(vitamin E, vitamin C, carotenoids)
Anti-cancer effect, Anti-aging (oxidative stress as the clock of aging)
1990s
B vitamins
(folaet, vitamin B12, vitamin B6)
Heart attack prevention (lowering of blood homocysteine concentration)
2000s
Vitamin D
Anti-cancer effect, etc.
clear proof for the causality of the increased intake of these
vitamins and the prevention of chronic diseases (Table 1).
In this comparison, however, we note that the initial profile
of vitamin D is completely different from the antioxidative
vitamins that were in focus during the 1980s or the B vitamins
during the 1990s. In contrast to the latter, the supply situation
of vitamin D can be assessed to be critical, which has been
shown in several population studies in both the U.S. and
Europe [3, 4]. Thus, in Germany according to the National
Nutrition Survey I, about 2 % of adults have severe and 15 %
moderate vitamin D deficiency [5]. If a 25-hydroxy vitamin D
(25-OHD) concentration of 50 nmol/l is specified as the
threshold for an adequate supply, only about 60 % of adults
are adequately supplied with vitamin D [5, 6]. In children and
adolescents, the supply situation is similarly unfavourable [7].
Data from other European countries show a similar picture [8].
Although it was possible to determine the 25-OHD3/25OHD2 concentration using HPLC, it was decided to use an
LC-MS/MS measurement. This was due to the ease of sample
preparation (simple precipitation of proteins and extract
of the supernatant versus solid-phase extraction on HPLC
with UV detection) and the more interference-free analysis.
Given the available mass spectrometer (API 2000 of AB
Sciex), the method-specific parameters (especially sensitivity)
had first to be determined. For that purpose, the variation
coefficient was determined at various concentrations and
used to establish a lower quantification limit of < 13 nmol/l
for 25-OHD2 and < 19 nmol/l for 25-OHD3. The relatively
low sensitivity is device-specific and the lower limit may be
significantly lower with more sensitive equipment.
After gathering the measurement data and comparing them
by Passing-Bablok regression, the y-value of 1.05 x - 6.77
and the limits of the 95 %-confidence interval of y = 0.97x - 10.98
and y = 1.14 x - 2.48 (Fig. 2) show a shift of the regression
lines. The cause of this shift and the outlier values can probably be attributed to the cross-reactivity of the antibody
used in RIA.
LC-MS/MS, 25-OHD3 + 25-OHD2, nmol/l
The reasons for the insufficient supply situation are the low
vitamin D intake with food as well as the limited exposure
to UVB radiation that causes vitamin D synthesis in the skin.
Data from the National Nutrition Survey II determined the
mean vitamin D intake at 2–3 µg per day. At the same
time, more than 40 % of the vitamin originates from fish
and seafood, so people who do not consume fish have an
even lower intake [9]. In other European countries, such
as the UK or the Netherlands, vitamin D intake amount is
comparably low. These figures show that already up to
early 2012, the majority of the population did not reach
the DGE recommendations for vitamin D intake in the
amount of 5 µg per day. According to the recent increase
in the intake recommendation to 20 µg per day, a safe
vitamin D intake with food alone is no longer feasible [10].
However, assessment of vitamin D status on the basis
of its intake from foods is problematic since, with adequate UVB exposure, enough vitamin D can be formed
in the skin, making nutritional intake largely unnecessary. Therefore, concentration of 25-OHD3/25-OHD2 in
serum/plasma is used for determining vitamin D status.
For analysis of this onefold hydroxylated transport form
of vitamin D, a number of different methods are available
whose measurement results can only be partly compared
with each other due to methodological differences [11].
This means that it is always necessary to have information regarding the assay supplied, which also suggests the need for laboratories to participate in external quality controls, such as the DEQAS or similar [12].
In our laboratory, a method was needed for establishing
25-OHD measurement. To select a suitable one, an immunological method and an LC-MS/MS method were compared.
For this purpose, the Vitamin D concentration was determined
in 82 serum samples and the efficiency of the tests were
subsequently compared. The following methods were tested:
RIA, 25-OHD, nmol/l
Figure 2: Determination of the 25-OHD concentrations by LC-MS/MS
and RIA in patient samples (n = 82) according to Passing and Bablok.
The outlier in RIA-values are probably traceable to the antibody‘s
cross-reactivity.
The method comparison in the Bland-Altman diagram shows
a mean difference of 4.7 nmol/l with a 95 %-confidence interval of the difference between 15 and 24.4 nmol/l (Fig. 3).
Difference (RIA, 25-OHD; LC-MS/MS, 25-OHD3 + 25-OHD2), nmol/l
Interest in vitamin D has been dramatically increasing in
both the scientific as well as lay press for some time. This is
illustrated by the number of scientific publications in PubMed
(www.pubmed.org), the database of the National Institute
of Health (Fig. 1), an increasing demand for requirements to
determine vitamin D status as well as numerous publications.
Mean of methods RIA and LC-MS/MS (nmol/l)
Figure 3: 25-OHD concentration in patient samples (n = 82) is determined using RIA and LC-MS/MS in a method comparison according
to Bland and Altman.
After establishing the Chromsystems MassChrom® reagent kit, a
control sample over 39 series with a mean value of 49 nmol/l,
and a coefficient variation of 7.8 % was determined for
further quality testing of the method. The high quality of
the method used was also confirmed by participation in six
consecutive DEQAS proficiency tests and by complying with
the intervals specified by the organiser (www.deqas.org).
DIALOG 2014/1
Apart from method-specific differences in determining 25OHD serum concentrations, the determination of a lower
limit for the definition of vitamin D deficiency is also not
easy. 25‑OHD serum levels below 25 nmol/l are generally
considered to be too low and, in many cases, it is believed
that the optimum 25-OHD concentration is reached at
50 nmol/l. Some well-known authors disagree with this view
and provide a value of 75 nmol/l for optimal supply [13].
However, the subjects‘ health status should be included in
the assessment. Since the 25-OHD levels undergo a significant annual rhythm, there is also the question of whether
this limit should be achieved throughout the year, or as a
peak concentration at the end of summer.
Generally, it is assumed that 50 nmol/l 25-OHD is sufficient and American and German professional societies recommend that this limit be maintained (Table 2).
Interestingly, there is conflicting evidence concerning the
required amount of vitamin D intake. American professional societies recommend an intake of 15 µg of vitamin D,
whereas 20 µg are considered optimal in Germany.
It is clear that no matching statements are available on this
issue. Generally, it is assumed that an additional vitamin D
intake of 2.5 µg (100 International Units) can increase
the 25-OHD serum level by an average of 1–2 nmol/l.
However, the effect probably depends strongly on initial
25-OHD levels. Moreover, vitamin D supplement intake
only reaches a new plateau in serum concentration after
an initial treatment period of 6–8 weeks. In addition, vitamin D3 increases 25-OHD serum levels significantly more
than vitamin D2 does [18, 19]. These and other individual
factors mean that it is difficult to conclude from the daily
amount of vitamin D intake the 25-OHD serum levels [20].
Page 6
Table 2: Comparison of vitamin D intake recommendations in different countries (all data in µg/day; the rickets prophylaxis for infants recommended in all countries is not included) [10, 14–17].
Scandinavia
(2004)
USA and Canada
(2010)
Germany
(2012)
United Kingdom
(1998)
Netherlands
(2000)
–
50
50
–
–
> 6 months: 10
10
10
0–6 months: 8.5
> 7 months: 7
5/10*
Children and
adolescents
7.5
15
20*
< 4 years: 7
ab 4 years: –/10*
1–3 years: 5/10*
4–18 years: 2.5/5*
Adult men and
women
7.5
15
20*
10*
< 50 years: 2.5/5*
51–60 years: 5/10*
61–70 years: 7.5/10*
> 61 years: 10*
20
20*
10
> 71 years: 12.5/15*
Target
concentration
25-OHD nmol/l
Infants
Seniors
* additional 10 µg as a
supplement in the absence of sun exposure
Effect of UVB radiation
In general, genuine vitamin D formation after UVB exposure
is the best and most important vitamin D source for humans.
If this is sufficient, then additional intake of supplements is
unnecessary. This is also taken into account in most recommendations for vitamin D intake (Table 2). The definition of
sufficient UVB exposure depends on many factors, which makes it very difficult to establish a generally valid time period.
Crucial factors include skin type, as people with darker skin
types produce less vitamin D than people with lighter skin,
use of sunscreens or skin care products with sun protection
* In case of insufficient
UVB exposure
* In the absence of sun
exposure
* In case of insufficient UVB
exposure
factors that can almost completely block vitamin D formation
in the skin, time of day, season and latitude. In Central and
Northern Europe, UVB radiation is so low from October to
March, that practically no vitamin D can be formed in the skin.
These considerations demonstrate that the current recommendations for protection of the skin against excessively
strong sunlight and skin cancer are contradicting the recommendations for vitamin D formation. Further research
is certainly required to provide precise recommendations
for sun exposure, and with results that are able to take both
endogenous vitamin D synthesis as well as skin protection
into account.
An integrated research project:
Vitamin D and cardiovascular health
From 2010 to 2013, a nutritional science collaborative project
was supported by the Federal Ministry for Education and
Research, in which an integrated approach to the importance of vitamin D for cardiovascular health was examined.
Integrated means that the project’s different approaches
in food science, nutritional, medical and epidemiological
research at different sites (Halle, Potsdam, Heidelberg) were
considered together and unified. Firstly, the vitamin D content of various foods, and in particular the variance of the
vitamin D concentration in animal foods, was investigated
as a function of the feeding and housing of animals and in
their natural environment [21]. Secondly, possibilities were
investigated on how vitamin D concentrations could be
increased, especially in soft water fish. Soft water fish and
fish from aquaculture contain vitamin D just as saltwater fish,
however, there is not much knowledge on the influence of
housing and feeding on vitamin D content.
Simplified illustration of the vitamin D metabolism in the body
Naturally skin harbours the 7-dehydrocholesterol from which vitamin D is formed when exposed to sunlight. The
vitamin D binding protein DBP transports the molecule further to the liver, where the first hydroxylation to 25-OH
vitamin D (25-OHD) takes place. This transportation and storage form circulates in the bloodstream, and therefore
25-OHD is used to determine the individual vitamin D status. In the kidney, 25-OHD is hydroxylated again as
needed and leads to the biologically active form 1,25-dihydroxy-vitamin D, which plays an important role among
others in bone metabolism.
In addition, the effects of vitamin D deficiency on lipid metabolism and atherosclerosis have been studied in model animals.
This was aimed at finding out how a vitamin D deficiency
might contribute to increased risk of heart disease and which
mechanisms are at work [22]. Moreover, in clinical trials with
human volunteers, the effects of vitamin D supplementation
on 25-OHD serum levels, on blood lipids and blood pressure
were tested. In this case, the effectiveness of the different
forms of vitamin D (vitamin D2 and vitamin D3) were compared with each other [18]. Furthermore, the relationship
between the amount of 25-OHD-serum concentration and
the risk of diabetes mellitus, heart attack and stroke was
examined in a sub-cohort of the EPIC study [23, 24]. EPIC
stands for “European Prospective Investigation into Cancer
and Nutrition” and is a European research project in which
approximately 500,000 people in ten European countries
participated between 1994 and 1998, and who have
been followed up since. In the two German EPIC centres
of Heidelberg and Potsdam, around 50,000 people participate in this long-term study. For the previously mentioned
DIALOG 2014/1
research questions, around 5,000 individuals of the 50,000
study participants were examined, of which around 1,000
had either been diagnosed with diabetes during the study
or had suffered a heart attack or stroke. The samples from
the EPIC study have been stored at -196 °C for 15 years
and are particularly valuable material. Consequently, it
was very important that determination of the 25-OHD be
effected from a minimal amount of sample. Chromsystems’
MassChrom® 25-OHD3/D2 reagent kit was therefore used to
determine the 25-OHD samples in all studies, which meant
the use of small amount of samples (100 µl) as well as
obtaining results from different studies that could be compared with each other. In addition, many studies underline
the advantages of the LC-MS/MS analysis for clarifying
vitamin D status, which is why all vitamin D determinations
within the large-scale EPIC study were performed by LCMS/MS using the MassChrom® 25-OHD3/D2 reagent kit.
In summary, it should be pointed out that vitamin D research
has not yet reached a plateau and interest in this field
continues.
Page 7
References
[1] Afzal S, Bojesen SE, Nordestgaard BG. (2013) Low 25-Hydroxyvitamin D and risk of
type 2 diabetes: A prospective cohort study and metaanalysis. Clin Chem 52(2): 381–91.
[2] Holick MF. (2007) Vitamin D deficiency. N Engl J Med 357(3): 266–81.
[3] Turer CB, Lin H, Flores G. (2013) Prevalence of vitamin D deficiency among over
weight and obese US children. Pediatrics 131(1): e152–61.
[4] Looker AC, Johnson CL, Lacher DA, Pfeiffer CM, Schleicher RL, Sempos CT. (2001)
Vitamin D status: United States, 2001–2006. NCHS Data Brief no. 59, National
Center for Health Statistics, Hyattsville, MD.
[14] Becker W, Lyne N, Pedersen A, Aro A, Fogelholm M, Thorsdottir I, Alexander J,
Anderssen S, Meltzer HM, Pedersen J. (2004) Nordic Nutrition recommendations
2004 – Integrating nutrition and physical activity. Scand J Nutr 48: 178–87.
[15] Ross AC, Institute of Medicine (US). Committee to review dietary reference intakes for
vitamin D and calcium. Dietary reference intakes: calcium, vitamin D. National Aca
demies Press, Washington/DC 2011.
[16] Department of Health (1998) Nutrition and Bone health. London, the stationary
office.
[17]Health council of the Netherlands: Dietary reference values. The Hague 2000,
publ. no. 2000/12.
[5] Hintzpeter B, Mensink GB, Thierfelder W, Müller MJ, Scheidt-Nave C. (2008a)
Vitamin D status and health correlates among German adults.
Eur J Clin Nutr 62(9): 1079–90.
[18] Lehmann U, Hirche F, Stangl GI, Hinz K, Westphal S, Dierkes J. (2013) Bioavailabi
lity of Vitamin D2 and D3 in Healthy Volunteers, a randomised placebo-controlled
trial. J Clin Endocrinol Metab doi: 10.1210/jc.2012–4287.
[6] Lip P. (2004) Which circulating level of 25-hydroxyvitamin D is appropriate?
J Steroid Biochem Mol Biol 89–90(1–5): 611–4.
[19] Tripkovic L, Lambert H, Hart K, Smith CP, Bucca G, Penson S, Chope G, Hyppönen E,
Berry J, Vieth R, Lanham-New S. (2012) Comparison of vitamin D2 and vitamin D3
supplementation in raising serum 25-hydroxyvitamin D status: a systematic review
and meta-analysis. Am J Clin Nutr 95(6): 1357–64.
[7] Hintzpeter B, Scheidt-Nave C, Müller MJ, Schenk L, Mensink GBM. (2008b)
Higher prevalence of vitamin D deficiency is associated with immigrant back
ground among children and adolescents in Germany. J Nutr 138(8): 1482–90.
[8]
Brouwer-Brolsma EM, Bischoff-Ferrari HA, Bouillon R, Feskens EJ, Gallagher CJ,
Hypponen E, Llewellyn DJ, Stoecklin E, Dierkes J, Kies AK, Kok FJ, Lamberg-Allardt C,
Moser U, Pilz S, Saris WH, van Schoor NM, Weber P, Witkamp R, Zittermann A,
de Groot LC. (2013) Vitamin D: do we get enough? A discussion between vitamin D
experts in order to make a step towards the harmonisation of dietary reference
intakes for vitamin D across Europe. Osteoporos Int 24(5): 1567–77.
[9] Max Rubner Institut (Hrsg). Ergebnisbericht Teil 2, Nationale Verzehrsstudie II.
Karlsruhe 2008.
[10]German Society for Nutrition (DGE). (2012) New reference values for vitamin D.
Ann Nutr Metab 60: 241–6.
[11] Binkley N, Krueger D, Cowgill CS, Plum L, Lake E, Hansen KE, DeLuca HF, Drezner MK.
(2004) Assay variation confounds the diagnosis of hypovitaminosis D: A call for
standardization. J Clin Endocrinol Metab 89(7): 3152–7.
[12] Carter GD, Carter R, Jones J, Berry J. (2004) How accurate are assays for
25-hydroxyvitamin D? Data from the international vitamin D external quality assessment
scheme. Clin Chem 50(11): 2195–7.
[13] Bischoff-Ferrari H, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. (2006)
Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health
outcomes. Am J Clin Nutr 84(1): 18–28.
[20]Cranney A, Weiler HA, O‘Donnell S, Puil L. (2008) Summary of evidence-based
review on vitamin D efficacy and safety in relation to bone health.
Am J Clin Nutr 88(2): 513S–19S.
[21] Lietzow J, Kluge H, Brandsch C, Seeburg N, Hirche F, Glomb M, Stangl GI. (2012)
Effect of short-term UVB exposure on vitamin D concentration of eggs and vitamin D
status of laying hens. J Agric Food Chem 60(3): 799–804.
[22] Max D, Brandsch C, Schumann S, Kühne H, Frommhagen M, Schutkowski A, Hirche F,
Staege MS, Stangl GI. (2013) Maternal vitamin D deficiency causes smaller muscle
fibers and altered transcript levels of genes involved in protein degradation, myo
genesis, and cytoskeleton organization in the newborn rat. Mol Nutr Food Res doi:
10.1002/mnfr.201300360.
[23]Kühn T, Kaaks R, Teucher B, Hirche F, Dierkes J, Weikert C, Katzke V, Boeing H,
Stangl GI, Buijsse B. (2013) Plasma 25-hydroxyvitamin D and its genetic determi
nants in relation to incident myocardial infarction and stroke in the European pro
spective investigation into cancer and nutrition (EPIC)-Germany study.
PLoS One 8(7): e69080.
[24] Buijsse B, Boeing H, Hirche F, Weikert C, Schulze MB, Gottschald M, Kühn T, Katzke VA,
Teucher B, Dierkes J, Stangl GI, Kaaks R. (2013) Plasma 25-hydroxyvitamin D and its
genetic determinants in relation to incident type 2 diabetes: a prospective case- cohort
study. Eur J Epidemiol 28(9): 743–52.
Two New Parameter Sets for MassTox TDM Series A
Antiarrhythmic and Anti-HIV Drugs
®
The modular MassTox® TDM Series A system has been extended by two more parameter sets. Now, more than
150 parameters can be reliably measured with just one
MasterColumn ® A, – no column change – and using
the same method of sample preparation. New to the
range is the parameter set for antiarrhythmic drugs, used
to treat heart rhythm disorders in order to reduce these
in frequency or strength. This parameter set allows the
fast and effective LC-MS/MS analysis of 27 analytes in
serum/plasma. By careful optimisation of all kit reagents
and chromatographic separation, matrix effects are minimised, and the robustness of the method is increased.
Antiarrhythmic Drugs Menu:
Acebutolol, Ajmaline, Amiodarone, Desethylamiodarone, Aprindine, Atenolol,
Bisoprolol, Chinidine, Hydrochinidine, Diltiazem, Disopyramide, Dronedarone,
Debutyldronedarone, Flecainide, Flunarizine, Gallopamil, Lidocaine, Metoprolol,
Mexiletine, Procainamide, N-Acetylprocainamide, Propafenone, Propranolol,
Sotalol, Tocainide, Verapamil, Norverapamil.
The series has also been expanded to include the parameter set for Anti-HIV Drugs, which embraces a total of
18 analytes. In HIV therapy the ongoing monitoring
of drug concentration may be of considerable relevance. An important precondition for the success of
therapy is sufficient plasma levels of the administered
antiretroviral drug, which may vary greatly from individual to individual.
Anti-HIV Drugs Menu:
Amprenavir, Atazanavir, Darunavir, Delavirdine, Efavirenz, Elvitegravir,
Etravirine, Indinavir, Lopinavir, Maraviroc, Nelfinavir, Nelfinavir-M8, Nevirapine,
Raltegravir, Rilpivirin, Ritonavir, Saquinavir, Tipranavir.
For all parameters of Series A the following applies: by careful optimisation of all kit
reagents and the chromatographic separation, matrix effects are minimised and the
robustness of the method is increased. The use of stable, isotopically labeled internal
standards and 3PLUS1® or 6PLUS1® multilevel calibrator sets ensures high precision
and reproducible and reliable quantification of all analytes.
DIALOG 2014/1
Page 8
Special vitamin D diagnosis in infants:
meaning of the C3-epimers
Dr Inga Unterieser, Dr habil. Richard Lukačin, Chromsystems GmbH
The clinical picture of rickets in children has been known
since the mid-17th century. Caused by vitamin D deficiency
it leads to bone deformities and growth retardation due to
a lack of mineralisation. Recent studies indicate that vitamin D and its active metabolites fulfil a number of additional
important functions in the human organism. These include
the promotion of epithelial cell differentiation in the skin, the
influence on the activity of the immune system, the regulation
of insulin secretion and the protection against cardiovascular
diseases [1]. Beyond that, vitamin D deficiency is linked to
many diseases such as breast and colon cancer, multiple
sclerosis, dementia, rheumatoid arthritis, diabetes, Parkinson‘s
and Alzheimer‘s disease, even though evidence for a direct
connection is often lacking.
Intake and metabolism
Vitamin D is a fat-soluble vitamin, whose basic structure
is derived from the cholesterol steroid skeleton. The main
representative occurring in humans and animals is cholecalciferol (vitamin D 3) whereas in plants and fungi
ergocalciferol (vitamin D 2) prevails. They only differ
slightly in the chemical structure of the side chain (Fig. 1).
Dietary intake plays a minor role for vitamin D supply. The
organism is supplied with vitamin D to a greater degree by
7-dehydrocholesterol stored in the skin that is converted
through sunlight into initially inactive vitamin D. Consequently, regular sun exposure is important for vitamin D intake
and the serum concentration is therefore also subjected to
H
H
25
3
HO
25-OH-Vitamin D3
25
H
3
25-OH-Vitamin D2
Figure 1: Chemical structures of the vitamins 25-OHD3, 25-OHD2, 3-epi-25-OHD3 and 3-epi-25-OHD2.
OH
25
H
H
H
H
HO
OH
OH
OH
seasonal fluctuations. Therefore, vitamin D supplementation for the western population is also often recommended
during the winter months, whereby in a few countries only
vitamin D2 preparations are available for this purpose [2].
The metabolising of vitamin D3/D2 is carried out in the liver,
where it is hydroxylated to 25-hydroxyvitamin D3 and D2
(25-OHD), respectively (Fig. 1). Both forms serve for transport
and storage, and have a half-life of a few weeks, which
is why the serum level of 25-OHD3/D2 is the recognised
parameter for determining vitamin D status. A further hydroxylation that can be carried out especially in the kidney,
but also locally and cell-specific, leads to the hormonally
active 1,25-dihydroxyvitamin D3 and D2, respectively.
HO
H
3
3-epi-25-OH-Vitamin D3
25
HO
3
3-epi-25-OH-Vitamin D2
DIALOG 2014/1
Page 9
References
[1] Deutsche Gesellschaft für Kinder- und Jugendmedizin e.V. (2011) Stellungnahme:
Vitamin D-Versorgung im Säuglings-, Kindes- und Jugendalter.
ISTD 1
5.90 min
[2] Holick MF. (2007) Vitamin D deficiency. N Engl J Med 357(3): 266-81.
ISTD 2
6.28 min
[3]
3--25-OHD3
6.30 min
17.8 µg/l
25-OHD3
5.91 min
14.9 µg/l
25-OHD2
6.32 min
3.4 µg/l
Singh RJ, Taylor RL, Reddy GS, Grebe SK. (2006) C3-epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. J Clin Endocrinol Metab
91(8): 3055-61.
[4] Strathmann FG, Sadilkova K, Laha TJ, LeSourd SE, Bornhorst JA, Hoofnagle AN,
Jack R. (2012) 3-epi-25-hydroxyvitamin D concentrations are not correlated with age
in a cohort of infants and adults. Clin Chim Acta 413(1–2): 203–6.
[5] Midasch O, Erlenfeld G, Yueksekdağ N, Fabian D, Burki D, Korall H, Wallner S, Lukačin R.
25-OHD 3 and its C3-epimer: Determination by LC-MS/MS. MSACL 2011.
3rd Annual Conference & Exhibits, San Diego, CA (PosterSplash Track 3/29).
[6]
3--25-OHD2
6.72 min
2.1 µg/l
Schleicher RL, Encisco SE, Chaudhary-Webb M, Paliakov E, McCroy LF, Pfeiffer CM.
(2011) Isotope dilution ultra performance liquid chromatography-tandem mass spectrometry method for simultaneous measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and epi-25-hydroxyvitamin D3 in human serum. Clin Chim Acta
412(17–18): 1594–9.
[7] Shah I, James R, Barker J, Petroczi A, Naughton DP. (2011) Misleading measures
in vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars.
Nutr J 10: 46.
[8]
van den Ouweland JMW, Beijers AM, van Daal H. (2011) Fast separation of 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3 in human serum by liquid chromatography-tandem mass spectrometry: Variable prevalence of 3-epi-25-hydroxyvitamin D3 in infants, children, and adults. Clin Chem 57(11): 1618–9.
[9] Stepman HMC, Vanderroost A, Stöckl D, Thienpont LM. (2011) Full-scan mass
spectral evidence for 3-epi-25-hydroxyvitamin D3 in serum of infants and adults.
Clin Chem Lab Med 49(2): 253–6.
5.2
5.4
5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
[10] Lensmeyer G, Poquette M, Wiebe D, Binkley N. (2012) The C-3 epimer of 25-hydroxy
vitamin D3 is present in adult serum. J Clin Endocrinol Metab 97: 163–8.
Time, min
Figure 2: An exemplary chromatogram of a serum sample from an infant that was analysed with the new epimer upgrade of the MassChrom® 25-OHvitamin D3/D2 reagent kit. In addition to 25-OHD3 and 25-OHD2, the epimeric forms 3-epi-25-OH-Vitamin D3 and 3-epi-25-OH-vitamin D2
were found in significant quantities.
The method of choice
In a study published in 2006, Singh and colleagues reported that in a significant proportion of infants and young
children up to one year 25-OHD can exist as C3-epimer.
The proportion of 3-epi-25-OH vitamin D can amount to
up to 60 % out of total 25-OHD concentration. Tendencies: The younger the child, the higher the average proportion of epimeric forms, with strong variance observed.
The C3-epimer of 25-OHD is only structurally different by
the spatial orientation of the hydroxylgroup in position C3
of the skeletal structure (Fig. 1).
Its active metabolite 3-epi-1,25-(OH)2-vitamin D3/D2 also
draws attention to differences in biological activities. For
example, the epimeric forms cause a comparable inhibition of parathyroid hormone secretion, however, calcium-induced effects in bone metabolism are significantly
reduced. Due to this difference in effectiveness, it is of
considerable importance to determine the concentration
levels of 25-OHD3/D2 of the epimeric forms separately
in order to identify the individual contribution of each
of the different molecules to the total vitamin D status.
At about one year, infants mostly have very low concentrations of 3-epi-25-OH-vitamin D [3–13] that are comparable
to adults. It is believed that epimerisation is primarily a
characteristic of a not yet matured vitamin D metabolism.
The new upgrade for the MassChrom® analysis allows not only
the main metabolites of vitamins D3 and D2, 25-hydroxycholecalciferol and 25-hydroxyergocalciferol to be determined, but also the rapid and simultaneous determination
of 3-epi-25-OH-vitamin D3/D2 by LC-MS/MS in serum/
plasma (Fig. 2). The manual sample preparation is limited
to a simple and effective protein precipitation. Analogous
to the 25-OHD3/D2-determination the analytes are systematically concentrated using a trap column and disturbing
matrix components are separated. The trap column is associated with a particularly high resolution analytical column
via a simple control valve, which enables chromatographic
separation and reliable quantification of the analytes in less
than 10 minutes (Fig. 2).
Thanks to systematic prophylaxis severe disease manifestations such as rickets and their side effects are rare. Nevertheless, a vitamin D deficiency is a problem which, even
today, is paid far too little attention to in children. A recent
extensive four-year representative U.S. study has shown that
61 % of 9- to 21-year-old Americans (about 50.8 million)
have a vitamin D deficiency (37–72 nmol/l 25-OHD) and
a further 9 % of this population (approximately 7.6 million)
even suffer from an undersupply of vitamin D (< 37 nmol/l
25-OHD) [14].
Especially in children, the level of 25-OHD 3/D2 and
3-epi-25-OH-Vitamin D3/D2 should be determined in
serum samples to allow for a differentiated assessment
of the supply situation in early childhood development.
At this point, it is expressly highlighted that diagnostic tests
that do not consider or inadequately mirror the contribution
of 3-epi-25-OH-Vitamin D3/D2 on total vitamin D status, at
least for infants under one year, are unsuitable.
[12] Wright MJP, Halsall DJ, Keevil BG. (2012) Removal of 3-epi-25-hydroxyvitamin D3
interference by liquid chromatography-tandem mass spectrometry is not required
for the measurement of 25-hydroyvitamin D3 in patients older than 2 years.
Clin Chem 58(12): 1719–20.
[13] Bailey D, Veljkovic K, Yazdanpanah, Adeli K. (2013) Review: Analytical measure
ment and clinical relevance of vitamin D3 C3-epimer. Clin Biochem 46(3): 190–6.
The C3-epimer
The supply situation in children and adolescents
[11] Baecher S, Leinenbach A, Wright JA, Pongratz S, Kobold U, Thiele R. (2012)
Simultaneous quantification of four vitamin D metabolites in human serum using high
performance liquid chromatography tandem mass spectrometry for vitamin D
profiling. Clin Biochem 45(16–17): 1491–6.
[14] DiMeglio LA. (2010) Pediatric endocrinology: Vitamin D and cardiovascular
disease risk in children. Nat Rev Endocrinol 6(1): 12-3.
[15] Thomas L. Labor und Diagnose. 7. Aufl, Verlag TH-Books Frankfurt/Main (2008).
For ionisation of the stable vitamin D molecules, the APCI
(Atmospheric Pressure Chemical Ionisation) technique is
used. The use of two isotope-labelled internal standards
adapted to the epimeric forms compensates matrix effects
and ensures the method’s high accuracy and robustness.
Currently, only Chromsystems offers multilevel serum calibrators (3PLUS1®) and serum controls for this analysis. In
addition, the calibrators and controls for the 3-epi-25-OHvitamin D3/D2 and 25-OHD3/D2 analysis are traceable to
the NIST-reference material 972.
Product Information
Specifications
Sample Preparation
Linearity: 1.0–250 μg/l
→ Place 100 μl sample into a reaction vial.
Limit of quantification:
→ Add 25 µl Precipitation Reagent.
Upgrade set also available
1.0 μg/l 3-epi-25-OHD2
→ Add 200 μl Internal Standard.
separately
2.0 μg/l 3-epi-25-OHD3
→ Vortex 20s.
Intraassay: CV < 5 %
→ Incubate 10 min at + 4°C.
Interassay: CV < 5 %
→ Centrifuge 5 min at 15 000 x g.
Analysis time: 8.5–10 min
→ Transfer 200 µl supernatant into an autosampler Upgrade 3-epi-25-OHD3/D2
vial.
→ Injection volume: 10–50 µl
DIALOG 2014/1
Page 10
Vitamin B1 and B6 analysis in veterinary
medicine – comparison of HPLC and UHPLC
Dr Anja Müller, Sebastian Raich, Samuel Bauder, Vet Med Labor GmbH, Ludwigsburg/Germany
The B vitamins are of vital importance for carbohydrate
metabolism in humans as well as in animals. Vitamin B undersupply due to incorrect feeding of animals can lead to
various deficiencies. It has been experimentally shown that
with reduced thiamine absorption vitamin B1 concentration
in the heart, liver and kidney decreases faster than in the
brain, and central nervous disorders occur already at a
vitamin B1 level that is reduced by 20 % [1, 2]. A suspicion
of a vitamin B1 or vitamin B6 deficiency can be clarified
by the veterinarian in the veterinary diagnostic laboratory.
The method of choice for determining B1 and B6 vitamins
from whole blood or serum/plasma is liquid chromatography
(HPLC), which has been established in human diagnostics
for decades. As an extension of the existing technology,
UHPLC is considered to offer many benefits, such as significantly reduced solvent consumption together with higher
sample throughput and improved sensitivity, and is therefore gaining more and more importance in the practice.
In the present comparative study, the values of B1 and B6
vitamins from EDTA-whole blood of different animal species
were determined by HPLC and UHPLC. In addition, it was
investigated whether commercially available complete kits
that are used in the human clinical diagnostics application,
could also provide reliable data in veterinary diagnostics.
Vitamin B1 deficiency in animals
Vitamin B1 deficiency in animals manifests itself in species-specific and varied symptoms, which may include
cramps, uncertainty in gait, paralysis („stargazer disease“
e.g. in young lions or cats), food refusal, growth disorders and bradycardia. In ruminants polioencephalomalacia (PEM; cerebrocortical necrosis, CCN), a metabolictoxic brain disease, is caused by vitamin B1 deficiency.
Vitamin B1 requirement is closely related to food intake, as it
is absolutely necessary for degradation of carbohydrates resulting in energy production. In addition, thiamine (vitamin B1)
plays a key role in the conversion of glucose into nucleic
acids, fatty acids and amino acids.
A common cause of deficiency is the accumulation of thiaminases in the digestive tract that are responsible for an
enzymatic breakdown of the absorbed or formed vitamin B1.
Thiaminases occur in the rumen of ruminants under conditions of ruminal acidosis. Furthermore, thiamine-containing
plants such as bracken and marsh horsetail can cause
real avitaminosis in horses. Deficiencies in dogs, cats and
pelted animals occur by frequent feeding with raw fish.
Thiaminases are largely inactive in heat-treated food. In
poultry, the coccidiostat Amprolium ® may be effective
as thiamine antagonist. This compound is used as feed
additive to prevent coccidioses, an enteritis caused by
protozoa, which occurs especially in poultry and rabbits.
Substitution with vitamin B1 supplements is carried out upon
thiamine deficiency as well as unusual disorders that indicate
insufficient formation, intensified degradation or increased
vitamin B1 consumption. As already mentioned above, this
can be the case by overfeeding with carbohydrates (rumen
acidosis), paralytic myoglobinuria (horse) and in cases of
poisoning by bracken and horsetail. A thiamine dose is also
indicated for neuritis, paralysis of the peripheral nerves and
impaired growth, and is usually treated in the form of vitamin B combination preparations parenterally or orally [3].
Vitamin B6 deficiency in animals
The active form of vitamin B6 is the pyridoxal-6-phosphate that
is required as a coenzyme for the activity of many enzymes.
Therefore, vitamin B6 deficiency leads to disturbances of all
the pyridoxal-6-phosphate-dependent catalytic reactions that
are mainly attributable to the protein metabolism. Sequelae
described are poor feed conversion, growth retardation,
immune deficiency and microcytic hypochromic anaemia
with an increased iron content in serum. In severe cases,
deficiency symptoms of the nervous system (polyneuritis),
cramps, dermatitis, and conjunctivitis are observed. Also,
paroxysmal convulsions in pigs and carnivores, liver and
kidney diseases, as well as cases of anorexia and delayed
growth in puppies and piglets may indicate deficiencies and
are handled by an appropriate vitamin B6 treatment. The
application is undertaken orally or parenterally [1].
Study implementation
EDTA blood samples of different animal species (dog,
cat, horse, bovine and dolphin) were processed using
Chromsystems kits for the determination of vitamin B1/B6
Dolphin
Dolphin
Dog
Dolphin
Cat
Figure 1: Chromatograms of processed dolphin samples (EDTA whole blood) by HPLC (top; vitamin B1 =
492 µg/l, vitamin B6 = 190 µg/l), and UHPLC (bottom; vitamin B1 = 377 µg/l, vitamin B6 = 155 µg/l). The
UHPLC separation of the three analytes, Vitamin B1, B6 and an internal standard was reached in less than
half the time.
Figure 2: UHPLC chromatograms of processed EDTA whole blood samples from different animal species.
Dolphin (top; vitamin B1 = 377 µg/l, vitamin B6 = 155 µg/l) with a very high value of vitamin B1, dog (centre,
vitamin B1 = 108 µg/l, vitamin B6 = 70 µg/l) with „normal“ levels of vitamin B1 and B6 and cat (bottom;
vitamin B1 = 31 µg/l, vitamin B6 = 1265 µg/l) with a very high vitamin B6 value.
DIALOG 2014/1
Page 11
140
y = 0.8748x + 4.8058
R2 = 0.964
120
200
y = 0.8395x + 3.7211
R2 = 0.983
180
160
140
B6 UHPLC (µg/l)
B1 UHPLC (µg/l)
100
80
60
120
100
80
60
40
40
20
20
0
0
0
0
20
40
60
80
100
120
20
40
60
80
100
120
140
160
180
200
B6 HPLC (µg/l)
140
B1 HPLC (µg/l)
Figure 3: Comparison of HPLC with the new UHPLC Chromsystems method for the analysis of vitamin B1
from EDTA whole blood samples of different animals (dogs, horses, dolphins and cattle). The correlation
coefficient of 0.964 indicates a very good agreement between both methods.
according to the manufacturer‘s instructions and analysed
with the corresponding HPLC or UHPLC method. The chromatographic separations were performed on a Dionex
ULTIMATE 3000 RS-LC UHPLC system with switchable FLD
(Fig. 1). There were only minor difficulties in establishing the
UHPLC method with respect to the gradient profile, which
required a custom change to mobile phase B using prompt
wavelength switching. Minimising the dead volume was
therefore achieved by removal of the gradient mixers. A
typical UHPLC run lasted about 3 minutes and was thereby
twice as fast as the chromatography on an HPLC system
(Fig. 1). In addition, solvent consumption decreased from
about 10 to 2 ml per sample, i.e. by about 80 %. Another
difference was found in regard to the peak form: UHPLC
signals appeared sharper and leaner.
Results
All animal samples as well as calibrators and controls could
be reproducibly measured. In contrast to human samples,
there were some large species-specific differences in vitamin B1
and B6 levels that needed to be taken into account. For
example, cats have very high levels of vitamin B6 compared
to dogs and other animals. Dolphins have high vitamin B1
levels (Fig. 2). In contrast, cattle, horses and dogs usually
have comparable vitamin B1 and B6 concentrations in serum.
Evaluation of the measured values of the UHPLC and HPLC
methods show a very good correlation of vitamin B1 in the
range of r2 = 0.964 (Fig. 3) and for vitamin B6 in the range
of r2 = 0.983 (Fig. 4). The studies on intra- and interassay
reproducibility were carried out for the Chromsystems controls
level I and II, and also for two dog samples, one cat and
one horse sample (Table 1, 2). The interassay reproducibility
in the animal samples was only determined 6-fold due to
the small sample size.
In summary, the study presented here shows that the Chromsystems commercial method for determining vitamin B1/B6
from serum and plasma as UHPLC version can also be used
not only in diagnostics for humans, but also in veterinary
medicine. At the same time, it provides similarly reliable
results compared to the established HPLC method, but
also with a significantly reduced solvent consumption and
a faster analysis speed.
Figure 4: Comparison of HPLC and the new UHPLC Chromsystems method for the analysis of vitamin B6
from EDTA whole blood samples of different animals (dogs, horses, dolphins and cattle). The correlation
coefficient of 0.983 indicates a very good agreement between both methods..
Reference
[1]
Butterworth RF. (1987) Thiamin malnutrition and brain development. Current topics in
Nutr and Dis 16: 287–304. Zitiert in Biesalski HK, Schrezenmeir J, Weber P, Weiß H
(Hrsg.). Vitamine. Physiologie, Pathophysiologie, Therapie. Georg Thieme Verlag
Stuttgart, 1997.
[2] Haas RH. (1988) Thiamin and the brain. Annu Rev Nutr 8: 483–515.
[3] Frey HH, Löscher W. (2002) Lehrbuch der Pharmakologie und Toxikologie für die
Veterinärmedizin. Enke Verlag Stuttgart, 2. Aufl, 1–609.
Table 1: Intra- and interassay reproducibility of the UHPLC methodology (Chromsystems) for the determination of vitamin B1 from EDTA whole
blood of different species; Chromsystems controls level I and II for comparison.
Vitamin B1
Intra-/Interassay
Mean value (µg/l)
Standard deviation
(µg/l)
CV (%)
Chromsystems control
Level I
Intra (n = 10)
22.2
0.7
3.0
Chromsystems control
Level I
Inter (n = 10)
23.4
1.0
4.4
Chromsystems control
Level II
Intra (n = 10)
92.7
1.0
1.1
Chromsystems control
Level II
Inter (n = 10)
90.8
3.3
3.6
Dog-EDTA
Intra (n = 10)
52.0
1.8
3.4
Dog-EDTA
Inter (n = 6)
52.9
2.2
4.1
Horse-EDTA
Intra (n = 10)
14.0
0.6
4.1
Horse-EDTA
Inter (n = 6)
14.4
0.7
4.9
Cat-EDTA
Intra (n = 10)
47.4
1.6
3.3
Cat-EDTA
Inter (n = 6)
47.1
3.0
6.4
Dog-EDTA
Intra (n = 10)
94.8
2.7
2.8
Dog-EDTA
Inter (n = 6)
99.3
3.8
3.8
Table 2: Intra- and interassay reproducibility of the UHPLC methodology (Chromsystems) for the determination of vitamin B6 from EDTA whole
blood of different species; Chromsystems controls level I and II for comparison.
Vitamin B6
Intra-/Interassay
Mean value (µg/l)
Standard deviation
(µg/l)
CV (%)
Chromsystems control
Level I
Intra (n = 10)
8.1
0.3
4.2
Chromsystems control
Level I
Inter (n = 10)
8.8
1.1
12.5
Chromsystems control
Level II
Intra (n = 10)
29.8
0.8
2.7
Chromsystems control
Level II
Inter (n = 10)
28.9
2.1
7.1
Dog-EDTA
Intra (n = 10)
76.9
2.9
3.8
Dog-EDTA
Inter (n = 6)
77.7
5.4
7.0
Horse-EDTA
Intra (n = 10)
37.2
1.7
4.5
Horse-EDTA
Inter (n = 6)
37.5
3.1
8.3
Cat-EDTA
Intra (n = 10)
619.4
19.2
3.1
Cat-EDTA
Inter (n = 6)
616.2
69.4
11.3
Dog-EDTA
Intra (n = 10)
186.7
4.3
2.3
Dog-EDTA
Inter (n = 6)
192.8
16.0
8.3
DIALOG 2014/1
Page 12
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Question 1: Which animal has been used as a model for a study to clarify vitamin D-enrichment in animal products?
Question 2: How much faster is UHPLC compared to HPLC for the measurement of vitamin B1/B6?
Question 3: Which chemical compound is isobaric to MMA?
Question 4: Why are MMA plasma levels elevated in subjects with bacterial overgrowth in the gut?
Question 5: What is the structural difference in the skeleton of the C3-epimer compared to 25-OHD?
Condition of participation
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IMPRINT
Publisher:
Chromsystems
Instruments & Chemicals GmbH
Am Haag 12
82166 Gräfelfing/Germany
Phone: +49 89 18930-300
Fax: +49 89 18930-399
E-Mail: [email protected]
Editors:
Dr Marc Egelhofer
Dr habil. Richard Lukačin
Dr Nihâl Yüksekdağ
Design:
Fred Lengnick Print- & Media Design
Edition January 2014