Vitamin D and Health

24,25(OH) 2 D 3 which can contribute to a positive bias in serum 25(OH)D concentrations relative to LCtandem
MS methods (Cashman et al., 2015) (described below). An important consideration is that
most samples collected over the past 20-30 years, which have provided the majority of current
evidence relating serum 25(OH)D concentration to health outcomes, have been analysed using
antibody-based assays.
4.24 LC-based assays which use a tandem mass spectrometer (LC-MS/MS) allow discrimination between
25(OH)D 2 and 25(OH)D 3 and other compounds by their unique molecular masses and mass fragments
(Makin et al., 2010). Since these methods use short LC retention times, and in some cases automated
robotic extraction and LC separation steps and computerised MS systems, they can be made relatively
operator-free and provide high throughput. Their potential advantages also include high specificity,
high sensitivity, and better reproducibility (< 10%). The consensus among analysts is that LC-MS/MS
assays will become the ‘gold standard’ for assay performance in the future (de la Hunty et al., 2010;
IOM, 2011).
Standardisation of the measurement of serum 25(OH)D concentration
4.25 While assay performance has been a concern of analysts and clinicians in the vitamin D field for some
time, the role of standard reference materials and inter-laboratory collaboration and quality
assurance schemes is an important aspect of overcoming the challenges that the assay methodologies
present.
4.26 The Vitamin D External Quality Assurance Scheme (DEQAS 35 ) serves as a quarterly monitor of
performance of analysts and 25(OH)D analytical methods for approximately 700 laboratories
worldwide (Carter et al., 2010). DEQAS has published performance reports regularly over the past
decade, which indicate some method biases in terms of accuracy and precision as well as variability as
high as 15-20%. However, some skilled analysts can perform better than this with a coefficient of
variation less than 10%. The introduction of the National Institute of Standards and Technology (NIST)
reference standards, calibrated using a “validated” LC-MS/MS method (Phinney, 2009), suggests that
the variability of all methods will be improved in the future and that an improvement is already
occurring (Carter & Jones, 2009).
4.27 The issue of international standardisation of serum 25(OH)D measurement is also being addressed by
the Vitamin D Standardization Program (VDSP), a collaborative initiative between the Office of Dietary
Supplements of the National Institutes of Health, the Centers for Disease Control and Prevention, the
NIST and a number of the national health surveys around the world (HHS, 2011; Binkley & Sempos,
2014). The International quality assurance/collaboration schemes, such as DEQAS and VDSP as well as
existing and next generation standard reference materials for 25(OH)D, will further help limit interlaboratory
assay-specific differences in this status marker.
Interpretation of measures of serum 25(OHD concentration
4.28 The normal range of serum 25(OH)D concentration is broad and the lower limit can vary among
populations (Weaver & Fleet, 2004). There is considerable and continuing debate on the suggested
threshold (cut-off) for serum 25(OH)D concentration used to define low vitamin D status, which has
ranged between 12.5 and 120 nmol/L (Zittermann, 2003). This is principally because different
functional endpoints/outcome indicators used have different serum 25(OHD) concentration
35 Based at Charing Cross Hospital, London, UK.
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