1) The study evaluated methods for measuring unbound 25-hydroxyvitamin D (25HD) in plasma, including a HPLC/MS/MS method and sample treatment methods like centrifugal filtration. 2) None of the sample treatment methods were able to extract detectable quantities of unbound 25HD from plasma. Experiments on 25HD partitioning suggested it does not distribute homogeneously in aqueous solutions without a carrier at low concentrations. 3) The results support that 25HD must rapidly bind to a carrier like vitamin D binding protein in physiological systems, as there appears no need for significant unbound 25HD given kidney uptake mechanisms.

1. Unbound 25-Hydroxyvitamin D:
Is it truly required for normal renal functions?
ABSTRACT
The measurement of unbound 25-hydroxyvitamin D has been gaining popularity
in clinical practice, however, the validity of the reported measurements as well as
their clinical implications still require further exploration . To this end, we sought
to conduct a series of experiments to:
1) evaluate the plausibility of a HPLC/MS/MS method for plasma free 25HD
measurements;
2) to evaluate the efficacy of select sample treatment methods, including
centrifugal filtration with various MWCO filters and dialysis, for the isolation
and recovery of unbound 25HD; and
3) to assess 25HD’s distributional behavior in aqueous environments.
The optimized LC/MS method had a detection limit of 40 femtogram on column,
with a response linearity up to 50 pictogram, and an inter-sample coefficient of
variation of 3.05%. None of the attempted sample treatment methods were capable
of extracting detectable quantities of unbound 25HD from plasma. Further
analysis of results generated from 25HD partitioning experiments suggest that at
pictogram per milliliter concentrations,25HD do not exhibit homogeneous
distribution in aqueous environments, without the presence of a carrier. However,
at nanogram per milliliter concentrations, a colloidal dispersion may be formed.
The results of this study is in favor of the hypothesis that in physiological systems,
if 25HD can become unbound while circulating in the bloodstream, it must be
rapidly recaptured by another carrier, due to its high hydrophobicity. Furthermore,
current knowledge of kidneys’ intake mechanism for liganded vitamin D binding
proteins portrays a system where there are no physiological need for unbound
25HD to exist in any significant quantity.
BACKGROUND
Vitamin D acquired from dietary sources and from subcutaneous synthesis both
require two hydroxylation steps before becoming functional. The first
hydroxylation occurs in the liver at the twenty-fifth carbon position, resulting in
the production of 25-hydroxyvitamin D [25HD]. The second hydroxylation occurs
in the proximal tubule of the kidneys at the first carbon position, resulting in the
activated hormone 1,25-dihydroxyvitmain D [1,25HD]. 25HD is the primary
vitamin D specie in circulation. Due to its highly hydrophobic nature, majority of
this pro-hormone is chaperoned by vitamin D binding protein [VDBP], and by
albumin to a much lesser extent. 25HD found in the plasma do not have any
known physiological function outside of serving as a circulating reservoir for the
active metabolite. Once activated, 1,25HD have been shown to play major roles in
maintaining calcium homeostasis and in immunomodulation.
Currently, there is a school of thought that only the unbound and the albumin
bound portions of 1,25HD are capable of affecting physiological functions. And
similarly, only those portions of 25HD, termed bioavailable fractions, can be
readily converted into the active hormone. The origin of this school of thought can
be traced back to the free hormone hypothesis, which suggests that only the
unbound portion of a given hormone is responsible for its attributed physiological
effects, as the protein bound portion are too large to efficiently interact with the
intended target sites.
Yuguan Liu, MS1; David Garby2; Lynn Burnell, PhD 1,2, Arlee Dulak, PhD1, Eugene Rogers, PhD1, and Garry J. Handleman, PhD1
1University of Massachusetts Lowell, 2New England Mayo Medical Laboratory,
Questions to Consider
• What are the mechanisms by which extra-renal 25HD activation sites take up
the ligand?
• What are the currently available ‘free’ 25HD ELISA kits actually measuring?
• Are there physiological needs for unbound 25HD to exist?
METHODS
25HD LC/MS/MS Method Development
RESULTS
25HD-Amplifex LC/MS/MS Methodology
25HD is extremely hydrophobic,
ergo it is highly unlikely that it will be able to exist in an aqueous
environment, without some form of a carrier.
The results of the 25
In mammals, majority of 25HD are chaperoned by vitamin D binding protein,
while a small percentage is carried by albumin.
As a part of normal kidney physiology, there are protein recovery mechanisms
in place to prevent the excretion of plasma proteins through urine.
Critical micellar concentration might have been reached.
All of these evidences seem to suggest that there is no physiological need for
the existence of free 25HD, at least with respect to kidney-driven activation.
However, since it is currently extra-renal activations/utilizations of 25HD may
still require the presence of unbound 25HD.
DISCUSSIONS
Acknowledgements
We’d like to thank _______ of AB Sciex [Framingham MA], for the generous
donation of the Amplifex diene-derivatization reagents. Also, we’d like to
express gratitude towards the staff at New England Mayo Medical Laboratory
for their continued support.
References
• Faix, J.D., “Principles and Pitfalls of Free Hormone Measurements”, Best Practice & Research, 27:631-645, 2013.
• Nykjaer, A., Dragun, D. et al, “An Endocytic Pathway Essential for Renal Uptake and Activation of the Steroid 25-
(OH) Vitamin D3”, Cell, 96:507-515, 1999.
• Nykjaer, A., Fyfe, J.C. et al, “Cubilin Dysfunction Causes Abnormal Metabolism of the Steroid Hormone 25(OH)
Vitamin D3”, PNAS, 98(24):13895-13900.
• Rowling, M., Kemmis, C.M. et al, “Megalin-Mediated Endocytosis of Vitamin D Binding Protein Correlates with 25-
Hydroxycholecalciferol Actions in Human Mammary Cells”, J Nutr., 136:2754-2759, 2006.
•ARTERY
•VEIN
•URINE
25HD Distribution in Aqueous Environments
Centrifugal Filtration for Unbound 25HD Extraction from Plasma
Recovery of Aqueous 25HD by Centrifugal Filtration
25HD Distribution in Aqueous Environments
Unbound 25HD Extraction from Plasma with CentriFree 10K MWCO
17%
19%
17%
21%
21%
5%
Average 25HD Distribution in PBS-Albumin
PBS-Alb X-1
PBS-Alb X-2
PBS-Alb X-3
PBS-Alb X-4
PBS-Alb X-5
PBS-Alb X-Rinse
Control Extracted Plasma
Recovery of Aqueous 25HD with CentriFree 10K MWCO Filters
2% 2% 2%2%
2%
90%
Average 25HD Distribution in PBS Aliquots
PBS [0% EtOH] X-1
PBS [0% EtOH] X-2
PBS [0% EtOH] X-3
PBS [0% EtOH] X-4
PBS [0% EtOH] X-5
PBS [0% EtOH] X-Rinse
25HD2
25HD3
d6-25HD3
d6-25HD3
25HD at 28ng/mL Concentrations
25HD Recovery at 1~63 pg/mL Concentrations
Method Parameters
Tandem Mass Spec Sciex 6500 Triple Quad
Ionization source ESI
Reaction Mode MRM
d6-25HD3 m/z 738.6679.4
25HD3 m/z 732.5673.6
25HD2 m/z 744.5685.7
HPLC Configuration Binary Gradient
Mobile Phase A Acetonitrile + 0.1%
Formic Acid
Mobile Phase B Water + 0.1% Formic
Acid
Column Phenomenex Kinetex
2.6u C-18
Flow Rate 0.3mL/Min
Retention Time 4.58 Min.
MP Gradient Graph
An aqueous solution of 50pg/mL 25HD2 and 25HD3 was prepared by diluting
From each donor [n=3], a 4-mL whole blood sample was collected using a lithium
heparin tube. Immediately upon collection, the sample was centrifuged at 3000 RPM
for 10 minutes, at the end of which the plasma supernatant was retained. Each plasma
sample was diluted 1/10 with sterile blood bank saline, prior to centrifugal filtration
with CentriFree [Millipore, ] 10k MWCO filters for 1 hour at room temperature.
500uL of the filtrate was collected from each filtration, to which 30uL of 0.5pg/uL
deuterated 25HD internal standard was added. Each sample was then extracted with
5mL of hexane, and the evaporated products were reacted with Amplifex Diene
derivatization reagent [Sciex, Framingham, MA], following manufacturer’s
instructions.
In order to assess the distribution behavior of 25HD in aqueous environments,
A 40mg/mL albumin solution was produced by adding the appropriate amount of
essentially fatty acid free human albumin [Sigma, ] to 0.1 mM phosphate buffered saline.
To create the 25 ng/mL 25HD2&3 solution in PBS-albumin, 50uL of the stock 0.5ng/uL
25HD solution was transferred into a 5-mL capacity glass vial using a positive
displacement pipette. The solution was evaporated under nitrogen gas with heat, prior to
the addition of 1 mL PBS-albumin.
1.95pg/mL 3.9pg/mL 7.8pg/mL
15.6pg/mL 31.25pg/mL 62.5pg/mL
No 25HD2 or 25HD3 Detected!
d6-25HD3 d6- 25HD3 d6-25HD3
d6-25HD3 d6-25HD3 d6-25HD3
25HD2 25HD2 25HD2
25HD2 25HD2 25HD2
25HD3 25HD3 25HD3
25HD3 25HD3 25HD3