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Parathyroid
hormone-related
protein in
cerebrospinal
uid in health and
disease
Parathyroid hormone-related protein (PTHrP) is involved in intracellular calcium
regulation. In this article, we describe PTHrP determinations in paired serum and
cerebrospinal uid samples by liquid chromatography-tandem mass spectrometry and
our recent studies evaluating associations between PTHrP and calcium concentrations,
age, sex, albumin index and biomarkers of Alzheimer’s disease.
by Dr Mark M. Kushnir, Dr Lisa K. Peterson and Dr Frederick G. Strathmann
January 2020 17
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Endocrinology
Background
Calcium ions (Ca2+) play important roles in cellular physiology. Among
other functions, calcium ions regulate contraction of muscle cells, serve
as second messengers in the signal transduction in the central nervous
system (CNS), and regulate release of neurotransmitters from neurons.
Parathyroid hormone-related protein (PTHrP), participates in intracellular
Ca2+ regulation and maintaining Ca2+ homeostasis in cells [1]. PTHrP is a
20 kDa protein discovered in the mid-1980s as a hormone secreted by
various types of tumour cells, which causes tumour-associated hyper-
calcemia [2]. Recently we developed and validated a liquid chromatogra-
phy-tandem mass spectrometry (LC-MS/MS) method for the measure-
ment of PTHrP, which is currently in routine use in a large clinical
diagnostic laboratory [3]. Before the availability of this LC-MS/MS
method, it was thought that PTHrP is present in circulation only in
pathology, whereas our data demonstrated that PTHrP is a normal
constituent of circulating blood; in addition our data demonstrated that
PTHrP concentrations in blood in health are comparable to concentra-
tions of parathyroid hormone (PTH) [3].
PTHrP and PTH have homologous amino acid sequences at their
N-termini, bind to the same receptor (PTHR1), and exert similar eects
on target cells in the bone and kidney [2]. In contrast, PTHrP is unique in
that its amino acid sequence is approximately twice as long as PTH with
domains responsible for its unique functions located in parts of the
sequence that do not share homology with PTH. PTH is produced only in
the parathyroid gland, whereas PTHrP is synthesized by most of the cell
types of the human body, including most of the cell types in the CNS.
PTHrP predominantly acts locally where it is synthesized (in paracrine and
autocrine fashion), but when present in circulating blood, it also acts as
an endocrine hormone [2, 4]. It has also been reported that in the CNS,
PTHrP is involved in neuronal synaptic transmission, integration and
modulation of pain, neural cell proliferation, cell dierentiation, cell
plasticity, and the inammatory response [5].
Measurement of PTHrP concentration in human cerebrospinal uid (CSF)
has been performed in two earlier studies using immunoassays [6, 7]. In
one study CSF-PTHrP was not detectable [6], and in the other study
CSF-PTHrP was shown to be a normal constituent of human CSF, with
concentrations up to 100 times lower than those observed in blood [7].
Based on the importance of calcium regulation in the CNS and the local
production of PTHrP at the sites of its action, we questioned the
conicting ndings related to PTHrP in CSF [5, 6, 8], and hypothesized
that poor specicity of immunoassays used in the studies [6, 7] may be at
fault.
We analysed PTHrP (using LC-MS/MS) and Ca2+ [using inductively coupled
plasma-MS (ICP-MS)] in paired serum and CSF samples collected from
individuals with concentrations of nine CSF diagnostic markers within
respective reference intervals (except when noted otherwise), and
assessed associations between concentrations of PTHrP and Ca2+ in CSF
and serum. In addition, we evaluated the association between PTHrP and
albumin index, a marker of blood-brain barrier (BBB) integrity.
Participants and methods
Patient samples
In the study, we analysed sets of paired serum and CSF samples collected
from 104 women and 36 men, aged 14 to 83 years [9]. The samples were
de-identied, residual aliquots of serum and CSF samples submitted for
routine diagnostic testing at ARUP Laboratories (Salt Lake City, UT, USA).
Concentrations of all measured CSF biomarkers were within the
corresponding reference intervals, except when noted otherwise. Use of
residual de-identied samples was approved by the Institutional Review
Board of the University of Utah (#00007275).
Measurement of PTHrP and Ca2+
PTHrP concentrations were determined using an LC-MS/MS method [3].
The assay was fully validated for plasma samples according to the Clinical
and Laboratory Standards Institute guidelines and performance character-
istics of the method for CSF and serum samples were established [10]. Our
data demonstrated acceptable stability of PTHrP in CSF samples, data on
PTHrP stability in serum samples are shown elsewhere [3]. Ca2+ was
measured by ICP-MS with quantitation performed using the Ca2+ isotope
m/z 43. Calibrators and controls for the Ca2+ assay were from an FDA (US
Food & Drug Administration)-approved assay for Ca2+ in serum (Roche
Diagnostics).
Results
Distribution of concentrations of PTHrP and Ca2+in serum and CSF samples
The combination of immunoanity-based enrichment of PTHrP, use of the
recombinant 15N-labelled PTHrP as the internal standard, and quantitative
analysis based on PTHrP-specic tryptic peptide ( 105YLTQETNK112
) using
LC-MS/MS, allowed high sensitivity and high specicity measurements.
Previously available PTHrP immunoassays were able to detect PTHrP only
in pathologic samples and were not suciently sensitive to measure PTHrP
in samples from healthy individuals [3]; the LC-MS/MS method used in this
study was adequately sensitive to quantify PTHrP in serum and CSF in all
samples included in this study [3].
We evaluated distributions of concentrations of PTHrP and Ca2+ in the
paired serum and CSF samples and assessed association between
concentrations of PTHrP and Ca2+ in the serum and the CSF samples. Ca2+
concentrations in the samples were on average 1.8 times lower in CSF than
in serum; whereas PTHrP concentrations in CSF were 20–150 times higher
in CSF than in serum. The median concentration of PTHrP in CSF was
51 times higher than median concentration of PTHrP in serum (Fig. 1).
Figure 1. Distribution of calcium (a) and PTHrP (b) concentrations in serum and
cerebrospinal uid (CSF) samples, n=140 [adapted from (9)]
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Measured serum Ca2+ concentrations were outside of the reference in-
terval in 22 individuals, after exclusion of these samples from the dataset
(n=118), the median (central 95% distribution) of the concentrations
of Ca2+-CSF, PTHrP-serum and PTHrP-CSF were 5.4 (4.5–6.1) mg/dL, 1.2
(0.5–2.5) pmol/L, and 62 (22–125) pmol/L, respectively.
It was previously reported that the PTHrP receptor (PTHR1) is expressed
in various cell types of central and peripheral nervous systems [11]. In
our study, the ratio of PTHrP-CSF/PTHrP-serum concentrations ranged
between 20 and 150, whereas earlier studies that measured PTHrP in CSF
using immune-based methods, either did not detect PTHrP in CSF [6],
or reported concentrations up to 100 times lower than concentrations
detected in blood [7]. Taken together, the earlier reported expression of
mRNAs encoding PTHrP receptor in various cell types in brain [8, 12], and
observation from this study of signicantly greater PTHrP concentrations
in CSF (as compared to blood), suggest that in health, and especially in
conditions where the BBB is compromised, CSF-derived PTHrP could be a
signicant source of the PTHrP present in circulation. The above observa-
tions also suggest importance of PTHrP in brain function and physiology.
Association of PTHrP and Ca2+ concentrations
with age and sex
Distribution of PTHrP concentration in serum was statistically dierent
between women and men, with higher concentrations observed in
women, whereas distribution of PTHrP-CSF was not [3]. PTHrP concen-
trations in serum were statistically signicantly associated with age; in
the subset of samples, containing serum Ca2+ concentrations within the
reference intervals (n=118), PTHrP-CSF were progressively increasing
with age [9]. Association between the concentrations of Ca2+ with age
approached statistical signicance in both serum and CSF [9].
A trend toward higher PTHrP-serum/PTHrP-CSF ratios was observed in ol-
der individuals; a similar trend was reported for the association between
albumin indices and age [9], suggesting that elevated PTHrP in serum in
older individuals may be associated with age-related changes in the brain.
In our recent study [13] on the association of PTHrP-CSF with Alzhei-
mer’s Disease (AD) biomarkers [total tau protein (T-tau), phosphorylated
tau protein (P-tau) and amyloid β (Aβ42)], we observed trends toward
positive associations between PTHrP-CSF and concentrations of T-tau and
P-tau; and a trend toward a negative association with Aβ42. Higher PTHrP
concentrations and lower Aβ42 concentrations were observed in older
AD patients, while the associations were not observed in the participants
of the control group, suggesting potential utility of the Aβ42/PTHrP ratio
for conrmation of the AD diagnosis and as a marker of disease progres-
sion in older AD patients. Results from the study [13] demonstrated that
PTHrP is not a specic marker for AD pathology, while its presence in
CSF could be a consequence of the release of intracellular PTHrP from
neurons, suggesting potential utility of PTHrP-CSF as a complementary
marker for monitoring neuronal function and integrity of cells in the CNS.
Conclusions
The results of our studies suggest that PTHrP is a normal constituent
of human CSF, which is present in CSF at concentrations 20–150 times
higher than in serum. Concentrations of serum PTHrP were statistically
signicantly associated with age, and with PTHrP concentrations in CSF,
with higher concentrations of PTHrP-serum and PTHrP-CSF observed
in older individuals. The observed higher PTHrP concentrations in CSF
as compared to serum, negative association between PTHrP-CSF and
albumin indices, as well as positive association between concentrations
of PTHrP-CSF and PTHrP-serum, suggest that CSF may be a signicant
contributor to the pool of PTHrP present in circulating blood. The CSF-de-
rived PTHrP, potentially could be a contributing factor to the disorders of
Ca2+ regulation in older individuals, and in individuals with neuropatholo-
gic conditions. Based on our data, PTHrP is not a specic marker for AD
pathology, although it has potential to serve as a complementary marker
for monitoring neuronal function and integrity. Observations from this
study suggest a need for continued research regarding the association of
PTHrP-CSF with age and various neurologic conditions.
Acknowledgements
We thank the ARUP® Institute for Clinical and Experimental
Pathology (Salt Lake City, USA) for nancial support.
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The authors
Mark M. Kushnir*1,2 PhD, Lisa K. Peterson1,2 PhD,
Frederick G. Strathmann3 PhD
1 ARUP Institute for Clinical and Experimental Pathology,
Salt Lake City, UT, USA
2 Department of Pathology, University of Utah,
Salt Lake City, UT, USA
3 NMS Labs, Horsham, PA, USA
*Corresponding author
E-mail: kushnmm@aruplab.com
www.clinlabint.com & search 27822
