Mark Kushnir (Presenter)
ARUP Institute for Clinical & Experimental Pathology
Bio: Mark Kushnir, Ph.D. Senior Scientist ARUP Institute for Clinical and Experimental Pathology Education: PhD in Analytical Chemistry from Uppsala University (Sweden). Affiliations: • Adjunct Assistant Professor, Department of Pathology, University of Utah Publications and presentations: • Author/coauthor of over 100 publications • Over 130 scientific presentations at scientific conferences • 8 patents • Author/coauthor of 7 book chapters • Citation index 26 Reviewer for Journals: Clinical Chemistry, Clinical Biochemistry, Clin Chim Acta, Rapid Communications in Mass Spectrometry, Journal of American Society of Mass Spectrometry, Journal of Chromatography, Bioanalysis.
Authorship: Mark M. Kushnir, Frederick G. Strathmann.
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, USA; Department of Pathology, University of Utah, Salt Lake City, USA.
| Short Abstract Calcium plays role in the nerve signaling and brain function, while PTHrP is involved in intracellular calcium regulation. Limited information is available related to PTHrP expression in brain. We analyzed PTHrP using LC-MS/MS and calcium using ICP-MS in sets of paired serum/CSF samples from individuals without neurologic diseases and patients with elevated albumin indexes, and assessed association between concentrations of PTHrP and calcium, and concentration of nine common CSF biomarkers. We observed association of PTHrP concentrations with age and albumin index, suggesting that PTHrP may play a role in age-related physiologic changes in the brain and in pathologic neurologic conditions. |
Long Abstract
Introduction: Parathyroid hormone related protein (PTHrP) is involved in intracellular calcium regulation and homeostasis. Calcium is necessary for the initiation of nerve signaling and release of neurotransmitters, and plays several key roles in normal brain function. PTH/PTHrP receptors were identified in various cells in brain and were demonstrated to be involved in mediation of the inflammatory response, transmission and modulation of pain, regulation of neural cell proliferation, cross-talk between glial cells and neurons, cell differentiation and functions. Limited information is available on PTHrP expression and concentrations in CSF and in brain.
Methods: Recently we developed and validated a clinical laboratory method for the measurement of PTHrP in plasma using LC-MS/MS, and evaluated the method’s performance for analysis of CSF samples (1). Sample preparation is performed as follows: 15N labeled internal standard is added to the samples and PTHrP is enriched using anti-PTHrP antibody conjugated to magnetic beads. After incubation, the beads are washed, PTHrP is digested with trypsin and the samples are analyzed by 2D LC-MS/MS in MRM mode of acquisition. The assay was fully validated according to the CLSI guidelines. Limit of quantitation of the assay was 0.3 pmol/L; the upper limit of linearity was 1100 pmol/L; the total imprecision of the assay at concentrations 1.3 and 600 pmol/L was 9.3% and 5.8%, respectively.
Ca was measured using ICP-MS (Agilent 7900, Santa Clara, CA) equipped with autosampler Teledyne MVX-7100 (Teledyne CETAC, Omaha, NB). Sample preparation consisted of diluting 50 ìL of CSF or serum with 450 ìL of 1% nitric acid containing EDTA, Triton-X, and internal standard (Iridium). The quantitation of Ca was performed using Ca isotope m/z 43. The imprecision of the assay was <5% at 6.0, 9.5 and 11.6 mg/dL.
Results: We analyzed PTHrP and Ca in sets of paired serum/CSF samples collected from individuals without neurologic diseases (n=140) and samples from patients with elevated albumin indexes (n=40), and assessed association between the measured concentrations in CSF and serum samples, and with concentrations of nine common CSF biomarkers.
In samples from individuals without neurologic diseases Ca concentrations were on average 1.8 times lower in CSF than in serum; median concentration of PTHrP in CSF was 51 times higher than in serum. Distribution of PTHrP concentrations in serum was statistically different between women and men (p=0.043), while distribution of the concentrations in CSF was not. PTHrP concentrations in serum were statistically significantly associated with age (p=0.0082); no statistically significant association with age was observed for concentrations of PTHrP in CSF. Association of the concentrations of Ca with age approached statistical significance in both, serum (p=0.064) and CSF (p=0.061). PTHrP concentrations in serum were associated with the concentrations in CSF (p=0.019). Statistically significant differences were observed in the ratios PTHrP-serum/ PTHrP-CSF between the age groups <30 and >60; and 50-59 and >60 years. The ratio of concentrations (PTHrP-CSF/albumin-CSF)/(PTHrP-serum/albumin-serum) ranged between 3,300 and 39,100. Statistically significant difference in the ratio was observed between the age groups <30 and >60 (p=0.0047), and 30-49 and >60 (p=0.031). Higher albumin index values were associated with lower concentrations of PTHrP in serum (p=0.0019), and with higher ratio of PTHrP-serum/PTHrP-CSF (p=0.0028).
Conclusions: Our data demonstrated that PTHrP is a normal constituent of CSF, with concentrations in CSF been 20–150 times higher than in serum. This is contrary to an earlier study that used RIA for the measurements, and reported PTHrP concentrations in CSF up to 100 times lower than in plasma (2). Concentrations of serum PTHrP were statistically significantly associated with age, and with PTHrP concentrations in CSF. Higher concentrations of serum PTHrP were observed in older individuals and in females when compared to males. Lower serum PTHrP concentrations and higher ratios PTHrP-serum/PTHrP-CSF were observed in individuals with higher albumin index. The data suggest that CSF may be a significant contributor to the pool of PTHrP present in circulating blood. The observed association of PTHrP concentrations with age and albumin index suggests that PTHrP may play a role in age-related physiologic changes in the brain and in pathologic neurologic conditions. The significance of PTHrP in CSF is poorly understood; ongoing studies are aimed at evaluation of the association of PTHrP concentrations in CSF with various neurologic conditions and brain function.
References & Acknowledgements:
References
1. Kushnir MM, Rockwood AL, Strathmann FG, Frank EL, Straseski JA, Meikle AW.LC-MS/MS Measurement of Parathyroid Hormone-Related Peptide. Clin Chem. 2016;62:218-26.
2. Bühler G, Balabanova S, Milowski S, Rosenthal J, Antoniadis G, Mohr K, Richter HP. Detection of immunoreactive parathyroid hormone-related protein in human cerebrospinal fluid. Exp Clin Endocrinol Diabetes. 1997;105:336-40.
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