Mark Kushnir (Presenter)
ARUP Institute
Bio: Dr. Mark Kushnir is Senior Scientist at ARUP Institute for Clinical and Experimental Pathology. He received PhD in Analytical Chemistry from Uppsala University in Sweden. Dr. Kushnir is an author of over 90 publications, and over 100 scientific presentations.
Authorship: Mark M. Kushnir (1,2,3), Canary Tennison (1), Alan L. Rockwood (1,2,3), Frederick G. Strathmann (1,2,3)
(1) ARUP Laboratories, Salt Lake City, UT; (2) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT; (3) Department of Pathology, University of Utah, Salt Lake City, UT
| Short Abstract Parathyroid hormone related protein (PTHrP) is involved in intracellular calcium regulation. Calcium is necessary for the nerve signaling and brain function, but limited information is available related to PTHrP expression in brain. Recently we developed LC-MS/MS method for the assessment of PTHrP in plasma. Using this method we analyzed PTHrP in a set of paired serum and CSF samples, and evaluated distribution of PTHrP concentrations, and the ratio of concentrations (PTHrPCSF/PTHrPserum) in adults. A trend to higher PTHrP concentrations with increasing age was observed in serum, while a trend to lower concentrations was observed for PTHrPCSF and the PTHrPCSF/PTHrPserum ratio. |
Long Abstract
Introduction: Parathyroid hormone related protein (PTHrP) is involved in intracellular calcium regulation and homeostasis. The main clinical utility of PTHrP as a biomarker is to assist with diagnosing patients clinically suspected of hypercalcemia. Calcium is necessary for the initiation of nerve signaling and release of neurotransmitters, and plays several key roles in normal brain function. PTHrP gene is expressed in various cells within the body, while limited information is available on PTHrP expression and concentrations in brain. Considering a putative role of calcium in brain, the question arises if PTHrP is involved in calcium regulation in the brain and if PTHrP can be measured in CSF.
Methods: Recently we developed and validated a clinical laboratory method for the measurement of PTHrP in plasma using LC-MS/MS. Sample preparation is performed as follows: stable isotope 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 lower limit of quantification and upper limit of linearity of the assay are 0.3 and 1100 pmol/L, respectively. Total imprecision of the method is < 10%. Clinical performance of the assay was evaluated by analysis of PTHrP in sets of plasma samples biochemically characterized as hypercalcemic (Ca> 10.5 mg/dL and PTH< 15 pg/mL; n=91), and samples from self-reported healthy adults with concentrations of Ca and PTH within established reference intervals (n=108). Logistic regression analysis resulted in ROC curve with area under the curve of 0.874.
Results: A set of paired serum and CSF samples, which were tested negative (by isoelectric focusing) for the presence of oligoclonal bands was analyzed for PTHrP using LC-MS/MS and for calcium (Ca) using ICP-MS methods. We evaluated the distribution of PTHrP and calcium concentrations and the ratio of the concentrations (PTHrPCSF/PTHrPserum and CaCSF/Caserum) in adult men and women (n=22), mean age (range) 45.5 years (17-79 years), standard deviation 16.1 years. Measurable concentrations of PTHrP were observed in all the samples. PTHrP concentrations [median (interquartile range)] in CSF and serum were 45 (31-72) pmol/L, and 1.1 (0.73-1.4) pmol/L, respectively. Calcium concentrations [median (interquartile range)] in CSF and serum were 5.3 (4.9-5.9) mg/dL and 11.6 (10.5-12.2) mg/dL, respectively. No statistically significant difference was observed between men and women, and no correlation was observed between PTHrP concentrations in CSF and serum. Calcium concentrations in serum were associated with PTHrPserum (p=0.02) and CaCSF/Caserum were associated with PTHrPserum (p=0.03). Different trends were observed between serum and CSF for the association of the PTHrP and Ca concentrations with age. A trend to higher PTHrP concentrations at advanced age was observed in serum (p=0.49), and a trend to lower PTHrP concentrations was observed in CSF (p=0.14). A trend to higher Ca concentrations at advanced age was observed in serum (p=0.03), and a trend to lower Ca concentrations was observed in CSF (p=0.68). Associations of age with PTHrPCSF/PTHrPserum and CaCSF/Caserum ratios approached level of statistical significance, p=0.16 and p=0.07, respectively.
Conclusions: These data demonstrated that the developed LC-MS/MS method allows the measurement of PTHrP in human CSF and that PTHrP is a normal constituent of CSF. The significance of PTHrP in CSF is poorly understood and ongoing studies are aimed at evaluation of the association of PTHrP concentrations in CSF with various neurologic conditions and brain function.
References & Acknowledgements:
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