Using a filtration technique to isolate platelet free plasma for assaying pyrophosphate

Abstract

Background: Vascular calcification (VC) is a strong prognostic marker of mortality from cardiovascular disease. Extracellular inorganic pyrophosphate (PPi) is a critical inhibitor of vascular calcification and it has been reported that hemodialysis patients have reduced plasma PPi levels, suggesting that altered PPi metabolism could contribute to VC in hemodialysis patients. Platelets are rich in PPi and release of PPi from platelets during storage or processing of plasma can lead to falsely elevated plasma PPi levels. To prepare plasma samples that are suitable for measuring PPi levels, ultracentrifugation has been used to remove platelets. Consequently, plasma PPi measurements have been limited to research laboratories since the majority of clinical laboratories do not have access to an ultracentrifuge. The purpose of the present study was to test the validity of an improved method of preparing platelet free plasma that uses filtration with a 300,000 Dalton molecular weight cut-off filter to exclude platelets, while minimizing their release of PPi.

Methods: In 20 maintenance hemodialysis patients, PPi levels were measured in plasma samples prepared by the conventional technique of low-speed centrifugation to remove red and white blood cells versus a novel filtration technique.

Results: Plasma prepared by filtration had significantly lower platelet counts (0 vs. 3 - 7 10(3)/microL) and PPi levels (1.39 +/- 0.30 microM vs. 2.74 +/- 1.19 microM; mean +/- SD, p < 0.01).

Conclusions: The filtration method appears effective in excluding platelets without causing trauma to platelets and can be used by clinical laboratories to prepare platelet-depleted plasma for PPi measurement.

Figure 1

Key chemical reactions in radiometric enzymatic assay for measurement of pyrophosphate (PPi). Abbreviations: UDPG = Uridine 5′-Diphosphoglucose, PPi = Inorganic Pyrophosphate, UTP = Uridine 5′-Triphosphate, β-NADP = β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form, β-NADPH = β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form, G-6-PDH = Glucose-6-Phosphate Dehydrogenase

Figure 2

Standard Curve for PPi assay. Linear Regression: y = 974.72x +1011 r² = 0.9989.

Figure 3

PPi assay performed in duplicate with (solid symbols) and without (open symbols) UDPG pyrophosphorylase. A single sample of normal human plasma was assayed before and after filtration as described in the methods.

Figure 4

Non-PPi signal in the PPi assay. Filtered human plasma samples were assayed with and without UDPG pyrophosphorylase and values in the absence of enzyme (non-PPi signal; ordinate) are compared with the difference between the values obtained with and without enzyme (PPi signal; abscissa). (n = 86).