A Preliminary Research On The Quantitative Detection Methods And Clinical Significance Of Plasma Polyphosphate

Background and PurposePolyphosphate (polyP), a linear polymer of orthophosphate residues linked by high-energy phosphoanhydride bonds, which exists in almost all living cells. Rich polyP also has been found in the dense granules of platelets and pathogenic microbes, which has significant coagulant and pro-inflammatory effect, and might be involved in new mechanisms of thrombosis and inflammation. There is not a simple, stable and reliable extraction and quantitative detection method of plasma polyP. Lorenz reported that human plasma contains about6μg polyP but its clinical pathological significance had not been reported and there is no related research of plasma polyP in the domestic.Based on these facts, the purposes of our study is to explore a simple, stable and reliable extraction, quantitative detection method of plasma polyp and to detect the plasma polyP level in patients with infection and patients with thrombosis.MethodsDAPI fluorescence quantitative assay was used in quantative detection of polyP.We detected the fluorescent signal interference of plasma, plasma components to evaluate whether DAPI fluorescence quantitative assay can detect the plasma polyP content directly;We used Martin extraction, glassmilk extraction to extract plasma which contained the polyP standards in order to analyze their effect of extraction and then detected the fluorescent signal of various components coming from the extraction process to analyze which one has the polyP standards;We used the extraction method of Lorenz combined with DAPI fluorescence quantitative assay to detect the level of plasma polyP from patients with infection and patients with venous thrombosis.ResultsIn tris buffer solution, the DAPI-polyP signal reached peak when the DAPI concentration is up to5μg/ml. DAPI-polyP fluorescence signal in the0.05to0.8μg/ml range of polyP concentration had a linear relationship with the concentration of polyP (P<0.01).In DAPI staining solution containing0.8μg polyP, the fluorescence value of no heparin group and heparin group had significant difference (P<0.01); No citrate group and citrate group had no significant difference on fluorescence value and statistics (P>0.05). In DAPI staining solution containing0.6μg polyP, the fluorescence value of no SDS group and SDS group had significant difference (P<0.01); The fluorescence value of no albumin and albumin group had no significant difference (P>0.05); The fluorescence value of no plasma and plasma group had significant difference (P<0.05).For all kinds of Martin extractions, in the solutions of SDS+boil+nuclease+PK, boil+nuclease+PK, boiling+PK, the fluorescence value between polyP standards adding group and no polyP standards group had statistical difference (P<0.05), the fluorescence value of boil+nuclease+PK+boil method in two groups had no significant difference (P>0.05). In the solutions of boil+nuclease+PK and boil+PK, the extraction ratio of polyP each was only5.7%and2.8%. In various components coming from the extraction process, only the protein precipitation detected a low polyP fluorescent signal.For all kinds of glassmilk solutions, in the solutions of SDS+ethanol+Glassmilk, SDS+Glassmilk, Glassmilk, the fluorescence value between0.6μg polyP standards adding group and no polyP standards group had no significant difference (P>0.05), the fluorescence value between6μg polyP standard adding group and no polyP standards group had no significant difference (P>0.05). In various components coming from the extraction process, only the abandoned liquid after glassmilk adsorption detected a high level signal of polyP.The plasma polyP level of infection group was15.301±6.106, was significantly different from (P<0.05) normal control group (3.939±1.112); The plasma polyP level of venous thrombosis group was3.841±1.575, was significantly different from (P>0.05) normal control group (3.939±1.112)Conclusion1.Heparin, SDS will disturb the DAPI-polyP signal which should avoid to use in experiment, while citrate anticoagulant tube can be used.2.Only the DAPI fluorescence quantitative assay cannot detect the plasma polyP, and methods which based on Martin extraction and glassmilk extraction still cannot effectively extract the plasma polyP.3.We can use the extraction method of Lorenz combined with DAPI fluorescence quantitative assay to detect the level of plasma polyp.4.Plasma polyP level in patients with infection was increased; Plasma polyP level in patients with venous thrombosis had no significant change.