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WO2014072545A1 - Procédé et kit de détection de polyphosphates inorganiques dans du plasma sanguin - Google Patents

Procédé et kit de détection de polyphosphates inorganiques dans du plasma sanguin Download PDF

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Publication number
WO2014072545A1
WO2014072545A1 PCT/ES2013/000235 ES2013000235W WO2014072545A1 WO 2014072545 A1 WO2014072545 A1 WO 2014072545A1 ES 2013000235 W ES2013000235 W ES 2013000235W WO 2014072545 A1 WO2014072545 A1 WO 2014072545A1
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polyphosphate
determination
blood plasma
procedure
plasma according
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Spanish (es)
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Felix Alejandro RUIZ RODRIGUEZ
Liliana Marcela MONTILLA RODRIGUEZ
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Universidad de Cadiz
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Universidad de Cadiz
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

  • An object of the present invention is a method for determining the levels of inorganic polyphosphates in the blood plasma. This procedure includes the use of the cryoprecipitated plasma fraction, which concentrates most of the polyphosphate and reduces the contamination of free phosphate in the sample.
  • polyP Inorganic polyphosphates
  • PolyP are polymers of various sizes that are present in all living things (Kornberg, A., J Bacteriol, 1995, 177, 491-6). As the name implies, polyP are made up of more than three phosphate residues, linked together by high energy phosphoanhydrous bonds ( Figure 1A). (Kornberg, A., J Bacteriol, 1995, 177, 491-6).
  • polyP have been widely used in various applications including food processing, use as a food additive and reduction of water hardness, among others (Kornberg, A., J Bacteriol, 1995, 177, 491- 6).
  • polyP can be used for the treatment of gum diseases (Yamaoka, M., Uematsu, T. et al., Gerodontology, 2008, 25, 10-7), to stimulate bone regeneration (Hacchou, Y., Uematsu , T. and coi, J Dent Res, 2007, 86, 893-7) and wound healing (Kim, HR, Kim, HY et al.),
  • polyphosphates can modulate blood coagulation both in vitro (Smith, SA, Mutch, NJ et al, Proc Nati Acad Sci USA, 2006, 103, 903-88) and in vivo (Muller, F., Mutch, NJ et al., Cell, 2009, 139, 1143-56), and also, that increase fibrinolysis (Smith, SA and Morrissey, JH, Blood, 2008, 112, 2810-6).
  • polyP can be used to promote coagulation and reduce bleeding (Morrissey, JH, Smith, SA et al., 2006, Patent US2006198837-A1;, 2010, Patent US 2010/0143492 Al; Smith, SA and Morrissey, JH, 2010, US2010 / 0297257 Al).
  • polyP stimulates the production of the vasoactive peptide bradikinin, with the consequent induction of edema (Muller, F., Mutch, NJ et al, Cell, 2009, 139, 1143-56). PolyP also induces an inflammatory response mediated by the nuclear factor "kappa beta" in endothelial cells (Bae, JS, Lee, W. et al, J Thromb Haemost, 2012, 10, 1145-51).
  • polyP bind and affect the activities of the following plasma proteins: factor XII (Muller, F., Mutch, NJ. Et al, Cell, 2009, 139, 1143-56), fibrin and fibrinogen (Smith, SA and Morrissey, JH, Blood,
  • cryoprecipitate is a high molecular weight protein concentrate that forms when frozen plasma is slowly thawed at temperatures between 1 to 6 ° C (Pool, JG, Gershgold, EJ et al, Nature, 1964, 203, 312).
  • the concentrate mainly contains factor VIII (antihemolytic factor), von Willebrand factor, fibrinogen, factor XIII, fibronectin, and small amounts of other proteins of the plasma (Sparrow, RL, Greening, DW et al., Methods Mol Biol, 2011, 728, 259-65).
  • cryoprecipitate was widely used to treat patients with von Willebrand factor deficiency (von Willebrand disease) or factor VIII deficiency (Hemophilia A), but in general the treatment of these pathologies has been replaced by transfusion of purified factor concentrates (Yang, L., Stanworth, S. et al., Transfus Med, 2012, 22, 315-20).
  • the use of cryoprecipitate is scarce, being common only to replace fibrinogen in the specific coagulopathies of patients deficient in this protein (hypofibrinogenemia), as well as to treat other coagulopathies in cases where isolated plasma factors are not available (Yang, L., Stanworth, S. et al, Transfus Med, 2012, 22, 315-20).
  • BRADFORD MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principie of protein-dye binding.
  • HERNANDEZ-RUIZ L. et al. Platelet inorganic polyphosphate decreases in patients with delta storage pool disease. J Thromb Haemost, v. 7, n. 2 P. 361-3, Feb 2009.
  • LORENZ, B. et al. A novel method for determination of inorganic polyphosphates using the fluorescent dye fura-2.
  • composition useful for e.g. promoting clott ⁇ ng and slowing fibrinolysis comprising a polyphosphate and a carrier: Patent US2006198837-A1 p. 2006
  • Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo. Cell, v. 139, n. 6, p. 1143-56, Dec 11 2009.
  • Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J Biol Chem, v. 279, n. 43, p. 44250-7, Oct 22, 2004.
  • RUIZ F.A. RODRIGUES, C.O .; DOCAMPO, R. Rapid changes in polyphosphate content within acidocalcisomes in response to cell growth, differentiation, and environmental stress in Trypanosoma cruzi. J Biol Chem, v. 276, n. 28, p. 26114-21, Jul 13 2001.
  • PROCOAGULANT Patent US2010 / 0297257 Al p. 2010
  • TAMMENKOSKI M. et al. Human metastasis regulatory protein H-prune is a short-chain exopolyphosphatase. Biochemistry, v. 47, n. 36, p. 9707-13, Sep 9 2008.
  • WANG L. et al. Inorganic polyphosphate stimulates mammalian TOR, a kinase involved in the proliferation of mammary cancer cells. Proc Natl Acad Sci USA, v. 100, n. 20, p. 11249-54, Sep 30 2003.
  • a first object of the present invention is a method for determining the levels of polyphosphates in the blood plasma that includes the following steps:
  • the biological sample includes blood and specifically blood plasma.
  • b) separate high molecular weight proteins from plasma.
  • a proposed method for this separation is that described for obtaining cryoprecipitate for transfusions.
  • c) determine, in this protein fraction, the content of the polyphosphate compound. This protein fraction contains most of the sample polyphosphate as well as minute amounts of contaminating phosphate.
  • Plasma polyPs are potent modulators of coagulation and / or thrombosis and inflammatory processes also play a role. Therefore, the contents of polyP in plasma can be used as an indirect measure of thrombosis and inflammation.
  • the biological sample is taken from humans, but in addition to humans, it can be used to determine the levels of polyP in other mammals such as dogs, cats, pigs, cattle, rats and mice. This can be of special importance to study diseases in animals of food interest, as well as to observe different animal models of pathologies.
  • the procedure comprises the following steps:
  • a) Obtaining the biological sample of the individual to determine their plasma polyP levels.
  • the plasma isolation stage is carried out by a simple centrifugation process.
  • the separation stage of high molecular weight proteins is carried out by freezing and subsequent slow thawing of the plasma, to form the fraction known as cryoprecipitate.
  • the separation of the polyphosphates from the cryoprecipitate fraction from the plasma is done by a procedure that includes extractions with phenol / chloroform solutions (total polyP).
  • the determination of polyphosphates can be performed directly in the cryoprecipitate protein fraction by precipitation of the proteins with a strong acid, such as perchloric acid or trichloroacetic acid, and subsequent neutralization with alkaline solution (polyP bound to proteins).
  • the quantitative determination of the solubilized and separated polyphosphate in the previous steps is carried out, directly by colorimetry or fluorimetry, preferably colorimetry.
  • Another object of the present invention is a kit for the determination of plasma polyphosphates by the aforementioned procedure.
  • This kit includes:
  • cryoprecipitate means for isolating plasma from blood and producing high molecular weight protein extracts (cryoprecipitate).
  • cryoprecipitate means for the detection and quantification of polyphosphate in the cryoprecipitate.
  • kit may include enzymes for the specific processing of polyphosphate in products detectable by colorimetry or by luminescence.
  • the means for producing acidic extracts of plasma cryoprecipitate proteins are acids that are selected from perchloric acid or trichloroacetic acid
  • the means for the detection and quantification of polyphosphate are reagents for colorimetric, fluorescence or luminescence, preferably colorimetric reagents that are selected from basic dyes such as toulidine blue or fluorophores such as Fura-2 or DAPI.
  • kits that include enzymes
  • these are selected from exopolyphosphatases, polyphosphate kinases, or polyphosphate glucokinases.
  • FIG. 1 Panel A shows a scheme of a polyP molecule (left) and a phosphate molecule (right). Each polyP molecule contains at least three phosphate residues. The symbol designated as "n” represents an integer greater than 1 and can be up to several hundred.
  • Panel B shows a scheme for obtaining cryoprecipitate from plasma. Plasma is frozen and then slowly thawed at temperatures of 1 to 6 ° C, which induces the precipitation of high molecular weight or cryoprecipitate (CRIO) proteins. The "Cryoprecipitate Free Plasma" (PLC) fraction was also analyzed.
  • CRIO cryoprecipitate Free Plasma
  • FIG. 2 Histograms of the measurements of total polyP (in panel A) and phosphate (in panel B), in the Cryoprecipitate (CRIO) and "Cryoprecipitate Free Plasma” (PLC) fractions are shown, according to the methodology explained in Example 1. In all cases the values obtained from 1 ml of plasma are offered.
  • Figure 3 Histograms of the measurements of total polyP (panel A), protein-bound polyP (panel B) and phosphate (panel C) are shown in the Plasma and Cryoprecipitate (CRIO) fractions, according to the methodology explained in Example 2.
  • FIG. 4 Histograms of the measurements of total polyP (panel A) and protein-bound polyP (panel B) are shown in the cryoprecipitated plasma fractions, according to the methodology explained in Example 3. Samples were obtained from healthy individuals. (Control) and of patients with dSPD (dSPD).
  • cryoprecipitate defrosting at 4 ° C, followed by simple centrifugation (Pool, JG, Gershgold, EJ et al, Nature, 1964, 203, 312; Edqm, 2010).
  • Other procedures can be used for the separation of cryoprecipitate, such as the use of separating devices, membranes and filters (Foster, PR, 1986, Patent US 4,638,048; Coelho, PH and Wolf, T., 1993, Patent US 5,261,55).
  • the total polyP are then separated from the sample by a procedure described above (Kumble, KD and Kornberg, A., J Biol Chem, 1995, 270, 5818-22) and which includes: a) enzymatic degradation of DNA, RNA and Proteins; b) extractions with phenol / chloroform and with buffer and with chloroform; and c) concentrate the extracted polyP for later determination.
  • Other procedures could also be used for the separation of polyP such as the separation of the long chain polyP described in bacteria (Ault-Riche, D., Fraley, CD et al., J Bacteriol, 1998, 180, 1841-7 ).
  • the phosphates contained in the CRIO and PLC samples are also extracted by collecting the supernatant, after precipitation with a strong acid as described in Example 1.
  • exopolifosafatase Wurst, H. and Kornberg, A., J Biol Chem, 1994, 269, 10996-1001; Ruiz, FA, Rodrigues, CO et al, J Biol Chem, 2001, 276, 26114-21
  • polyphosphate kinase Robotson, NA, Clark, JE et al, J Biol Chem, 1987, 262, 5216-22; Ault-Riche, D., Fraley, CD et al, J Bacteriol, 1998, 180, 1841-7
  • glycokinase polyphosphate Clark, JE, Beegen, H.
  • Example 1 polyP has been quantified by the use of the recombinant and purified form of inorganic yeast exopolyphosphatase (Ruiz, FA, Rodrigues, CO et al, J Biol Chem, 2001, 276, 26114-21).
  • the phosphate of the samples has been evaluated colorimetrically by the use of molybdenum blue and malachite green (Lanzetta, PA, Alvarez, LJ et al., Anal Biochem, 1979, 100, 95-7).
  • Example 2 we performed measurements in the complete Plasma and in the cryoprecipitate fraction of healthy individuals (Figure 3).
  • the levels of total polyP (panel A) and phosphate (panel C) were determined as explained in Example 1.
  • the measurement of protein bound polyP (panel B) obtained by precipitation of the samples with samples is also included.
  • a strong acid in this case, perchloric acid
  • the proteins are resuspended and neutralized, to then determine the polyP attached to them according to the procedure described in Example 1, which is based on the specific degradation with the enzyme exopolyphosphatase.
  • the cryoprecipitate fraction has the highest levels of total polyP and protein-bound polyP, as well as minute amounts of phosphate compared to plasma ( Figure 3).
  • Example 4 we performed a proof of concept in Example 3, using the plasma polyP measurement procedure in healthy individuals and in patients with a coagulation disorder called "delta reservoir disease” (abbreviated as dSPD) ( Figure 4) .
  • dSPD delta reservoir disease
  • 4.5 ml of blood is taken in tubes with 3.2% sodium citrate.
  • the samples were centrifuged at 1800 x g for 20 min at 4 ° C, to obtain the plasma in the supernatant.
  • Plasma is separated into 1 ml aliquots and frozen at -80 ° C for a minimum period of 24 hours. Defrosting is performed at 4 ° C, which takes approximately 30-45 min.
  • the thawed plasmas are then centrifuged at 1800 x g for 10 min at 4 ° C to separate two fractions: the Cryoprecipitate (CRIO) in the pellet and the Cryoprecipitate Free Plasma (PLC) in the supernatant.
  • the Cryoprecipitate is resuspended in 300 ⁇ of saline solution (150mM NaCl, pH 7).
  • Total polyphosphate is isolated, from Cryoprecipitate and PLC, according to the procedure described by Kumble and Kornberg (Kumble, KD and Kornberg, A., JBiol Chem, 1995, 270, 5818-22) with modifications. Briefly, the DNAase and RNAase enzymes (at a final concentration of 40 ⁇ g / ml each) and MgC12 are added to the sample (to obtain a final concentration of 5mM MgC12). Then the sample is incubated at 37 ° C for 30 minutes. Then add the enzyme Proteinase K (350 ⁇ g / ml and incubate at 37 ° C for 1 hour.
  • the sample is extracted with a phenol / chloroform solution (1: 1 equilibrated with Tris-HCL, pH 7.5 ), in which the phases are obtained by centrifugation at 14000 xg for 5 min.
  • the aqueous phase is extracted twice with 50 mM Tris-HCl, pH 7.5, 10 mM EDTA and then three times with Chloroform saturated in water (The phases are obtained by centrifugation at 14000 xg for 5 min.)
  • the polyP of the sample is precipitated by adding 2.5 more volumes of very cold ethanol and 10% of the volume of 5M NaCl. It is centrifuged at 14000 xg for 15 min, The supernatant is removed.The resulting pellet is resuspended with 20 ⁇ of water and used for the determination of polyP.
  • PolyP levels were determined in neutralized extracts by measurement of the inorganic phosphate (Pi) resulting after treatment with an excess of the purified enzyme recombinant yeast exopolyphosphatase (Saccharomyces cerevisiae) (ScrPPXl Enzyme).
  • the ScrPPXl Enzyme was obtained by expressing a plasmid (assignment from Stanford University) with the gene (rPPXl) with histidine tails of the recombinant exopolyphosphatase of S. cerevisiae (Wurst, H., Shiba, T. et al., J Bacteriol, 1995, 177, 898-906), in the strain of E.
  • the polyP determination of the neutralized extracts was performed as described below: In a final volume of 75 ⁇ adjusted to final concentrations of 60 mM Tris-HCl, pH 7.5, and 15 mM MgC12, aliquots of the extracts are mixed together with 0.5 to 2 ⁇ g of purified ScrPPXl Enzyme. Incubations are performed for at least 20 min at 37 ° C and the resulting Pi production was monitored calorimetrically by the phosphate detection method described by Lanzetta et al., Which uses malachite and malachite green molybdate (Lanzetta, PA, Alvarez, LJ et al, Anal Biochem, 1979, 100, 95-7).
  • the phosphate is isolated, from the Cryoprecipitate and from the PLC, from the resulting supernatant after precipitation with a perchloric acid. Briefly, 1/5 of its volume of 3M perchloric acid (HC104) 3M is added to the samples. After an incubation on ice for 30 min, the acidic extracts were centrifuged at 14,000 xg for 5 min. The resulting supernatants were neutralized by the addition of a solution of 1M KOH, 100mM TrisHCl, which results in the formation of KC104 and its precipitation. The precipitated KC104 was separated by centrifugation at 14,000 xg for 5 min, and the resulting supernatant was used for phosphate determination.
  • 3M perchloric acid HC104
  • the phosphate in the sample was monitored colorimetrically by the phosphate detection method described by Lanzetta et al., Which uses ammonium and malachite green molybdate (Lanzetta, PA, Alvarez, LJ et al, Anal Biochem, 1979, 100, 95 -7).
  • Example 1 Plasma and Cryoprecipitate Plasma (CRIO) are obtained according to procedures detailed in Example 1. Extraction and detection of polyP ( Figure 3, panel A) and phosphate ( Figure 3, panel C), were performed according to the procedures indicated in Example 1.
  • the resulting polyP in the precipitate was measured after precipitation with a perchloric acid.
  • a perchloric acid 1/5 of their volume of perchloric acid (HC104) 3 M is added. After an incubation on ice for 30 min, the acidic extracts were centrifuged at 14,000 xg for 5 min at 4 ° C The resulting pellets are resuspended in 100 ⁇ of saline solution (150mM NaCl, pH 7).
  • Plasma was obtained from blood samples as explained in Example 1.
  • the preparation of cryoprecipitate as well as the extraction and detection of total polyP in the samples was performed as explained in Example 1.
  • the extraction and determination of protein-bound polyP was performed in a similar manner as detailed in Example 2.
  • plasma polyP are potent modulators of coagulation and / or thrombosis
  • the method object of the present invention is optimal for the accurate and rapid estimation of these processes. Therefore, it can be very effective as an alternative to evaluate patients with anticoagulant treatments (such as in the administration of heparins and / or coumarins), or as an aid to diagnose congenital coagulation defects (such as dSPD or hemophilia) or acquired (such as it happens with patients with liver and kidney diseases, myeloid leukemias, myelodysplasia or proliferative syndromes).
  • anticoagulant treatments such as in the administration of heparins and / or coumarins
  • congenital coagulation defects such as dSPD or hemophilia
  • acquired such as it happens with patients with liver and kidney diseases, myeloid leukemias, myelodysplasia or proliferative syndromes.
  • the procedure object of the present invention can be effective in estimating the severity of different pathologies with an inflammatory component (such as diabetes, infections, allergies or autoimmune diseases, among others).
  • an inflammatory component such as diabetes, infections, allergies or autoimmune diseases, among others.
  • kits for the evaluation and testing of polyP levels in human and / or animal blood plasma.
  • the kits could include enzymes and / or colorimetric assays that allow quantification of polyP in the plasma.
  • Appropriate kits are expected to include, but are not limited to, means to prepare the cryoprecipitate from plasma and to separate the protein fraction, means to detect and / or quantify polyP, and also control samples of polyP to compare with the values obtained.

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Abstract

La présente invention concerne un procédé et un kit de détection de polyphosphates inorganiques dans du plasma sanguin. Un objet de la présente invention concerne un procédé permettant de déterminer les niveaux de polyphosphates inorganiques dans le plasma sanguin. Ce procédé consiste à utiliser la fraction du plasma cryoprécipité qui concentre la majorité des polyphosphates et réduit la contamination du phosphate libre dans l'échantillon. Un autre objet de la présente invention concerne un kit permettant de déterminer les niveaux de polyphosphates inorganiques dans le plasma sanguin.
PCT/ES2013/000235 2012-11-09 2013-10-21 Procédé et kit de détection de polyphosphates inorganiques dans du plasma sanguin Ceased WO2014072545A1 (fr)

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ES201201137A ES2465970B2 (es) 2012-11-09 2012-11-09 Procedimiento y kit para la determinación de polifosfatos inorgánicos en plasma sanguíneo

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104655857A (zh) * 2015-02-10 2015-05-27 南京大学 一种微生物细胞内多聚磷酸盐的定量检测方法
WO2018191582A1 (fr) * 2017-04-13 2018-10-18 The Board Of Trustees Of The University Of Illinois Analyse permettant de quantifier des polyphosphates

Citations (1)

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ES2325520A1 (es) * 2008-03-04 2009-09-07 Universidad De Cadiz Procedimiento y kit para el diagnostico de transtornos de la coagulacion.

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ES2325520A1 (es) * 2008-03-04 2009-09-07 Universidad De Cadiz Procedimiento y kit para el diagnostico de transtornos de la coagulacion.

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Title
CLARK, J. E. ET AL.: "Isolation of intact chains of polyphosphate from Propionibacterium shermanii grown on glucose or lactate.", JOURNAL OF BACTERIOLOGY., vol. 168, no. 3, December 1986 (1986-12-01), pages 1212 - 1219 *
JIMENEZ-NUNEZ, M. D. ET AL.: "Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription.", HAEMATOLOGICA., vol. 97, no. 8, August 2012 (2012-08-01), pages 1264 - 1271 *
KUMBLE, K. D. ET AL.: "Inorganic polyphosphate in mammalian cells and tissues.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 11, March 1995 (1995-03-01), pages 5818 - 5822 *
LORENZ, B. ET AL.: "A novel method for determination of inorganic polyphosphates using the fluorescent dye fura-2.", ANALYTICAL BIOCHEMISTRY, vol. 246, no. 2, P, March 1997 (1997-03-01), pages 176 - 184 *
LORENZ, B. ET AL.: "Anti-HIV-1 activity of inorganic polyphosphates.", JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES AND HUMAN RETROVIROLOGY: OFFICIAL PUBLICATION OF THE INTERNATIONAL RETROVIROLOGY ASSOCIATION, vol. 14, no. 2, February 1997 (1997-02-01), pages 110 - 118 *
MONTILLA, M. ET AL.: "Polyphosphate binds to human von Willebrand factor in vivo and modulates its interaction with glycoprotein Ib.", JOURNAL OF THROMBOSIS AND HAEMOSTASIS., vol. 10, no. 11, November 2012 (2012-11-01), pages 2315 - 2323. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104655857A (zh) * 2015-02-10 2015-05-27 南京大学 一种微生物细胞内多聚磷酸盐的定量检测方法
WO2018191582A1 (fr) * 2017-04-13 2018-10-18 The Board Of Trustees Of The University Of Illinois Analyse permettant de quantifier des polyphosphates
US11519917B2 (en) 2017-04-13 2022-12-06 The Regents Of The University Of Michigan Assay for quantifying polyphosphates

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