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WO2014072545A1 - Method and kit for determining inorganic polyphosphates in blood plasma - Google Patents

Method and kit for determining inorganic polyphosphates in blood plasma 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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French (fr)
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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

The invention relates to a method and kit for determining inorganic polyphosphates in blood plasma. More specifically, the invention relates to a method for determining the levels of inorganic polyphosphates in blood plasma. The method comprises the use of the fraction of the cryoprecipitated plasma that contains most of the polyphosphates and reduces the contamination of the free phosphate in the sample. The invention also relates to a kit for determining polyphosphates in a blood sample.

Description

PROCEDIMIENTO Y KIT PARA LA DETERMINACIÓN DE POLIFOSFATOS INORGÁNICOS EN PLASMA SANGUÍNEO.  PROCEDURE AND KIT FOR THE DETERMINATION OF INORGANIC POLYPHOSPHATES IN BLOOD PLASMA.

OBJETO DE LA INVENCIÓN OBJECT OF THE INVENTION

Constituye un objeto de la presente invención un procedimiento para la determinación de los niveles de los polifosfatos inorgánicos en el plasma sanguíneo. Este procedimiento comprende el empleo de la fracción del plasma crioprecipitado, que concentra la mayoría del polifosfato y reduce la contaminación del fosfato libre en la muestra.  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.

ESTADO DE LA TECNICA STATE OF THE TECHNIQUE

Los polifosfatos inorgánicos (polyP) son polímeros de variado tamaño que están presentes en todos los seres vivos (Kornberg, A., J Bacteriol, 1995, 177, 491-6). Como su nombre lo indica, los polyP están formados por más de tres residuos de fosfato, unidos entre sí por enlaces fosfoanhidro de alta energía (Figura 1A). (Kornberg, A., J Bacteriol, 1995, 177, 491-6).  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).

En la industria los polyP han sido ampliamente usados en variadas aplicaciones que incluyen el procesamiento de alimentos, el uso como aditivo alimentario y la reducción de la dureza del agua, entre otros (Kornberg, A., J Bacteriol, 1995, 177, 491-6).  In the industry, 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).

En años recientes, muy variadas funciones han sido asociadas a los polyP relacionadas con la medicina por ejemplo la modulación de la proliferación celular (Wang, L., Fraley, C.D. y col, Proc Nati Acad Sci USA, 2003, 100, 11249-54), la angiogénesis (Han, K.Y., Hong, B.S. y col., Biochem J, 2007, 406, 49-55), la mineralización de los huesos (Schroder, H.C., Kurz, L. y col, Biochemistry (Mosc), 2000, 65, 296-303), el metabolismo energético (Pavlov, E., Aschar-Sobbi, R.y coL, J Biol Chem, 285, 9420-8) y metástasis de tumores (Tammenkoski, M., Koivula, K. y col, Biochemistry, 2008, 47, 9707-13), entre otros. Se ha propuesto que los polyP pueden ser utilizados para el tratamiento de enfermedades de las encías (Yamaoka, M., Uematsu, T. y col., Gerodontology, 2008, 25, 10-7), para estimular la regeneración de los huesos (Hacchou, Y., Uematsu, T. y coi, J Dent Res, 2007, 86, 893-7) y en la cicatrización de las heridas (Kim, H.R., Kim, H.Y. y col.), In recent years, very varied functions have been associated with polyP related to medicine such as cell proliferation modulation (Wang, L., Fraley, CD et al, Proc Nati Acad Sci USA, 2003, 100, 11249-54 ), angiogenesis (Han, KY, Hong, BS et al., Biochem J, 2007, 406, 49-55), bone mineralization (Schroder, HC, Kurz, L. et al, Biochemistry (Mosc), 2000, 65, 296-303), energy metabolism (Pavlov, E., Aschar-Sobbi, Ry coL, J Biol Chem, 285, 9420-8) and tumor metastases (Tammenkoski, M., Koivula, K. and col, Biochemistry, 2008, 47, 9707-13), among others. It has been proposed that polyP they 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.),

En este sentido, nuestro grupo de investigación en el año 2004, describió que los polyP se acumulan en las plaquetas humanas y que son secretados por las plaquetas activadas en los procesos de coagulación de la sangre (Ruiz, F.A., Lea, C.R. y col., J Biol Chem, 2004, 279, 44250-7). A partir de ese momento, ha surgido un marcado interés por el estudio de las posibles funciones de los polyP relacionadas con la hematología. En consecuencia, se ha encontrado que los polifosfatos pueden modular la coagulación de la sangre tanto in vitro (Smith, S.A., Mutch, N.J. y col, Proc Nati Acad Sci USA, 2006, 103, 903-8) como in vivo (Muller, F., Mutch, N.J. y col., Cell, 2009, 139, 1143-56), y también, que incrementan la fibrinólisis (Smith, S.A. y Morrissey, J.H., Blood, 2008, 112, 2810-6). Por tanto se ha propuesto que los polyP pueden ser usados para promover la coagulación y disminuir el sangrado (Morrissey, J.H., Smith, S.A. y col., 2006, Patent US2006198837-A1; , 2010, Patent US 2010/0143492 Al; Smith, S.A. y Morrissey, J.H., 2010, Patent US2010/0297257 Al). In this sense, our research group in 2004, described that polyP accumulate in human platelets and that they are secreted by activated platelets in blood clotting processes (Ruiz, FA, Lea, CR et al. , J Biol Chem, 2004, 279, 44250-7). From that moment, there has been a marked interest in the study of the possible functions of polyP related to hematology. Consequently, it has been found that polyphosphates can modulate blood coagulation both in vitro (Smith, SA, Mutch, NJ et al, Proc Nati Acad Sci USA, 2006, 103, 903-8) 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). Therefore it has been proposed that 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).

Estos estudios, han servido de origen a recientes descubrimientos acerca de funciones para de los polyP relacionadas con la inflamación. Los polyP estimulan la producción del péptido vasoactivo bradikinina, con la consecuente inducción de edemas (Muller, F., Mutch, N.J. et al, Cell, 2009, 139, 1143-56). Los polyP también inducen una la respuesta inflamatoria mediada por el factor nuclear "kappa beta" en las células endoteliales (Bae, J.S., Lee, W. y col, J Thromb Haemost, 2012, 10, 1145-51). Más aún, nuestro grupo ha encontrado que las células de mastocitos y basófilos, determinantes en la inflamación y en los procesos de alergia, contienen estos polyP pro-inflamatorios en su granulos secretores (Moreno-Sánchez, D., Hernandez-Ruiz, L. y col, J Biol Chem, 2012, 287, 28435-44). A pesar de la relevancia de los polyP en la coagulación y en la inflamación, los niveles presentes de este polímero en el plasma de la sangre no han podido ser determinados de manera precisa hasta el momento. Dependiendo los niveles encontrados dentro de las plaquetas, hasta ahora sólo se había estimado que debería estar en unos 3 Μ (Smith, S.A., Mutch, NJ. et al, Proc Nati Acad Sci U SA, 2006, 103, 903-8). La invención que aquí se presenta permite, por primera vez, la estimación precisa de los polyP en el plasma. These studies have given rise to recent discoveries about functions for polyP related to inflammation. The polyP stimulate 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). Moreover, our group has found that mast cell and basophil cells, determinants in inflammation and allergy processes, contain these pro-inflammatory polyP in their secretory granules (Moreno-Sánchez, D., Hernandez-Ruiz, L. et al, J Biol Chem, 2012, 287, 28435-44). Despite the relevance of polyP in coagulation and inflammation, the levels of this polymer present in the blood plasma have not been determined precisely so far. Depending on the levels found within the platelets, until now it had only been estimated that it should be around 3 Μ (Smith, SA, Mutch, NJ. Et al, Proc Nati Acad Sci U SA, 2006, 103, 903-8). The invention presented here allows, for the first time, the precise estimation of polyP in the plasma.

Una de las razones que dificulta la medida de polyP en al plasma sanguíneo es la alta concentración de fosfato inorgánico presente (figura IB). Los niveles normales de fosfato en un adulto están entre 0,8 y l,45mM (2,5- 4.5 mg/dL) (Yu, A.S.L., Cecil Medicine, 2011, chap. 121), que son concentraciones 250-500 veces más altas que la estimadas para los polyP.  One of the reasons that makes it difficult to measure polyP in the blood plasma is the high concentration of inorganic phosphate present (Figure IB). Normal phosphate levels in an adult are between 0.8 and 1, 45mM (2.5-4.5 mg / dL) (Yu, ASL, Cecil Medicine, 2011, chap. 121), which are 250-500 times higher concentrations than estimated for polyP.

Recientemente se ha propuesto que algunas de las funciones del polyP en la sangre ocurren a través de su unión con las proteínas presentes en el plasma sanguíneo. Hasta el momento se ha descrito que los polyP se unen y afectan las actividades de las siguientes proteínas del plasma: factor XII (Muller, F., Mutch, NJ. et al, Cell, 2009, 139, 1143-56), fibrina y fibrinógeno (Smith, S.A. y Morrissey, J.H., Blood, Recently it has been proposed that some of the functions of polyP in the blood occur through its union with the proteins present in the blood plasma. So far it has been described that 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,

2008, 112, 2810-6; Mutch, N.J., Engel, R. y col, Blood, 2010, 115, 3980-8), trombina (Mutch, N.J., Myles, T. y col, J Thromb Haemost, 2010, 8, 548-55), Proteasa activadora de Factor VII (FSAP) (Muhl, L., Galuska, S.P. y col, Febs J,2008, 112, 2810-6; Mutch, NJ, Engel, R. et al, Blood, 2010, 115, 3980-8), thrombin (Mutch, NJ, Myles, T. et al, J Thromb Haemost, 2010, 8, 548-55), Activating protease of Factor VII (FSAP) (Muhl, L., Galuska, SP et al, Febs J,

2009, 276, 4828-39) y Factor von Willebrand (Montilla, M., Hernandez-Ruiz, L. y col, J Thromb Haemost, 2012). 2009, 276, 4828-39) and Factor von Willebrand (Montilla, M., Hernandez-Ruiz, L. et al, J Thromb Haemost, 2012).

Un procedimiento ampliamente usado para concentrar algunas de las proteínas del plasma, para donaciones, es la elaboración del crioprecipitado (EDQM, 2010) (figura IB). El crioprecipitado es un concentrado de proteínas de alto peso molecular que se forma cuando el plasma congelado es descongelado lentamente a temperaturas entre 1 a 6°C (Pool, J.G., Gershgold, E.J. y col, Nature, 1964, 203, 312). El concentrado contiene principalmente factor VIII (factor antihemolítico), factor von Willebrand, fibrinógeno, factor XIII, fibronectina, y pequeñas cantidades de otras proteínas del plasma (Sparrow, R.L., Greening, D.W. y col., Methods Mol Biol, 2011, 728, 259- 65). A widely used procedure to concentrate some of the plasma proteins, for donations, is the preparation of cryoprecipitate (EDQM, 2010) (figure IB). The 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).

En el pasado, el crioprecipitado fue usado ampliamente para tratar pacientes con deficiencia del factor von Willebrand (Enfermedad de von Willebrand) o con deficiencia del factor VIII (Hemofilia A), pero en general el tratamiento de éstas patologías se ha ido remplazando por la transfusión de concentrados de los factores purificados (Yang, L., Stanworth, S. y col., Transfus Med, 2012, 22, 315-20). Actualmente, el uso del crioprecipitado es escaso, siendo común sólo para suplir el fibrinógeno en las coagulopatías específicas de los pacientes deficientes en esta proteína (hipofibrinogenemia), así como para tratar otras coagulopatías en los casos donde no se disponga de los factores del plasma aislados (Yang, L., Stanworth, S. et al, Transfus Med, 2012, 22, 315-20).  In the past, 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). Currently, 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).

La invención que aquí se presenta está basada en el hallazgo de que la mayoría de los polyP presentes en el plasma de la sangre están unidos a las proteínas que se acumulan en la fracción del crioprecipitado (Figura 2A).  The invention presented here is based on the finding that the majority of polyP present in the blood plasma are bound to the proteins that accumulate in the cryoprecipitate fraction (Figure 2A).

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EXPLICACION DE LA INVENCIÓN EXPLANATION OF THE INVENTION

Constituye un primer objeto de la presente invención un procedimiento para la determinación de los niveles de polifosfatos en el plasma sanguíneo que incluye las siguientes etapas:  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:

a) obtener una muestra biológica de un individuo. La muestra biológica incluye sangre y específicamente plasma sanguíneo. a) obtain a biological sample from an individual. The biological sample includes blood and specifically blood plasma.

b) separar las proteínas de alto peso molecular del plasma. Un método propuesto para esta separación es el descrito para la obtención del crioprecipitado para transfusiones. c) determinar, en esta fracción de proteínas, el contenido del compuesto polifosfato. Esta fracción de proteínas contiene la mayoría del polifosfato de la muestra a la vez que cantidades ínfimas de fosfato contaminante. 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.

Los polyP plasmáticos son potentes moduladores de la coagulación y/o trombosis y también tienen también un papel determinante los procesos inflamatorios. Por tanto, los contenidos de polyP en plasma pueden ser utilizados como medida indirecta de la trombosis y de la inflamación.  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.

Por otra parte, para la presente invención se aportan ejemplos en los cuales la muestra biológica se toma de humanos, pero además de los humanos, se puede utilizar para determinar los niveles de polyP en otros mamíferos como por ejemplo, perros, gatos, cerdos, ganado vacuno, ratas y ratones. Esto puede ser de especial importancia para estudiar enfermedades en animales de interés alimentario, así como para observar diferentes modelos animales de patologías.  On the other hand, examples are provided for the present invention in which 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.

Específicamente, el procedimiento comprende las siguientes etapas:  Specifically, the procedure comprises the following steps:

a) obtención del una muestra biológica del individuo a determinar sus niveles de polyP plasmáticos. b) aislamiento del plasma de los otros componentes de la muestra biológica. c) separación de las proteínas de alto peso molecular del plasma. d) separación de los polifosfatos inorgánicos del resto de componentes de la fracción de proteínas de alto peso molecular del plasma. e) determinación cuantitativa del polifosfato solubilizado y separado en las etapas anteriores. a) Obtaining the biological sample of the individual to determine their plasma polyP levels. b) plasma isolation from the other components of the biological sample. c) separation of high molecular weight proteins from plasma. d) separation of inorganic polyphosphates from the other components of the high molecular weight protein fraction of plasma. e) quantitative determination of solubilized and separated polyphosphate in the previous stages.

La etapa de aislamiento del plasma se lleva a cabo mediante un proceso de centrifugación simple.  The plasma isolation stage is carried out by a simple centrifugation process.

La etapa de separación de proteínas de alto peso molecular se realiza mediante la congelación y posterior descongelación lenta del plasma, para formar la fracción conocida como crioprecipitado.  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.

La separación de los polifosfatos de la fracción de crioprecipitado del plasma se realiza mediante un procedimiento que incluye extracciones con soluciones de fenol/cloroformo (polyP total). Alternativamente, la determinación de polifosfatos puede realizarse directamente en la fracción de proteínas del crioprecipitado mediante la precipitación de las proteínas con un ácido fuerte, como ácido perclórico o ácido tricloroacético, y la posterior la neutralización con solución alcalina (polyP unido a proteínas).  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). Alternatively, 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).

La determinación cuantitativa del polifosfato solubilizado y separado en las etapas anteriores se efectúa, directamente mediante colorimetría o fluorimetría, preferentemente colorimetría.  The quantitative determination of the solubilized and separated polyphosphate in the previous steps is carried out, directly by colorimetry or fluorimetry, preferably colorimetry.

Alternativamente, la determinación cuantitativa del polifosfato solubilizado y separado incluye:  Alternatively, quantitative determination of solubilized and separated polyphosphate includes:

a) un ataque enzimático para procesar el polifosfato, liberándose los productos resultantes de dicha reacción enzimática. a) an enzymatic attack to process the polyphosphate, freeing the products resulting from said enzymatic reaction.

b) determinación cuantitativa de alguno de los productos resultantes de la reacción enzimática mediante colorimetría o por luminiscencia, preferentemente mediante colorimetría. b) quantitative determination of any of the products resulting from the enzymatic reaction by colorimetry or by luminescence, preferably by colorimetry.

Constituye otro objeto de la presente invención un kit para la determinación de los polifosfatos plasmáticos mediante el referido procedimiento. Dicho kit incluye: Another object of the present invention is a kit for the determination of plasma polyphosphates by the aforementioned procedure. This kit includes:

- medios para aislar el plasma de la sangre y producir los extractos de proteínas de alto peso molecular (crioprecipitado). - medios para la detección y cuantificación del polifosfato en el crioprecipitado.- means for isolating plasma from blood and producing high molecular weight protein extracts (cryoprecipitate). - means for the detection and quantification of polyphosphate in the cryoprecipitate.

- muestras de control de polifosfato y/o plasma para comparar con los valores obtenidos. - polyphosphate and / or plasma control samples to compare with the values obtained.

Adicionalmente, el kit puede incluir enzimas para el procesamiento específico de polifosfato en productos detectables mediante colorimetría o por luminiscencia. Additionally, the kit may include enzymes for the specific processing of polyphosphate in products detectable by colorimetry or by luminescence.

En realizaciones preferentes del kit, los medios para producir extractos acídicos de las proteínas del crioprecipitado del plasma son ácidos que se seleccionan entre ácido perclórico o ácido tricloroacético, los medios para la detección y cuantificación del polifosfato son reactivos para ensayos colorímétricos, de fluorescencia o de luminiscencia, preferentemente reactivos colorimétricos que se seleccionan entre colorantes básicos como el azul de toulidina o fluoróforos como Fura-2 o DAPI.In preferred embodiments of the kit, 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.

En el caso de kits que incluyan enzimas, éstas se seleccionan entre las exopolifosafatasas, la polifosfato kinasas, o la polifosfato glucokinasas. In the case of kits that include enzymes, these are selected from exopolyphosphatases, polyphosphate kinases, or polyphosphate glucokinases.

BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES

Figura 1: En el panel A se muestra un esquema de una molécula de polyP (izquierda) y de una molécula de fosfato (derecha). Cada molécula de polyP contiene al menos tres residuos de fosfato. El símbolo designado como "n" representa un número entero superior a 1 y puede ser hasta varios cientos. El panel B muestra un esquema de obtención del crioprecipitado del plasma. El Plasma es congelado y luego descongelado lentamente a temperaturas de 1 a 6°C, lo que induce la precipitación de las proteínas de alto peso molecular o Crioprecipitado (CRIO). También se analizó la fracción de "Plasma Libre de Crioprecipitado" (PLC).  Figure 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.

Figura 2: Se muestran histogramas de las medidas de polyP total (en el panel A) y de fosfato (en el panel B), en las fracciones de Crioprecipitado (CRIO) y de "Plasma Libre de Crioprecipitado" (PLC), según la metodología explicada en el Ejemplo 1. En todos los casos se ofrece los valores obtenidos a partir de 1 mi de plasma. Figura 3: Se muestran histogramas de las medidas de polyP total (panel A), del polyP unido a proteínas (panel B) y de fosfato (panel C), en las fracciones de Plasma y de Crioprecipitado (CRIO), según la metodología explicada en el Ejemplo 2. Figure 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.

Figura 4: Se muestran histogramas de las medidas de polyP total (panel A) y del polyP unido a proteínas (panel B), en las fracciones de plasma crioprecipitado, según la metodología explicada en el Ejemplo 3. Las muestras fueron obtenidas de individuos sanos (Control) y de pacientes con dSPD (dSPD). Figure 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).

DESCRIPCIÓN DETALLADA Y MODO DE REALIZACION DE LA INVENCIÓN DETAILED DESCRIPTION AND MODE OF EMBODIMENT OF THE INVENTION

Se ha identificado que la mayoría de los polyP se concentran en la fracción de crioprecipitado del plasma. Esta fracción posee niveles muy bajos de fosfato contaminante lo que permite la determinación precisa del polyP presente en el plasma. A pesar de la relevancia de los polyP en la coagulación y en la inflamación, los niveles presentes de este polímero en el plasma de la sangre no han podido ser determinados de manera precisa hasta el momento y el método propuesto resuelve este problema.  It has been identified that the majority of polyP are concentrated in the cryoprecipitate fraction of the plasma. This fraction has very low levels of contaminating phosphate, which allows the precise determination of the polyP present in the plasma. Despite the relevance of polyP in coagulation and inflammation, the levels of this polymer in the blood plasma have not been determined precisely so far and the proposed method solves this problem.

Utilizando la metodología descrita más detalladamente en el Ejemplo 1, se ha determinado las concentraciones de polyP y de fosfato en las dos fracciones resultantes del plasma después su congelación y descongelación lenta: El Crioprecipitado (CRIO) y el "Plasma Libre de Crioprecipitado" (PLC) (Figura 2). La mayoría del polyP se concentra en la fracción del CRIO y la mayoría del fosfato se acumula en la fracción PLC (Figura 2). Brevemente, el método se inició con la recogida de sangre de individuos sanos que no sufrían trastornos en la coagulación y que una semana antes de la donación de sangre no habían tomado medicaciones que pudieran afectar sus niveles de coagulación. Seguidamente, el plasma se aisló de la muestra de sangre por centrifugación simple. A continuación se obtuvieron las fracciones de CRIO y de PLC, mediante una congelación a -80°C y posterior descongelación a 4°C, seguido de una centrifugación simple (Pool, J.G., Gershgold, E.J. et al, Nature, 1964, 203, 312; Edqm, 2010). Otros procedimientos pueden ser usados para la separación del crioprecipitado, como el uso de aparatos separadores, membranas y filtros (Foster, P.R., 1986, Patent US 4,638,048; Coelho, P.H. y Wolf, T., 1993, Patent US 5,261,55). Using the methodology described in more detail in Example 1, the concentrations of polyP and phosphate in the two resulting fractions of the plasma have been determined after freezing and slow thawing: Cryoprecipitate (CRIO) and "Cryoprecipitate Free Plasma" (PLC ) (Figure 2). Most polyP is concentrated in the CRIO fraction and most phosphate accumulates in the PLC fraction (Figure 2). Briefly, the method began with the collection of blood from healthy individuals who did not suffer from coagulation disorders and who a week before the blood donation had not taken medications that could affect their coagulation levels. Next, the plasma was isolated from the blood sample by simple centrifugation. The CRIO and PLC fractions were then obtained by freezing at -80 ° C and later. 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).

Luego se separan los polyP totales de la muestra por un procedimiento descrito anteriormente (Kumble, K.D. y Kornberg, A., J Biol Chem, 1995, 270, 5818-22) y que incluye: a) la degradación enzimática del ADN, el ARN y las Proteínas; b) extracciones con fenol/cloroformo y con tampón y con cloroformo; y c) concentrar los polyP extraídos para su posterior determinación. Otros procedimientos también podrían ser utilizados para la separación de los polyP como por ejemplo la separación de los polyP de cadena larga descritos en bacteria (Ault-Riche, D., Fraley, C.D. y col, J Bacteriol, 1998, 180, 1841-7). En paralelo, se extraen también los fosfatos contenidos en las muestras de CRIO y de PLC mediante la recogida del sobrenadante, luego de la precipitación con un ácido fuerte como se describe en el Ejemplo 1. 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 ). In parallel, 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.

Varios métodos pueden ser utilizados para la detección de polyP, que incluyen, pero no están limitados a, reacciones metacromáticas con colorantes básicos como azul de toluidina (Lorenz, B. y Schroder, H.C., Prog Mol Subcell Biol, 1999, 23, 217-39), la detección de los cambios de fluorescencia de fluoróforos como Fura-2 o DAPI en presencia de polyP (Lorenz, B., Munkner, J. y col, Anal Biochem, 1997, 246, 176- 84; Aschar-Sobbi, R., Abramov, A. Y. y col, J Fluoresc, 2008, 18, 859-66), o la determinación colorimétrica de los productos resultantes después de la incubación de los extractos con enzimas específicas que procesan el polyP. Como ejemplos de las enzimas utilizadas en este útimo tipo de detección están la exopolifosafatasa (Wurst, H. y Kornberg, A., J Biol Chem, 1994, 269, 10996-1001; Ruiz, F.A., Rodrigues, C.O. y col, J Biol Chem, 2001, 276, 26114-21), la polifosfato kinasa (Robinson, N.A., Clark, J.E. y col, J Biol Chem, 1987, 262, 5216-22; Ault-Riche, D., Fraley, C.D. et al, J Bacteriol, 1998, 180, 1841-7), o la polifosfato glucokinasa (Clark, J.E., Beegen, H. y col, J Bacteriol, 1986, 168, 1212-9). Los productos de las reacciones enzimáticas son marcados con cromóforos y son detectados y cuantificados con la ayuda de un espectrofotómetro o colorímetro o un luminómetro. En el ejemplo 1, el polyP ha sido cuantificado por el uso de la forma recombinante y purificada de la exopolifosfatasa inorgánica de levadura (Ruiz, F.A., Rodrigues, C.O. et al, J Biol Chem, 2001, 276, 26114-21). Así mismo, el fosfato de las muestras, así como el producido por la reacción enzimática de la exopolifosfatasa ha sido evaluado colorimétricamente por el uso de azul de molibdeno y verde de malaquita (Lanzetta, P.A., Alvarez, L.J. y col., Anal Biochem, 1979, 100, 95-7). Several methods can be used for the detection of polyP, which include, but are not limited to, metachromatic reactions with basic dyes such as toluidine blue (Lorenz, B. and Schroder, HC, Prog Mol Subcell Biol, 1999, 23, 217- 39), the detection of fluorescence changes of fluorophores such as Fura-2 or DAPI in the presence of polyP (Lorenz, B., Munkner, J. et al, Anal Biochem, 1997, 246, 176-84; Aschar-Sobbi, R., Abramov, AY et al, J Fluoresc, 2008, 18, 859-66), or the colorimetric determination of the resulting products after incubation of the extracts with specific enzymes that process the polyP. Examples of the enzymes used in this last type of detection are 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 (Robinson, 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), or glycokinase polyphosphate (Clark, JE, Beegen, H. et al, J Bacteriol, 1986, 168, 1212-9). The products of the reactions Enzymatic are labeled with chromophores and are detected and quantified with the help of a spectrophotometer or colorimeter or a luminometer. In 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). Likewise, the phosphate of the samples, as well as that produced by the enzymatic reaction of the exopolyphosphatase, 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).

En el ejemplo 2, realizamos medidas en el Plasma completo y en la fracción de crioprecipitado de individuos sanos (Figura 3). Los niveles de polyP total (panel A) y de fosfato (panel C) fueron determinados como se explicó en el Ejemplo 1. También se incluye la medida del polyP unido a proteínas (panel B) que se obtiene mediante la precipitación de las muestras con un ácido fuerte (en este caso, ácido perclórico) y el posterior análisis de las proteínas precipitadas. Después de la precipitación, las proteínas son resuspendidas y neutralizadas, para seguidamente determinar los polyP unidos a éstas según el procedimiento descrito en el Ejemplo 1, que se basa en la degradación específica con la enzima exopolifosfatasa. En estas mediciones comprobamos que la fracción de crioprecipitado tiene los mayores niveles de polyP total y de polyP unido a proteínas, así como unas cantidades ínfimas de fosfato en comparación con el plasma (Figura 3). In 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) and the subsequent analysis of the precipitated proteins. After precipitation, 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. In these measurements we verify that 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).

Por último realizamos una prueba de concepto en el Ejemplo 3, utilizando el procedimiento de medida de polyP del plasma en individuos sanos y en pacientes con un trastorno de la coagulación denominado "enfermedad de los reservorios delta" (abreviado como dSPD) (Figura 4). Finally, 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) .

En algunos individuos, sus plaquetas están deficientes lo que resulta en un trastorno de coagulación denominado "enfermedad de los reservorios delta" o "delta-storage pool disease". Nuestro grupo describió anteriormente que los pacientes con dSPD tienen menos polyP en sus plaquetas (Hernandez-Ruiz, L., Saez-Benito, A. y col, J Thromb Haemost, 2009, 7, 361-3; Ruiz, F.A., Hernandez-Ruiz, L. y col, 2009, WIPO Patent Application WO/2009/109676). Como la mayoría del polyP presente en el plasma debe tener su un origen en las plaquetas, se cree que los pacientes con dSPD deben tener también menor contenido de polyP en el plasma. In some individuals, their platelets are deficient which results in a coagulation disorder called "delta reservoir disease" or "delta-storage pool disease". Our group previously described that patients with dSPD have less polyP in their platelets (Hernandez-Ruiz, L., Saez-Benito, A. et al, J Thromb Haemost, 2009, 7, 361-3; Ruiz, FA, Hernandez- Ruiz, L. et al, 2009, WIPO Patent Application WO / 2009/109676). Like most polyP present In plasma should have an origin in platelets, it is believed that patients with dSPD should also have lower content of polyP in the plasma.

Efectivamente, la medición de los niveles de polyP total y del polyP unido a proteínas (Figura 4), nos mostró niveles muy inferiores del polímero en los crioprecipitados de los pacientes con dSPD en comparación con los medidos individuos sanos (Figura 4).  Indeed, the measurement of the levels of total polyP and protein-bound polyP (Figure 4), showed us much lower levels of the polymer in the cryoprecipitates of patients with dSPD compared to the measured healthy individuals (Figure 4).

Ejemplo 1: Example 1:

Este ejemplo describe la metodología utilizada para medir los niveles de polyP total y de fosfato libre de las fracciones de crioprecipitado del plasma y del plasma libre de crioprecipitado (Figura 2).  This example describes the methodology used to measure the levels of total polyP and free phosphate of the cryoprecipitate plasma and cryoprecipitate free plasma fractions (Figure 2).

Los experimentos fueron realizados en muestras de sangre de 10 individuos voluntarios. Los donantes confirmaron, que según su conocimiento, no tenían trastornos en la coagulación. Así mismo, los individuos del estudio confirmaron que no habían tomado alguna medicación que afectase a la coagulación de la sangre en la semana antes de la donación.  The experiments were performed on blood samples from 10 volunteer individuals. Donors confirmed that, according to their knowledge, they had no bleeding disorders. Likewise, the study individuals confirmed that they had not taken any medication that affected blood clotting in the week before the donation.

Se extraen 4.5 mi de sangre en tubos con citrato de sodio al 3,2%. Las muestras se centrifugaron a 1800 x g por 20 min a 4°C, para la obtención del plasma en el sobrenadante. Se separa el plasma en alícuotas de 1 mi y se congela a -80°C por un período mínimo de 24 horas. Se realiza la descongelación a 4°C, que tarda aproximadamente 30-45 min. A continuación, se centrifugan los plasmas descongelados a 1800 x g por 10 min a 4°C para separar dos fracciones: el Crioprecipitado (CRIO) en el pellet y el Plasma Libre de Crioprecipitado (PLC) en el sobrenadante. El Crioprecipitado se resuspende en 300 μΐ de solución salina (150mM NaCl, pH 7).  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 μΐ of saline solution (150mM NaCl, pH 7).

El polifosfato Total se aisla, del Crioprecipitado y del PLC, según el procedimiento descrito por Kumble y Kornberg (Kumble, K.D. y Kornberg, A., JBiol Chem, 1995, 270, 5818-22) con modificaciones. Brevemente, a la muestra se le añade las enzimas ADNasa y RNAasa (a una concentración final de 40 μg/ml de cada una) y MgC12 (para obtener una concentración final de 5mM MgC12). Luego la muestra se incuba a 37°C por 30 minutos. A continuación añade la enzima Proteinasa K (350 μg/ml y se incuba a 37°C por 1 hora. Seguidamente, la muestra se extrae con una solución de fenol/cloroformo (1 :1 equilibrada con Tris-HCL, pH 7,5), en el que las fases se obtienen por centrifugación a 14000 x g por 5 min. La fase acuosa se extrae dos veces con 50 mM Tris-HCl, pH 7,5, 10 mM EDTA y luego tres veces vez con Cloroformo saturado en agua (las fases se obtienen por centrifugación a 14000 x g por 5 min). Se precipita los polyP de la muestra añadiendo 2,5 volúmenes más de etanol muy frió y 10% del volumen de NaCl 5M. Se centrifuga a 14000 x g por 15 min, se extrae el sobrenadante. El pellet resultante se resuspende con 20 μΐ de agua y se utiliza para la determinación de polyP. 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 μg / 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 μg / ml and incubate at 37 ° C for 1 hour. Then, 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 μΐ of water and used for the determination of polyP.

Los niveles de polyP fueron determinados en los extractos neutralizados por la medición del fosfato inorgánico (Pi) resultante después del tratamiento con un exceso de la enzima purificada exopolifosfatasa recombinante de levaduras (Saccharomyces cerevisiae) (Enzima ScrPPXl). La Enzima ScrPPXl fue obtenida por la expresión de un plásmido (cesión de la Universidad de Stanford) con el gen (rPPXl) con colas de histidina de la exopolifosfatasa recombinante de S. cerevisiae (Wurst, H., Shiba, T. y col, J Bacteriol, 1995, 177, 898-906), en la cepa de bacteria E. coli strain CA38 pTrcPPXl (Crooke, E., Akiyama, M. y col., J Biol Chem, 1994, 269, 6290-5). El aislamiento de la Enzima ScrPPXl fue realizado como se describe en Ruiz y col. (Ruiz, F.A., Lea, C.R. et al, JBiol Chem, 2004, 279, 44250-7). La determinación de polyP de los extractos neutralizados, se realizó como se describe a continuación: En un volumen final de 75 μΐ ajustado a concentraciones finales de 60 mM Tris-HCl, pH 7.5, y 15 mM MgC12, se mezclan alícuotas de los extractos junto con 0.5 a 2 μg de Enzima ScrPPXl purificada. Las incubaciones se realizan por al menos 20 min a 37 °C y la producción de Pi resultante fue monitorizada calorimétricamente por el método de detección de fosfato descrito por Lanzetta y col., que utiliza molibdato de amonio y verde de malaquita (Lanzetta, P.A., Alvarez, L.J. et al, Anal Biochem, 1979, 100, 95-7). El fosfato se aisla, del Crioprecipitado y del PLC, del sobrenadante resultante tras la precipitación con un ácido perclórico. Brevemente, a las muestras se les añade 1/5 de su volumen de ácido perclórico (HC104) 3 M. Después de una incubación en hielo de 30 min, los extractos acídicos fueron centrifugados a 14,000 x g por 5 min. Los sobrenadantes resultantes se neutralizaron por la adición de una solución de 1M KOH, 100 mM TrisHCl, lo que produce la formación de KC104 y la precipitación del mismo. El KC104 precipitado fue separado por centrifugación a 14,000 x g por 5 min, y el sobrenadante resultante fue utilizado para la determinación de fosfato. 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. coli strain CA38 pTrcPPXl (Crooke, E., Akiyama, M. et al., J Biol Chem, 1994, 269, 6290-5). Isolation of the ScrPPXl Enzyme was performed as described in Ruiz et al. (Ruiz, FA, Lea, CR et al, JBiol Chem, 2004, 279, 44250-7). The polyP determination of the neutralized extracts was performed as described below: In a final volume of 75 μΐ 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 μg 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.

El fosfato de la muestra fue monitorizado colorimétricamente por el método de detección de fosfato descrito por Lanzetta y col., que utiliza molibdato de amonio y verde de malaquita (Lanzetta, P.A., Alvarez, L.J. et al, Anal Biochem, 1979, 100, 95-7). 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).

Ejemplo 2: Example 2:

Este ejemplo describe la metodología utilizada para medir los niveles de polyP total, de polyP unido a proteínas y de fosfato libre de las fracciones de plasma congelado y en crioprecipitado del plasma (Figura 3).  This example describes the methodology used to measure the levels of total polyP, protein-bound polyP and free phosphate of the frozen plasma and cryoprecipitate plasma fractions (Figure 3).

Los experimentos fueron realizados en muestras de sangre de 10 individuos voluntarios obtenidos como se explica en el Ejemplo 1. La obtención del Plasma y del Crioprecipitado del Plasma (CRIO), se realiza según procedimientos detallados en el Ejemplo 1. La extracción y detección de los polyP (Figura 3, panel A) y del fosfato (Figura 3, panel C), se realizaron según los procedimientos indicados en el Ejemplo 1. The experiments were performed on blood samples from 10 voluntary individuals obtained as explained in 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.

Para la extracción del polyP unido a proteínas se midió el polyP resultante en el precipitado tras la precipitación con un ácido perclórico. A las muestras, de Plasma o de Crioprecipitado, se les añade 1/5 de su volumen de ácido perclórico (HC104) 3 M. Después de una incubación en hielo de 30 min, los extractos acídicos fueron centrifugados a 14,000 x g por 5 min a 4°C. Los pellets resultantes se resuspenden en 100 μΐ de solución salina (150mM NaCl, pH 7). De la muestra resuspendida se separan alícuotas de 5 μΐ para la determinación de cantidad de proteínas por el método de Bradford, que utiliza el colorante "Brilliant Blue G" (Bradford, M.M., Anal Biochem, 1976, 72, 248-54). El resto de la muestra respondida, se neutraliza con una solución de 1M KOH, 100 mM TrisHCl (hasta lograr un pH entre 6 y 8). Luego se centrifugan a 13000 x g a 4°C durante 5 minutos. En el sobrenadante se determina la concentración de polyP, utilizando la enzima ScrPPXlcomo se explica en el Ejemplo 1. For the extraction of the protein-bound polyP, the resulting polyP in the precipitate was measured after precipitation with a perchloric acid. To the samples, of Plasma or Cryoprecipitate, 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 μΐ of saline solution (150mM NaCl, pH 7). Of the resuspended sample, Aliquots of 5 μΐ are separated for the determination of protein quantity by the Bradford method, which uses the "Brilliant Blue G" dye (Bradford, MM, Anal Biochem, 1976, 72, 248-54). The rest of the sample answered is neutralized with a solution of 1M KOH, 100mM TrisHCl (until a pH between 6 and 8 is achieved). They are then centrifuged at 13000 xga at 4 ° C for 5 minutes. The concentration of polyP is determined in the supernatant, using the enzyme ScrPPXl as explained in Example 1.

Ejemplo 3: Example 3:

Este ejemplo describe la metodología utilizada para medir los niveles de polyP del Crioprecipitado de plasma en controles sanos y en pacientes con el trastorno de coagulación "enfermedad de los reservorios delta" o dSPD (Figura 4). This example describes the methodology used to measure polyP levels of plasma cryoprecipitate in healthy controls and in patients with coagulation disorder "delta reservoir disease" or dSPD (Figure 4).

Los experimentos fueron realizados utilizando plasma de 10 individuos voluntarios sanos y de cinco pacientes con dSPD. Los donantes del grupo control confirmaron, que según su conocimiento, no tenían trastornos en la coagulación. Así mismo, todos los individuos del estudio confirmaron que no habían tomado alguna medicación que afectase a las plaquetas en la semana antes de la donación de sangre. El diagnóstico de dSPD en los cinco pacientes utilizados se confirmó con anterioridad mediante el análisis de la incorporación de mepacrina por citometría de flujo y por el estudio de la secreción de ATD utilizando luminescencia (Hernandez-Ruiz, L., Saez-Benito, A. et al, J Thromb Haemost, 2009, 7, 361-3). The experiments were performed using plasma of 10 healthy volunteer individuals and five patients with dSPD. Donors in the control group confirmed that, according to their knowledge, they had no coagulation disorders. Likewise, all the individuals in the study confirmed that they had not taken any medication that affected platelets in the week before the blood donation. The diagnosis of dSPD in the five patients used was previously confirmed by analyzing the incorporation of mepacrine by flow cytometry and by studying the secretion of ATD using luminescence (Hernandez-Ruiz, L., Saez-Benito, A. et al, J Thromb Haemost, 2009, 7, 361-3).

El plasma se obtuvo a partir demuestras de sangre según se explica en el Ejemplo 1. La preparación de crioprecipitado así como la extracción y detección de los polyP totales en las muestras (Figuras 4, panel A), se realizó como se explica en el Ejemplo 1. La extracción y determinación del polyP unido a proteínas (Figura 4, panel B) se realizó de manera similar como se detalla en el Ejemplo 2. APLICABILIDAD INDUSTRIAL 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 (Figures 4, panel A), was performed as explained in Example 1. The extraction and determination of protein-bound polyP (Figure 4, panel B) was performed in a similar manner as detailed in Example 2. INDUSTRIAL APPLICABILITY

Como los polyP plasmáticos son potentes moduladores de la coagulación y/o trombosis, el procedimiento objeto de la presente invención es óptimo para la estimación precisa y rápida de estos procesos. Por tanto, puede ser muy efectivo como alternativa para evaluar pacientes con tratamientos anticoagulantes (como en la administración de heparinas y/o cumarínicos), o como ayuda para diagnosticar defectos en la coagulación congénitos (como la dSPD ó la hemofilia) o adquiridos (como sucede con los pacientes de enfermedades hepáticas y renales, leucemias mieloides, mielodisplasias o síndromes proliferativos).  Since 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).

Por otra parte, como los polyP plasmáticos tienen también un papel determinante los procesos inflamatorios, mediante la modulación de la producción de bradiquinina, el procedimiento objeto de la presente invención puede ser efectivo para estimar la gravedad de diferentes patologías con componente inflamatorio (como por ejemplo diabetes, infecciones, alergias o enfermedades autoinmunes, entre otras).  On the other hand, since plasma polyP also plays a role in inflammatory processes, by modulating the production of bradykinin, 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).

El procedimiento de la invención sirve de base para el desarrollo de kits para la evaluación y ensayo de los niveles de polyP en plasma de sangre humana y/o de animales. Por ejemplo, los kits podrían incluir enzimas y/o los ensayos colorimétricos que permitan la cuantificación del polyP en el plasma. Se espera que los kits apropiados incluyan, pero no estén limitados a, medios para preparar el crioprecipitado de plasma y para separar la fracción proteica, medios para detectar y/o cuantificar polyP, y también muestras control de polyP para comparar con los valores obtenidos.  The process of the invention serves as the basis for the development of kits for the evaluation and testing of polyP levels in human and / or animal blood plasma. For example, 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.

Claims

REIVINDICACIONES 1. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo que incluye las siguientes etapas: 1. - Procedure for the determination of polyphosphate in blood plasma that includes the following stages: a) obtener una muestra biológica del individuo a determinar. a) obtain a biological sample of the individual to be determined. b) separar una fracción de la muestra, que contiene la mayoría del polifosfato. c) determinar en dicha muestra el contenido de polifosfatos. b) separate a fraction of the sample, which contains most of the polyphosphate. c) determine in said sample the content of polyphosphates. 2. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según la reivindicación 1, caracterizado porque el individuo del que se obtiene la muestra biológica es un mamífero. 2. - Procedure for the determination of polyphosphate in blood plasma according to claim 1, characterized in that the individual from whom the biological sample is obtained is a mammal. 3. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según la reivindicación 2, caracterizado porque el mamífero del que se obtiene la muestra biológica es un humano. 3. - Procedure for the determination of polyphosphate in blood plasma according to claim 2, characterized in that the mammal from which the biological sample is obtained is a human. 4. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 1-3, caracterizado porque la muestra biológica incluye sangre. 4. - Procedure for the determination of polyphosphate in blood plasma according to claims 1-3, characterized in that the biological sample includes blood. 5. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 1-4, caracterizado porque la muestra biológica incluye plasma. 5. - Procedure for the determination of polyphosphate in blood plasma according to claims 1-4, characterized in that the biological sample includes plasma. 6. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según la reivindicación 5, caracterizado porque cuando la muestra incluye plasma, el procedimiento comprende las siguientes etapas: a) obtención de una muestra biológica del individuo a diagnosticar 6. - Method for the determination of polyphosphate in blood plasma according to claim 5, characterized in that when the sample includes plasma, the method comprises the following steps: a) obtaining a biological sample of the individual to be diagnosed b) aislamiento del plasma de los otros componentes de la muestra biológica c) separación de la fracción de proteínas de alto peso molecular. d) separación del polifosfato inorgánico del resto de componentes de la fracción. e) determinación cuantitativa del polifosfato solubilizado y separado en las etapas anteriores. b) plasma isolation from the other components of the biological sample c) separation of the high molecular weight protein fraction. d) separation of inorganic polyphosphate from the rest of the components of the fraction. e) quantitative determination of solubilized and separated polyphosphate in the previous stages. 7. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según la reivindicación 6, caracterizado porque la etapa de aislamiento del plasma se lleva a cabo mediante un proceso de centrifugación simple. 7. - Procedure for the determination of polyphosphate in blood plasma according to claim 6, characterized in that the plasma isolation step is carried out by a simple centrifugation process. 8. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 6 y 7, caracterizado porque la separación de las proteínas de alto peso molecular se realiza mediante la congelación y descongelación lenta del plasma y posterior centrifugación simple (crioprecipitado). 8. - Procedure for the determination of polyphosphate in blood plasma according to claims 6 and 7, characterized in that the separation of high molecular weight proteins is carried out by freezing and slow thawing of the plasma and subsequent simple centrifugation (cryoprecipitate). 9. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 6-8, caracterizado porque la separación del polifosfato se realiza por extracción con soluciones de fenol/cloroformo, o bien mediante la precipitación de las proteínas en un ácido fuerte como ácido perclórico o ácido tricloroacético. 9. - Procedure for the determination of polyphosphate in the blood plasma according to claims 6-8, characterized in that the separation of the polyphosphate is carried out by extraction with phenol / chloroform solutions, or by precipitation of the proteins in a strong acid such as perchloric acid or trichloroacetic acid. 10. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 6-9, caracterizado porque la separación del polifosfato solubilizado se lleva a cabo mediante centrifugación. 10. - Procedure for the determination of polyphosphate in the blood plasma according to claims 6-9, characterized in that the separation of the solubilized polyphosphate is carried out by centrifugation. 11. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 6-10, caracterizado porque la determinación cuantitativa del polifosfato solubilizado y separado en las etapas anteriores se efectúa, tras neutralización con solución alcalina, directamente mediante colorimetría o fluorimetría, preferentemente colorimetría. 11. - Procedure for the determination of polyphosphate in blood plasma according to claims 6-10, characterized in that the quantitative determination of the polyphosphate solubilized and separated in the previous stages is carried out, after neutralization with alkaline solution, directly by colorimetry or fluorimetry, preferably colorimetry 12. - Procedimiento para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 6-10, caracterizado porque la determinación cuantitativa del polifosfato y solubilizado y separado en las etapas anteriores incluye tras la neutralización con solución alcalina a) un ataque enzimático para procesar el polifosfato, para liberar los productos resultantes de dicha reacción enzimática. b) determinación cuantitativa de alguno de los productos resultantes de la reacción enzimática mediante colorimetría o por luminiscencia, preferentemente mediante colorimetría. 12. - Procedure for the determination of the polyphosphate in the blood plasma according to claims 6-10, characterized in that the quantitative determination of the polyphosphate and solubilized and separated in the previous stages includes after neutralization with alkaline solution a) an enzymatic attack to process the polyphosphate, to release the products resulting from said enzymatic reaction. b) quantitative determination of any of the products resulting from the enzymatic reaction by colorimetry or by luminescence, preferably by colorimetry. 13. - Kit para la determinación del polifosfato en el plasma sanguíneo mediante un procedimiento según las reivindicaciones 1-12, caracterizado porque incluye: 13. - Kit for the determination of polyphosphate in blood plasma by a method according to claims 1-12, characterized in that it includes: - medios para aislar la fracción de crioprecipitado del plasma. - means for isolating the cryoprecipitate fraction from plasma. - medios para la separación, detección y cuantificación del polifosfato.  - means for the separation, detection and quantification of polyphosphate. - muestras de control de polifosfato y/o plasma para comparar con los valores obtenidos.  - polyphosphate and / or plasma control samples to compare with the values obtained. 14. - Kit para la determinación del polifosfato en el plasma sanguíneo según la reivindicación 13, caracterizado porque incluye adicionalmente enzimas para el procesamiento específico de polifosfato en productos detectables mediante colorimetría o por luminiscencia. 14. - Kit for the determination of polyphosphate in blood plasma according to claim 13, characterized in that it further includes enzymes for the specific processing of polyphosphate in products detectable by colorimetry or by luminescence. 15. - Kit para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 13 y 14, caracterizado porque los medios para producir extractos acídicos de las proteínas del crioprecipitado son ácidos que se seleccionan entre ácido perclórico o ácido tricloroacético. 15. - Kit for the determination of polyphosphate in blood plasma according to claims 13 and 14, characterized in that the means for producing acidic extracts of cryoprecipitate proteins are acids that are selected from perchloric acid or trichloroacetic acid. 16. - Kit para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 13 - 15, caracterizado porque los medios para la detección y cuantificación del polifosfato son reactivos para ensayos colorímétricos, de fluorescencia o de luminiscencia, preferentemente reactivos colorímétricos que se seleccionan entre colorantes básicos como el azul de toulidina o fluoróforos como Fura-2 o DAPI. 16. - Kit for the determination of polyphosphate in blood plasma according to claims 13-15, characterized in that the means for the detection and quantification of polyphosphate are reagents for colorimetric, fluorescence or luminescence assays, preferably colorimetric reagents selected from basic dyes such as toulidine blue or fluorophores such as Fura-2 or DAPI. 17. - Kit para la determinación del polifosfato en el plasma sanguíneo según las reivindicaciones 14 - 16, caracterizado porque las enzimas se seleccionan entre las exopolifosafatasas, la polifosfato kinasas, o la polifosfato glucokinasas. 17. - Kit for the determination of polyphosphate in blood plasma according to claims 14-16, characterized in that the enzymes are selected from exopolyphosphatases, polyphosphate kinases, or polyphosphate glucokinases.
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CN104655857A (en) * 2015-02-10 2015-05-27 南京大学 Method for quantitatively detecting polyphosphates in microbial cells
WO2018191582A1 (en) * 2017-04-13 2018-10-18 The Board Of Trustees Of The University Of Illinois Assay for quantifying polyphosphates
US11519917B2 (en) 2017-04-13 2022-12-06 The Regents Of The University Of Michigan Assay for quantifying polyphosphates

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