Method for determining the propensity for calcification

Abstract

The present invention relates to a method for determining the propensity of a fluid for calcification characterized by the following steps: (i) adding a soluble calcium salt and a soluble phosphate salt to a sample of the fluid; (ii) incubating the sample at conditions allowing the formation of calciprotein particles (CPPs); and (iii) determining one or more of the following: (a) the rate of the formation of primary and/or secondary CPPs; (b) the amount of primary and/or secondary CPPs; and/or (c) the rate of the transition of primary CPPs into secondary CPPs, wherein an increase in one or more of (a), (b) and/or (c) of step (iii) indicates an increased propensity of the fluid for calcification.

Claims

1. A method for determining the propensity of a body fluid obtained from an individual for calcification comprising: (i) adding a soluble calcium salt and a soluble phosphate salt to a sample of said body fluid; (ii) incubating said sample at conditions allowing the formation of calciprotein particles (CPPs); and (iii) determining one or more of the following: (a) a rate of the formation of primary and/or secondary CPPs; (b) an amount of primary and/or secondary CPPs; and/or (c) a rate of transition of primary CPPs into secondary CPPs, wherein an increase over what is determinable for a fluid with known calcification in one or more of (a), (b) and/or (c) of step (iii) indicates an increased propensity of said body fluid for calcification.

2. The method of claim 1, wherein step (iii) is performed by an optical method.

3. The method of claim 2, wherein excitation light used in the optical method is a laser beam.

4. The method of claim 2, wherein the optical method is performed by detecting light scattering.

5. The method of claim 4, wherein the method of detecting light scattering is selected from the group consisting of: dynamic light scattering, cross-correlation dynamic light scattering, three-dimensional cross-correlation dynamic light scattering, or nephelometry.

6. The method of claim 2, wherein the optical method is a method selected from the group consisting of: absorptiometry, detection of light scattering, correlation spectroscopy, or a combination of two or more thereof.

7. The method of claim 1, wherein step (iii) is performed by any method selected from the group consisting of: sedimentation techniques, filtration analysis, size exclusion chromatography, granulometry, acoustic spectroscopy, or a combination of two or more thereof.

8. The method of claim 1, wherein the fluid is a body fluid obtained from a patient that has developed calcification and/or is at risk of developing calcification.

9. The method of claim 8, wherein the patient suffers from vascular, valvular and/or soft tissue calcification.

10. The method of claim 9, wherein said patient further suffers from a rheumatoid disease, a malignant disease and/or an infectious disease, or wherein the patient shows at least one of the syndromes selected from the group consisting of: renal dysfunction, hypertension, diabetes mellitus, dyslipidemia, a lack of adequate mineralization, and atherosclerosis.

11. The method of claim 10, wherein the lack of adequate mineralization is due to osteoporosis, osteomalacia, or a combination thereof.

12. The method of claim 8, wherein said patient is a dialysis patient.

13. The method of claim 1, wherein said method is performed at a constant temperature and/or at a constant pH.

14. The method of claim 1, wherein said method is performed in one of the following: (a) a multiwell format; (b) a flow-through cell; or (c) a microfluidic device.

15. The method of claim 1, wherein at least step (iii) is automated, or wherein at least steps (ii) and (iii) are automated, or wherein all of the steps (i), (ii) and (iii) are automated.

16. The method of claim 1, wherein the primary CPPs have an average diameter smaller than 100 nm and the secondary CPPs have an average diameter of larger than 100 nm.

17. The method of claim 1, wherein one or more of (a), (b) and/or (c) of step (iii) is/are compared with one or more control sample(s).

18. The method of claim 1, wherein (c) of step (iii) is determined by determining a time point of half maximal transition time (T.sub.50) of the transition of primary CPPs into secondary CPPs.

19. The method of claim 1, wherein the body fluid is blood, blood plasma, blood serum, lymph, or urine.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Detection of CPP transition. (A) 3D-DLS detection of CPP transition in the presence of fetuin-A and human serum. (B) Nephelometry detection of CPP transition in the presence of human serum. (C) Gross visual appearance of the standard nephelometer assay serum solutions containing CPPs in solution (above) and after sharp centrifugation of the solutions (below). Experiment was performed at 3rC in standard photometry vials and with the same solutions used in the respective proportions as in the final nephelometer assay. (D) Pellets after centrifugation of the solutions.

(2) FIG. 2. Determination of nephelometer assay conditions. (A) Impact of varying calcium and phosphate concentrations. Calcium 10 mM and phosphate 6 mM were finally chosen as standard concentrations for the assay. (B) Temperature-dependence of pH. Calcium or phosphate solutions buffered by Hepes or Tris were heated from room temperature to 40 C. and the pH values recorded. The less temperature sensitive Hepes buffer was chosen for the assay. (C) Impact of pH. Calcium and phosphate solutions adjusted to pH values from 7.1 to 7.6 were tested. A pH of 7.40 at 37 C. was chosen for the assay. (D) Impact of amount of serum. Serum amounts from 60 l to 100 l were tested (with NaCI 140 mM replenishing the missing volume to an amount 200 A in all vials), and 80 A serum finally chosen for the assay.

(3) FIG. 3. Nephelometer assay conditions. (A) Assay results when performed with pooled serum from healthy volunteers with the Nephelostar instrument run at room temperature and the internal radiator set at 37 C. (B) Assay results when performed with pooled serum from healthy volunteers with the Nephelostar radiator turned off and the assay run in a temperature controlled room set at 34.5 C. The resulting measurement temperature within the Nephelostar plate holder bay under these conditions was 36.5 to 37 C.

(4) FIG. 4. Nephelometer assay in calcifying animal models. (A) Illustrating representative x-rays of 10 to 16 week old dba2 fetuin-A knock-out (/) and wildtype (+/+) mice showing excessive pathological calcifications of fetuin-A knock-out mice. Heterozygous mice have the same phenotype as wildtype mice. (B) Discrimination of wild type (wt), heterozygous (het) and knock out (ko) mouse sera. (C) Results of nephelometer assay performed with serum from fetuin-A knock-out, heterozygous and wildtype mice. (D) Illustrating representative histology of aortas from 16 week old adenine-treated uremic rats with calcifications of the vessel media (Alizarin stain for calcium), and healthy rats without calcifications. (E) Comparison between samples from uremic and non-uremic animals. (F) Nephelometry assay with sera from 20 hemodialysis patients (black) and 20 healthy volunteers (grey).

(5) FIG. 5. Particle characterization, microscopic appearance and molecular composition of the CPPs. (A) Scanning electron microscopy (SEM) (left) and transmission electron microscopy (TEM) (right) of primary CPPs. (B) SEM (left) and TEM (right) of secondary CPPs. (C) Coomassie blue stain of protein contents of primary and secondary CPPs (left) and albumin and fetuin-A western blots of primary and secondary CPPs (right). (D) Disappearance of phosphate from the solution upon formation of CPPs.

(6) FIG. 6. Assay dependence of spiked serum components. (A) Nephelometry in the absence of serum: only spiking of fetuin A, the strongest intrinsic serum calcification inhibitor significantly influences the assay. (B) Nephelometry in the presence of serum: all spiked substances influence the assay. (C) Alternative presentation of the data shown in B. Substance concentrations used in (a) and (B) were the same as given in the legend of (C).

(7) FIG. 7. Correlation of one-halfmaximal relative nephelometric units (RNU.sub.50) and T.sub.50 with fetuin-A serum concentrations. Fetuin-A concentrations were measured in sera obtained from 20 hemodialysis patients and plotted against the RNU.sub.50 and T.sub.50 values obtained from the assay of the present invention. Fetuin-A concentrations highly correlated with (A) RNU.sub.50 (p=0.0006) and (B) T.sub.50 (p=0.0413). Patient sera were the same as used for the experiment shown in FIG. 4F. Fetuin-A serum concentrations were measured by ELISA as described by Ketteler M, et al. (Kettler et al., 2003).

EXAMPLES

Methods

(8) Sampling and Preparation of Serum Specimens

(9) Venous blood from eight healthy volunteers was taken in Sarstedt Monovette vials. After clotting for 30 min, the samples were centrifuged at 3,000g for 10 minutes at room temperature. Serum from all individuals was pooled and aliquoted. Blood from 10 to 16 week old DBA/2 fetuin-A knock-out, heterozygous and wildtype mice (Sch fer et al., 2003; Jahnen-Dechent et al., 1997), was sampled from the heart at the time of sacrifice.

(10) Blood from male Wistar rats (Charles River, Sulzfeld, Germany), which had received food supplemented with 0.75% adenine and calcium 1.05%, phosphorus 0.8%, protein 18.5% for four weeks to induce uremia and vascular calcifications, was taken at sacrifice at age 16 weeks from the inferior vena cava (Pasch et al., 2008). Likewise, control blood was taken from healthy, nonuremic, non-calcified rats of the same age and gender, which had been treated with sodium thiosulfate (0.4 g/kg body weight) in normal (0.9%) saline i.p. three times a week for 6 weeks. Of note, sodium thiosulfate (Na.sub.2S.sub.2O.sub.3) did not have any impact on the assay when spiked to serum in amounts of up to 40 mM.

(11) After clotting at room temperature, blood samples from humans, mice or rats were spun at 3,000g for 10 minutes at room temperature to separate serum from blood cells. The serum was shock frozen in liquid nitrogen and stored at 80 C. Before use in the nephelometer assay, samples were thawed, and centrifuged at 10,000g for 30 min at room temperature to remove potential small particles which might have formed during the freezing and thawing of samples (cryoprecipitates) and which might interfere with the assay by providing precipitation-accelerating niduses.

(12) Devices, Plastic Materials and Chemicals

(13) The Nephelostar nephelometer was purchased from bmg labtech, Offenburg, Germany, the Liquidator96 bench-top pipetting system was purchased from Mettler Toledo GmbH, Giessen, Germany. 96-well plates were from Brand GmbH, Wertheim, Germany, and 96-well plastic Covers from Carl Roth GmbH, Karlsruhe, Germany. All chemicals (e.g., NaCI, Hepes, CaCl.sub.2, NaH.sub.2PO.sub.4, Na.sub.2HPO.sub.4 and NaOH) were purchased from AppliChem, Darmstadt, Germany, in pro analysis grade quality.

(14) Protein Quantification

(15) For quantification of proteins in solutions, the Pierce BCA Protein Assay Kit was used according to the manufacturer's instructions. BSA (2 mg/ml, Pierce) was used as a standard. Western blots were performed according to standard protocols with SDS-PAGE (4%-12%), with 1 mg protein or 0.4 mg pure fetuin-A or albumin loaded per lane. The following primary antibodies against fetuin-A and albumin were used: polyclonal rabbit anti-human fetuin-A antiserum 5359 (Behring AG, Marburg, Germany) and mouse anti-human albumin (1:2500, catalog number 0300-0080; AbD Serotec). For fluorescence detection, the following horseradish peroxidase-coupled secondary antibodies were used: swine anti-rabbit IgG (1:5000, catalog number P0217; Dako) and rabbit anti-mouse IgG (1:2000, catalog number P0260; Dako). Protein stains were performed with the Imperial Protein Stain according to the manufacturer's instructions (Thermo Scientific); 6.0 mg total protein or 2.5 mg pure fetuin-A or albumin was loaded per lane.

(16) Three-Dimensional Cross-Correlation Dynamic Light Scattering (3D-DLS)

(17) Multiple scattering in Solutions with high particle density prevents the characterization by Standard dynamic lightscattering methods. Therefore we used a 3D cross-correlation dynamic light scattering (3D-DLS) setup for the analysis of turbid CPP samples. 50-52 Measurements were performed using a Standard light scattering device (ALV GmbH, Langen, Germany) with HeNe-laser (JDS Uniphase, Koheras GmbH, 632.8 nm, 25 mW, Type LGTC 685-35), two avalanche photodiodes (Perkin Eimer, Type SPCM-AQR-13-FC) and an ALV 5000 correlator. The scattered light was detected at 90 geometry. The sample temperature was adjusted by an external thermostat equipped with a Pt-100 temperature sensor. The hydrodynamic radius Rh was calculated from second-order cumulant fits via the Stokes-Einstein equation. Measurements covered a time span of 1400 minutes in 2 minute intervals. Previous TEM investigations revealed that aged, secondary CPPs have an ellipsoidal shape with an axes ratio of approximately b/a z 0.3. For the sake of clarity, we calculated the hydrodynamic radii, not the semi-axes, to characterize the individual CPP stages.

(18) Nephelometer Assay

(19) Three-dimensional cross-correlation dynamic light scattering (3D-DLS) is a method, which detects laser scatter in solutions and integrates these data to yield Information about the development of particle size over time.

(20) Stock Solutions: 1. NaCl-solution: NaCl 140 mM, 2. Calcium solution: CaCl.sub.2 40 mM+Hepes 100 mM+NaCI 140 mM, pH adjusted with NaOH 10 mM to 7.40 at 37 C., 3. Phosphate solution: Na.sub.2HP0.sub.4 19.44 mM+NaH.sub.2PO.sub.4 4.56 mM+Hepes 100 mM+NaCI 140 mM, pH adjusted with NaOH 10 mM to 7.40 at 37 C. Preparation of 96-well plates: all Solutions were pre-warmed to 34.5 C. in a thermo constant room where also all pipetting steps were performed with the liquidator96 bench-top pipetting System using a set of new pipetting tips for every pipetting step. These pipetting steps were performed in the following order: 1. NaCl-solution: 20 l/well, 2. serum 80 l/well, 3. shaking for 1 minute, 4. phosphate solution 50 l/well, 5. shaking for 1 minute, 6. calcium solution 50 l/well, shaking for 1 minute. Air bubbles in the wells were disintegrated with a pocket lighter and the 96-well covered with a ThinSeal adhesive sealing film for microplates. As line, A of the 96-well plate often showed unreliable results, it was generally left out. Assay conditions and Nephelostar settings: measurement in a thermo constant room at 34.5 C. with the internal radiation of the device turned off. This led to an infernal measurement temperature of 36.5 C. to 37 C. The Nephelostar was operated and controlled via the Nephelostar provider's Galaxy Software on a Windows Computer platform. The assay was performed with 200 cycles of 1.5 seconds measurement time per well and a position delay of 0.1 seconds in horizontal plate reading mode, adding up to a cycle time of 180 seconds/cycle for our Standard assay. This adds up to a total assay run time of 10 hours per assay. For some measurements, the cycle time was extended to 360 or 540 seconds, which adds up to assay times of 20 and 30 hours, respectively. The gain and laser adjustment was set at 90% required value, gain 50 with a laser beam focus of 1.5 mm and a laser intensity of 50%.

(21) Data Processing

(22) After completion of the run, data were transferred to Excel and transposed from lines into columns. Data columns were copied into the GraphPad Prism program to generate an XY-graph. Data were then processed by calculating nonlinear regression in the log(agonist) vs. responsevariable slope (four Parameters) mode using the robust fit fitting method. The resulting values obtained for T.sub.50 and RNU.sub.T50 were further processed as required.

(23) Results

(24) Here, we tested whether primary CPPs would also be generated when human serum instead of fetuin-A solution was used (FIG. 1A, lower part). Indeed, in both cases primary CPPs of comparable size (diameter about 50 nm) were generated, which underwent spontaneous transition to secondary CPPs (diameter about 150 nm), albeit within very different time frames (FIG. 1A). Given these grossly different transition times, we reasoned that the delay of the transition might reflect the stability of primary CPPs and that measuring this step might provide a quantitative estimate for the calcification inhibitory propensity inherent in serum.

(25) As 3D-DLS is not widely available and can measure only one sample per day, we aimed to establish a practical and broadly applicable alternative assay for the detection of the mentioned transition step. Nephelometry is based on the same principles as DLS and quantifies the amount of laser light scatter in turbid solutions. Consequently, the transition was also detectable by nephelometry (FIG. 1B), and it is of note even visible to the naked eye (FIG. 1C).

(26) For the establishment of the nephelometry-based assay, we took advantage of the automated laser-based microplate nephelometer Nephelostar which we run in the 96-well-plate mode. The resulting data were analyzed using Excel and GraphPad Prism Software to yield nonlinear regression curves and the resulting values of half maximal transition time (T.sub.50, FIG. 1A)) and of Relative Nephelometric Units (RNU.sub.T50, FIG. 1B) at this point in time were determined.

(27) We chose physiological conditions for temperature (37 C.) and pH (7.40 at 37 C.), and designed the assay for a final volume of 200 l per well. These 200 l consisted of 20 l NaCl 140 mM, 80 l serum, 50 l phosphate 24 mM and 50 l calcium 40 mM solution which were mixed in this order. The phosphate and calcium Solutions were supplemented with NaCl 140 mM and Hepes 100 mM, the pH was adjusted to 7.40 at 37 C. This mixture resulted in the final concentrations as depicted below:

(28) Ca.sup.2+: 10 mM

(29) PO.sub.4.sup.2: 6 mM

(30) NaCl: 140 mM

(31) Hepes: 50 mM

(32) at a pH 7.40 and 37 C.

(33) The 20 l NaCl were introduced as an extra volume usable for spiking experiments. A wide range of calcium and phosphate concentrations were systematically tested (FIG. 2A) and final concentrations of calcium 10 mM (i.e. 40 mM in stock solution) and phosphate 6 mM (i.e. 24 mM in stock solution) finally chosen in our assay for three reasons: (i) the transition occurred in a central position in the time- and RNU coordinate system leaving space for changes into all directions, (ii) these concentrations represent the numerically optimal relation of calcium and phosphate with regard to the formation of hydroxylapatite, (iii) our previous experiments investigating CPPs have been performed with these concentrations.

(34) Unfortunately, first attempts to standardize the assay showed an enormous variation even within a given 96-well plate (FIG. 3A), indicating that the transition step is an extremely sensitive physicochemical process, which is sensitive towards subtle variations in pH (FIG. 2C) and serum amounts used (FIG. 2D).

(35) Stabilization of the assay (FIG. 3B) was achieved by introducing three important modifications: (i) assay temperature was stabilized by running the assay in a Special constant temperature room with the intrinsic radiator of the Nephelostar turned off, (ii) pipetting volumes were stabilized by using a high precision 96-well pipetting device (the Liquidator96) instead of multi-channel pipettes, and (iii) temperature-sensitivity of the test was diminished by using Hepes instead of the more temperature-sensitive Tris buffer (FIG. 2B). Under these conditions, the assay was stable and yielded highly reproducible results (FIG. 3B) with an intra-day variability of +/5.2% and an inter-day variability of +/11.6% in our hands.

(36) To confirm the correlation between the assay results and calcifications in vivo, we compared serum of fetuin-A knock-out (ko), heterozygous (het) and wildtype (wt) mice (FIGS. 4A, B and C) and found that T.sub.50 was shorter in serum from the knockout mouse (ko), when compared to heterozygous and wildtype mice. The same pattern was found when serum from calcifying uremic rats and from healthy non-calcifying rats was compared (FIGS. 4D and E). Here again the transition (T.sub.50) occurred earlier in the calcifying than in the non-calcifying animals.

(37) We also tested sera from healthy volunteers and hemodialysis patients. Again, the test discriminated the calcification-prone from the noncalcificationprone individuals (i.e., the hemodialysis patients from the healthy volunteers (FIG. 4F), indicating that the test reflects calcification propensity in serum.

(38) These results confirm that the nephelometer assay presented here provides an estimate of intrinsic serum-related calcification propensity.

(39) The test of the present invention increases supersaturation of serum by adding Ca (10 mM) and phosphate (6 mM). The specific effect of supersaturation depends on the intrinsic concentrations of fetuin-A, albumin, phosphate etc. in a given serum. As a general rule, the higher the calcium and phosphate supersaturation, the lower T.sub.50 and the higher RNU.sub.50. This applies to sera from HD patients and healthy volunteers alike. RNU.sub.50 largely depends on the protein content (fetuin-A, albumin) of the CPPs with some contribution of phosphate. T.sub.50 largely depends on Mg and phosphate with some contribution of fetuin-A and albumin. A low T.sub.50 is therefore often associated with a high RNU.sub.50 and vice versa. A universal RNU.sub.50-to-T.sub.50-ratio can however, not be determined as both variables depend on differentalbeit overlappingdeterminants.

(40) In summary, we present a nephelometer-based assay, which measures calcification propensity of a body fluid, exemplarily shown in blood serum. Given the wide area of potential applications of this assay, this method is a useful tool for the investigation and elucidation of biomineralization-related issues in clinical as well as scientific research and in diagnosis in vivo and ex vivo.

REFERENCES

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