Abstract:
A process and a reagent for the specific and direct determination of the LDL-fraction in the presence of other serum lipoproteins by adding a polymeric LDL-aggregating agent, followed by direct turbidimetric measurement of the LDL aggregate. Preferred are polymers which have a comb or brush type structure with the side groups having acid character, such as branched alkane sulfonic acids.

Description:
This is a continuation application of application Ser. No. 08,375,078, filed Jan. 17, 1995, now abandoned, which was a continuation of application Ser. No. 08/092,205, filed Jul. 15, 1993, now abandoned. 
    
    
     FIELD OF THE INVENTION 
     The invention addresses a process for specific and direct determination of LDL in biological fluids. This is accomplished by adding an LDL-aggregating or agglutinating agent, which specifically aggregates LDL, even in the presence of other serum lipoproteins. Preferably, the LDL aggregating or agglutinating agent is a polymer has a comb or brush type structure. 
     BACKGROUND AND PRIOR ART 
     The determination of the LDL level in plasma and, especially, in serum is of great clinical importance: up to 80% of the total cholesterol contents is transported in the form of so-called low-density lipoproteins (LDLs of the β-fraction), and are, hence, part of those particles which today are considered to be paramount atherogenic component in lipid metabolism. Moreover, it is known that elevated plasma LDL concentrations have a direct endothelial damaging effect, which can be compared to a number of other problems caused by hypertension, hyperinsulinemia or endotoxins, and that cholesterol which was deposited in an astherosclerosis plaque was originally present in the form of LDL cholesterol in plasma. 
     Today, a number of processes exist for the quantitative determination of LDL and β-cholesterol in plasma and, especially, in serum (Mills, G. L., Lane , P. A., Weech, P. K., A Guidebook to Lipoprotein Technique, Elsevier, Amsterdam, 1984; Cremer, P., und Seidel, D., Deutsch. Gesell. Klin. Chem. Mitl. 21 (1990), 21-232). 
     A basic distinction is made between differentiating techniques such as ultracentrifugation and electrophoresis on the one hand, and precipitation techniques, on the other hand. The problem with the former is that they require expensive instrumentation and are also time-consuming. Today they are primarily used for reference and confirmation. Precipitation of LDL with high molecular substances containing mostly negative changes is, however, part of the routine work in laboratories. In clinical diagnostics, polyvinyl sulfate, polycyclical surface-active anions or heparin are frequently used as LDL precipitation reagents. They are, however, marked by several drawbacks. When heparin is precipitated in an acidic medium or with polycyclical anions in serum samples with a preceding or existing lipolytic activity, the LDL cholesterol measurements are falsely elevated. When dextran sulfate or polyvinyl sulfate, the agents of choice, are used, because they exhibit the highest possible precision in the LDL determination, serum samples with a preceding existing lipolytic activity show falsely depressed results. Moreover, measurements with dextran sulfate-containing precipitation reagents are also falsely depressed when high levels of free fatty acids or triglycerides are present in the sample to be analyzed. This problem occurs even more frequently with patients treated with heparin. Further, a disadvantages of all precipitation procedures is that in addition to LDL, the LDL-like lipoprotein Lp(a) is completely precipitated as well. The procedure is, hence, not entirely LDL-specific. 
     It is thus, an object of the invention to provide a simple and rapid process for the specific determination of LDL where there is no interference by other lipoprotein particles nor by elevated concentrations of triglycerides or free fatty acids or by heparinized samples. 
     SUMMARY OF THE INVENTION 
     The invention is a method for determining LDLs in a sample which contains other lipoproteins. This is accomplished by adding an LDL-specific aggregating agent to the sample and then determining the aggregation as a measurement of LDLs in the sample. In particularly preferred embodiments, the aggregating agent is a &#34;comb&#34; or &#34;brush type&#34; polymer, i.e., a poly(meth)acryl polymer having anionic side branches extending from the polymer backbone. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1: Linearity of the measuring signal for the turbidimetric determination of LDL cholesterol. 
     FIG. 2: pH dependency of the process of the invention, buffer concentration: 0.15 mg/ml sodium acetate/0.07 mol/l citric acid. 
     FIG. 3: Normal serum samples spiked up with LDL (ultra centrifugation (UC). 
     FIG. 4: Triglyceride-rich serum samples spiked up with LDL (UC). 
     FIG. 5: Method comparison: one embodiment of the invention (PAMPS) and prior art ultracentrifuge (Uc). 
     FIG. 6: LDL cholesterol determination in accordance with one embodiment of the invention (PAMPS) and with prior art dextran sulfate. 
     FIG. 7: LDL cholesterol levels determined with 
     (a) the PAMPS embodiment of the invention (0.05 mol/l sodium acetate/citric acid, pH 5.20) 
     (b) dextran sulfate precipitation reagent (Quantolip®). 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     The comb or brush type polymers of the preferred embodiments of the invention are derived from acrylic acid ester monomers of formula: ##STR1## where R 1  is hydrogen or C 1  -C 6  alkyl, preferably methyl; X is oxygen or --NH; A is a linear or branched alkyl chain of C 1  -C 10 , branched chains being preferred, and Z is selected from the group consisting of COO - , SO 3   - , and HPO 3   - . 
     The individual units of the resulting polymer will lack the double bond of the monomer depicted supra. Thus, the polymers as described herein are represented by the formula: ##STR2## where each of R 1  X, A and Z are as above, and &#34;n&#34; is any number as long as it is 2 or more. 
     Particularly suitable are homopolymers of 2-acrylamido-2-methyl-1-propane sulfonic acid, 2-acrylamido glycolic acid and/or acrylic acid (2-phospho-1, 1-dimethylethylamide and/or copolymers of the said monomers. 
     The brush anionic comb polymers of the invention have a preferred molecular weight of 2×10 4  to 5×10 6  dalton, the particularly preferred weight being approximately 5×10 5  daltons as determined by gel permeation chromatography. 
     The anionic comb polymers of the invention can be produced by employing procedures known to the expert such as solution, suspension or emulsion polymerization (Meth. d. Organ. Chemie (Houben-Weyl), Bd. E 20, Makrom. Stoffe, Thieme Verlag, Stuttgart, 1987). The preferred procedure is solvent polymerization as described, for example, in Polymer 31 (1990), 1269-1276 (Huglin and Rego). The polymerization procedure can be accelerated by conventional initiators which are included in aqueous systems and form free radicals, especially peroxides, persulfates or persulfate/bisulfite or corresponding azo combinations. These procedures are also known to the expert. The preferred initiator for the preparation of the anionic comb polymer of the invention is ammonium peroxodisulfate. 
     The concentration of polyanions used in accordance with the invention is between 0.01 to 0.50 mg/ml, preferably between about 0.05 and about 0.30 mg/ml. Particularly suitable is a range between 0.07 to 0.20 mg/ml. 
     A buffering substance can also be used in the invention. Generally, those buffering substances can be used for the determination of the invention whose buffering capacity is in the weakly acid, neutral or weakly alkaline pH range. Particularly suitable substances are sodium acetate and/or citric acid. The pH value for the determination when one of these buffer substance is used preferably ranges between pH 5 and 6, a range between 5.10-5.50 being particularly preferred, and a range between 5.20-5.30 being especially preferred. The concentration of the buffer should range between 0.03-0.15 mol/l. A particularly preferred concentration range is between 0.05 to 0.07 mol/l. 
     Another preferred embodiment includes the use of a Tris-HCl buffer, a bis 2-hydroxyethyl!imino-tris hydroxymethyl!methane (bis-tris) or a 1- N,N-bis(2-hydroxyethyl)-amino!-2-propanol (BAP) buffer in the presence of bivalent metal ions, the concentration of the buffer ranging between 0.01 to 0.15 mol/l, the concentration of the metal salt generating the bivalent ion between 0.01-0.20 mol/l and a pH range between 5.5 to 9.0. In a particularly preferred embodiment, the concentration of the buffer ranges from about 0.02 to about 0.1 mol/l, the concentration of the metal salts from about 0.02 to about 0.15 mol/l and the pH range between 7.0-8.5. Generally, it is possible to use the salts of all known bivalent metal ions, particularly preferred ions being Mg 2+ , Ca 2+ , Mn 2+   and Cu 2+ . More particularly preferred ions are Mg 2+  and Ca 2+ . The concentration of the polyanions must be selected as indicated above. 
     Another subject matter of the invention is a reagent which contains the polymers of the invention and a buffer substance in a pH range between 5 and 9. The amount of polymer ranges between 0.01 to 0.50 mg/ml reagent solution; preferably there is added poly-(2-acrylamido-2-methyl-1-propane sulfonic acid) (PAMPS), poly-acrylic acid- (2-phospho-1, 1-dimethyl ethylamide) (PAP), poly-2-acrylamido glycollic acid (PAAG), poly-(2-acrylamido-2-methyl-1-propane sulfonic acid-CO-2-acrylamido glycollic acid)  P(AMPS-AAG)! and/or corresponding copolymers and/or corresponding poly(meth) acrylic acid ester in a concentration range of 0.05-0.30 mg/ml, particularly preferred in a concentration range between 0.07-0.20 mg/ml. Sodium acetate, TRIS-HCl, bis-TRIS, BAP and/or citric acid have proven to be particularly suitable buffer substances. The preferred concentration of the buffer ranges between 0.01-0.15 mol/l. 
     For the determination of LDL, the pH range of the reagent is between 5.1 and 9.0, preferably between pH 5.1 and 5.5 or between pH 7.0 and 8.5. The assay temperature can vary between 10° and 40° C.; a preferred temperature is approximately 37° C. 
     The volume ratio between sample to be analyzed and reagent solution is variable. Ratios of 1:1 to 1:100 have proven to be suitable. In a preferred manner, approximately 10 volume parts of serum sample (e.g. 10 μl) are mixed with 350 volume parts reagent solution (e.g. 350 μl). 
     As compared to known precipitation reagents, the LDL-specific aggregation or agglutionation that was realized via the anionic comb polymers of the invention has the following advantages: 
     1. Rapid determination without requiring sample pretreatment (approximately 1-10 min). 
     2. Formed aggregate is present in a stable condition and can, hence, be directly and reproducibly measured. 
     3. The determination of LDL cholesterol is linear in a very broad range (50-350 mg/dl LDL cholesterol). 
     4. Except for LDL, other apoprotein B-containing lipoproteins (very low density lipoproteins (VLDL), Lp(a)) and/or chylomicrons) do not interact with the polyanions of the invention and are, hence, not detected. 
     In a manner which has so far been unknown, the process and the reagent of the invention, lead to a complete agglutination of the LDL particles within 1 to 5 minutes without separation of the other lipoprotein fractions. It is, therefore, also possible to employ an analyzer or a simple photometer (turbidimetric) for the determination of the LDL concentration or the LDL cholesterol, which is desirable in routine analysis work. Moreover, it is also possible to determine the apoprotein B-100 and/or other molecular components of the LDL particles that are present in the LDL agglutinate. 
     The broad measuring range in which the method of the invention generates a linear measuring signal includes the diagnostically relevant range, in particular the one for elevated LDL values (&gt;190 mg/dl), which includes the advantage of requiring only a one-point calibration procedure. As another consequence of the extended linear range, elevated LDL levels can also be quantitatively monitored in a more precise way without requiring corresponding complicated reference procedures such as lipoprotein electrophoresis or ultracentrifugation. 
     The process and the reagent of the invention agglutinates LDL, but not LDL-like Lp(a). This is surprising as all known LDL precipitation reagents also precipitate the Lp(a) fraction almost quantitatively. With the present invention it is, hence, also possible to determine the Lp(a) fraction in a simple manner by reductively cleaving the Lp(a) portion of the Lp(a) particle which is connected via a disulfide bridge after LDL agglutination and determination in accordance with the invention in a manner which is known to the expert. By forming the difference, it is then possible to determine the contents of lipoprotein(a). 
     Moreover, in order to implement the process of the invention on an analyzer, the individual reaction components can also be impregnated on or in a carrier material or be covalently bound. The carrier material can be material which is absorbent, capable of swelling or forming a film, e.g. carrier materials that are known to be used with test strips such as paper or similar fleece materials such as tea bag paper. The reaction components can be distributed on several carriers that are interconnected or may themselves serve as carrier material. 
     The following examples illustrate the invention in greater detail: 
     EXAMPLE 1 
     (a) Preparing poly-(2-acrylamido-2-methyl-l-propane sulfonic acid) (PAMPS) by radical polymerization 8.412 g of 2-acrylamido-2-methyl-l-propane sulfonic acid (AMPS) are dissolved in 100 ml redistilled water, and the solution is exposed to nitrogen for a period of 30 min or treated in an ultrasonic bath. A solution of 22.8 mg ammonium peroxodisulfate in 100 ml redistilled water which had previously been exposed to nitrogen for a period of 30 min or was treated in an ultrasonic bath is added to this batch, while being heavily stirred, and heated up to 50° C. After 30 min, the temperature is raised to 70° C. and the reaction mixture is reduced to a volume of 20 ml. Using a dialysis tube with an exclusion limit of 12 to 14,000 dalton, this reaction mixture is dialyzed against redistilled water for a period of 4 days. The dialysate is evaporated until dry and produces crystalline PAMPS with an average molecular weight of 5×10 5  dalton (gel permeation chromatography). 
     Analogously, the following anionic comb polymers were produced: 
     (b): Poly-methacrylic acid-(2-sulfopropyl ester) (PMAS), 
     (c): Poly-2-acrylamido glycollic acid (PAAG), 
     (d): Poly-acrylic acid as determined by 2-phospho-1,1-dimethyl-ethylamide (PAP) and 
     (e): Poly-(2-acryamido-2-methyl-1-propane sulfonic acid-CO-2-acrylamido glycollic acid)  P(AMPS-AAG)!, from AMPS and AAG in a ratio of 1:1. 
     EXAMPLE 2 
     Determination of LDL cholesterol 
     Reagent solution: 
     0.15 mg/ml PAMPS (free acid) 
     0.05 mol/l sodium acetate/citric acid, pH 5.20 (acetic acid or citric acid (0.05 mol/l ) were used to adjust the pH). 
     Sample: serum 
     10 μl serum sample were mixed with 350 μl reagent solution at 37° C. After 1-2 minutes, the absorbance increase which is a consequence of the turbidity is determined at 505 nm. Calibration for LDL cholesterol is carried out with a serum sample of a known LDL cholesterol contents (standard). Sample blank correction should be carried out particularly when lipemic serum samples are analyzed. 
     
                       TABLE 1______________________________________Absorbance       LDL cholesterol  mg/dl!______________________________________0.145             810.2864           1500.4217           2340.6433           348______________________________________Regression analysis:______________________________________Constant          0.528Standard deviation Y             5.577R squared         0.998Number of measurements             4Degrees of freedom             2X-coefficient     541.891Standard deviation d.             15.185R = 0.999______________________________________ 
    
     2.1 LDL cholesterol determination (PAMPS) depends upon the pH value 
     The processes were carried out as described in example 2. However, three additional reagent mixtures, the pH values being 5.30, 5.40 and 5.50, respectively, were prepared. 
     The corresponding results are given in Table 2 and FIG. 2. In addition, Table 2 also contains the measurement results that were obtained with the prior art process (Quantolip®, manufactured by Immuno GmbH, Heidelberg, FRG) and the respective triglyceride (TG) contents. 
     
                       TABLE 2______________________________________                    Turbidimetric          LDL       LDL cholesterol determinationTotal          cholesterol                    (0.15 mg/ml 0.07 M sodium acetate)cholesterol  TG      Quantolip ®                    pH 5.2                          pH 5.3                                pH 5.4                                      pH 5.5 mg/dl!   mg/dl!  mg/dl!    mg/dl!                           mg/dl!                                 mg/dl!                                       mg/dl!______________________________________236    142     153       134   134   135   138204    235     124       118   121   122   125280    126     177       183   190   200   180299    161     200       169   182   186   194269    131     208       162   168   206   207372    241     261       253   280   275   309215    141     146       151   162   179   185______________________________________ 
    
     LDL cholesterol determination: dependency upon the LDL contents (spiked up with LDL 
     The samples with human serum were spiked up with LDL (density fraction between 1.006 and 1.063 g/ml), which was isolated with the aid of an ultracentrifuge according to Wieland, H. and Seidel, D. (Clin. Chem., 28 (1982), 1335-1337). The determination was performed as described in example 2. 
     
                       TABLE 3______________________________________Normal serum sample spiked up with LDL (UC)                          PAMPS (P1) Total                    turbidim. LDL cholesterol       TG     chol.Sample  mg/dl!  Diff. to IV                    mg/dl!                           mg/dl!  Diff. to IV______________________________________IV    201                92     92+ LDL 221       20       96    114       22+ LDL 248       47       94    131       39+ LDL 273       72       98    164       72+ LDL 295       96      101    195      103+ LDL 345      144      106    2332     140+ LDL 401      200      113    274      182______________________________________          Expected                 Actual          value                    value______________________________________ 
    
     Recovery (LDL cholesterol) 
     Mean value: 98% 
     Pl: 0.05 mol/l sodium acetate/citric acid buffer, pH 5.20 
     IV=initial value 
     2.3 LDL cholesterol determination: dependency upon the LDL contents (spiked up with LDL) with a triglyceride (TG)-rich serum sample 
     The serum samples were spiked up with LDL as indicated under 2.1. 
     
                       TABLE 4______________________________________TG-rich serum samples spiked up with LDL (UC)                         PAMPSTotal                         Turbidim. LDL chol.cholesterol          TG       determin.  mg/dl!Sample  mg/dl!   DIff. to IV                     mg/dl!                           Buffer P1                                  Diff./IV______________________________________IV    166                859    102+ LDL 219        47      836    160     58+ LDL 249        83      828    198     96+ LDL 266       100      773    208    106+ LDL 282       116      787    227    125+ LDL 317       151      736    257    155+ LDL 379       213      682    315    213______________________________________           Expected               Actual           value                  value______________________________________ 
    
     Recovery (LDL cholesterol) 
     Mean value: 108% 
     EXAMPLE 3 
     Method comparison 
     3.1 The method of the invention (PAMPS) was compared to an ultracentrifugation method (UC. reference method) 
     The procedure was carried out according to Armstrong, V. W. and Seidel, D. (Arztl. Lab. 31 (1985), p. 325-330). 
     Results for LDL cholesterol are given in Table 5 and FIG. 5. 
     
                       TABLE 5______________________________________Method comparison: the method of the invention (PAMPS)/ultracentrifugation (UC)______________________________________UC            PAMPSLDL cholesterol         (buffer as in example 2.2) mg/dl!       LDL cholesterol  mg/dl!______________________________________ 74            78111           108112           115125           116121           128144           139143           140139           140139           157160           162176           181181           188193           195______________________________________Regression analysis:______________________________________Constant         -1.166Standard deviation Y            6.959R squared        0.961Number of measurements            13Degrees of freedom            11X-coefficient    1.024Standard deviation d.            0.061R = 0.980______________________________________ 
    
     3.2 The method of the invention (RAMPS) was compared to the dextrane sulfate method 
     The determination of LDL cholesterol with PAMPS was carried out as described under example 2. For the determination of LDL cholesterol with dextrane sulfate, Quantolip® manufactured by Immuno GmbH, Heidelberg, was used as specified by the manufacturer. 
     The results of the comparative determination of LDL cholesterol are given in Table 6 and FIG. 6. 
     
                       TABLE 6______________________________________             LDL cholesterol                          Turbidimetric LDL             after dextrane                          cholesterol determ.Total cholesterol     TG      sulfate precipitation                          buffer PAMPS (P1) mg/dl!    mg/dl!  mg/dl!       mg/dl!______________________________________ 88       115      52           32175       449      69           74126       184      77           85136       177      78           76147       159      80           87195       137      88           90154       128      89           94200       177      93          111181        79     101          104190        58     105          111185       150     124          119206        77     146          150242        98     155          141266       148     173          182280       134     209          190327       112     214          200______________________________________Regression analysis:______________________________________Constant     9.758Standard deviation Y        10.438R squared    0.952Number of measurements        16Degrees of freedom        14X-coefficient        0.911Standard deviation d.        0.054R = 0.976______________________________________ 
    
     3.3 Influence of free fatty acids (FFA) on the determination of LDL cholesterol 
     When dextran sulfate (DS, Quantolip®, manufactured by Immuno GmbH, Heidelberg) or the polyvinyl sulfate precipitation reagent (Boehringer Mannheim) were used undesired interference occurs in the LDL cholesterol determination when increased concentrations of free fatty acids (FFA) are present in the material to be analyzed. Serum levels of free fatty acids greater than 2 mmol/l (normal range: 0.3 to 1.0 mmol/l) interfere with the interaction between the LDL particles and these precipitation reagents thus leading to an incomplete LDL precipitation and, hence, to falsely depressed measurements. This disadvantageous property also occurs when the material to be analyzed had been stored too long or in too warm an environment due to an increased in vitro lipolysis (Seidel, D., Armstrong, V. W., Cremer, P., Internist 28 (1987), 606-614). 
     The samples with human serum were enriched with free fatty acids (FFA: stearic acid, palmitic acid) according to Spector and Hoak, Anal. Biochem. 32 (1969), 297-302. 
     
                       TABLE 7______________________________________       LDL cholesterol                   LDL cholesterolFFA         DS precipitation                   PAMPS mmol/l!     mg/dl!      mg/dl!______________________________________0.45        123         1310.87        126         1341.2         119         1321.48        120         1391.81        116         1332.84        111         1363           103         1363.8          88         1394.24         59         1404.88         14         1385.28         3          137______________________________________ 
    
     3.4 Coagglutination of Very Low Density Lipoproteins (VLDL) 
     In order to investigate the selective agglutination properties of the reagent of the invention with respect to LDL particles in the presence of apoprotein B-containing VLDL particles, one volume part of VLDL (cholesterol: 153 mg/dl; triglycerides: 488 mg/dl), which has been isolated in an ultracentrifuge in a known manner, was added to 1 volume part of a serum sample (total cholesterol: 334 mg/dl; triglycerides: 107 mg/dl; LDL cholesterol 241 mg/dl). 
     A subsequent turbidimetric determination of the LDL cholesterol concentration with the reagent of the invention (PAMPS) yielded a value of 125 mg/dl. The method of the invention is, consequently, very selective with respect to the agglutination of apoprotein B-containing LDL particles. 
     When using the dextran sulfate precipitation method (Quantolip®), the initial value for the LDL cholesterol concentration was determined as 240 mg/dl. The serum to which VLDL was added in a ratio of 1:1 (v/v) yielded a value of 49 mg/dl. This falsely low LDL cholesterol value is a result of the incomplete LDL precipitation in triglyceride and VLDL-rich serum when using a dextrane sulfate precipitation reagent, which is known to the expert. 
     3.5 Coagglutination of Lp(a) 
     1 volume part of a Lp(a)-free normaglyceridemic serum (triglycerides: 120 mg/dl with a total cholesterol concentration of 104 mg/dl and a LDL cholesterol concentration of 104 mg/dl and a LDL cholesterol concentration of 71 mg/dl determined with the method of the invention was added to 1 volume part of isolated Lp(a)  cholesterol concentration: 120 mg/dl; Armstrong, V. W., Walli, A. K., Seidel, D. J. Lipid Research, 1985, 26, 1314-1323!. The subsequent turbidimetric determination of the LDL cholesterol concentration with the reagent of the invention (PAMPS) yielded a value of 34 mg/dl. 
     When using the dextran sulfate precipitation method (Quantolip®), the initial value for the LDL cholesterol concentration was determined as 72 mg/dl. The serum to which Lp(a) was added in a ratio of 1:1 (v/v) yielded a value of 96 mg/dl, demonstrating that the dextrane sulfate reagent also quantitatively precipitates the Lp(a) fraction in an undesired manner. The method of the invention, hence, agglutinates advantageously no Lp(a) particles and does not erroneously detect these as LDL particles.