DD273164A3 - PROCESS FOR ENRICHING LIPOPROTEINS - Google Patents

Abstract

Serum lipoproteins are especially needed in laboratory diagnostic studies, especially in the form of control sera or reference materials. The enrichment of the lipoproteins is carried out according to the procedure outlined in such a way that the lipoproteins themselves are dissolved in solution and the accompanying substances are removed by precipitation with ammonium sulfate in a final concentration of 1.40 mol / l and subsequent ultrafiltration. This method captures most of the serum lipoproteins and is easily feasible on a larger scale. The resulting concentrates are used either directly or after addition to serum for calibration or control purposes in lipid analysis.

Description

Characteristic of the known technical solutions Dio lipoproteins of the blood are a group of molecules, which in addition to the protein portion a relatively large Lipidanteil

but have qualitatively and quantitatively in the protein and lipid composition may have greater differences. The

Molecular weights range from about 2 χ 10 ⁵ to about 2 χ 10 ⁷ . The protein content h'jt different functions, among others is

It is responsible for the solubility of the molecules with the largely water-insoluble lipid components, and it also causes precipitability with protein precipitants. The lipid content gives the molecules a density that is below that of pure

Proteins and glycoproteins lies, he is responsible for the relatively easy aggregation of molecules, as z. In the Framework of the serum is expressed, and it does not interfere with the interaction with reagents or with proteins

only much weaker reugieren.

All of these properties of the lipoproteins become, in one form or another, different from the methods for their recovery Plasma or serum used. The previous methods are mainly based on their highest purity on a laboratory scale, on

the production of the ß-lipoprotelne and the isolation of lipoprotein fractions as part of a wider

Fractionation. Although the use of the ultracentrifuge provides very pure lipoproteins, it allows only smaller throughputs and comes

therefore hardly in question for the technical scale.

The one-step precipitation methods essentially detect only the β-lipoproteins but not the high-density lipoprotein fraction

(HDL fraction). - If the precipitation is nonspecific, z. R. with alcohol or other organic solvents, is usually relatively relatively 9 parts of unwanted serum proteins, which in the production of high Lipidkonicentrationen in

Kontrollmy disturbing notice. On the other hand, if more specific precipitating agents are used, e.g. B. ' Dextran sulfate or heparin require high accelerations for centrifugation, which are difficult on an industrial scale

to be realized.

In the laboratory production of lipoproteins, there are methods in which the lipoproteins are precipitated initially

and then after addition of certain substances, the precipitant from the precipitate by simple dialysis in

Hoses is removed, whereby a substantial clarification occurs. Such methods are not on the required for larger batches ultrafiltration with tangential flow over Mombran, because only clear solutions can be used from the outset. The adaptation of another method to ultrafiltration, namely the precipitation of the lipoproteins with rivanol, solution of the Precipitation and removal of the precipitant by dialysis, was not possible because the lipoproteins after

accumulated in a short time to droplets and thus were no longer usable for its intended purpose.

The classical plasma fractionation is relatively complex and would be particularly useful to use if the Major plasma components such as albumin, immunoglobulins or other fractions and the lipoproteins

could be isolated from one of the resulting fractions with relatively little effort. Their sole use for obtaining the lipoproteins on a larger scale is too expensive.

Object of the invention

The object of the invention was therefore the development of a simple and robust method for use on an industrial scale, with the majority of the serum lipoproteins including the HDL fraction can be obtained with relatively low admixtures of other serum components.

-? - 273164 Explanation of the essence of the invention

Apart from ultrafiltration, in the previous methods the principle is used to separate the lipoproteins by precipitation from the accompanying substances of the serum. Surprisingly, it has been found that the reverse principle, namely the release of the lipoproteins and the separation of the undesired accompanying substances, results in a process which is well suited for industrial purposes, if very specific conditions are met. For this purpose, most non-lipoproteins are precipitated in the first stage with molecular weights above 1.5 x 10, and in the second stage, the precipitant and other low molecular weight substances sowio proteins up to a molecular weight of about β χ 10 ⁴ , especially albumin, by ultrafiltration separated. For the precipitation in the first stage, a final ammonium sulfate concentration of 1.40 ± 0.05 mol / l must be strictly observed. At higher concentrations, the lipoproteins are already goschädigt or brought into an aggregated form, which is not visible to the naked eye, but which can hinder the ultrafiltration very much, apparently by clogging the pores. At lower concentrations of ammonium sulfate, on the other hand, too few of the unwanted accompanying proteins are separated off. The very narrow concentration range for the ammonium sulfate is required in particular by the fact that the molecular weights of accompanying proteins to be separated - about 1.5 × 10 ^s to more than 2 × 10 × in immunoglobulins or immune complexes - and low molecular weight lipoproteins - from about 2 χ 10 ^s - clearly overlap. Precipitation is counteracted by the fact that at 1.4 mol / l of ammonium sulfate some of the immunoglobulins precipitate along with the other proteins, while in pure form they precipitate higher concentrations of the precipitate.

On the one hand, the membranes required for ultrafiltration must allow albumin to pass well; on the other hand, the low molecular weight lipoproteins, namely the least abundant HDL anyway, must not be allowed to pass through. Therefore, a pore size with the nominal molecular weight of 10 ^{s is} the most favorable.

Since the ultrafiltration of the proteins in relation to the water seh; is slowly going on, a larger amount of aqueous solution must be added to the concentrate to be cleaned in the course of the procedure. These washing solutions can be arbitrarily composed, they must not have any adverse effect on the lipoproteins or the accompanying proteins. Appropriately, therefore, one uses dilute salt solutions, for. B. 1% saline. Finally, if necessary, it is also possible to wash with a solution of the composition which one wishes to have finally in the lipoprotein concentrate, e.g. a solution equivalent to the serum of low molecular weight substances.

The degree of concentration of the lipoproteins and the leaching of the accompanying proteins depends on the requirements. The concentration can not be increased arbitrarily, since the simultaneously increasing viscosity of the solution causes technical problems. A concentration of 10 g cholesterol / l, which corresponds to a concentration of lipoproteins more than fivefold, is about the limit at which one can still work under normal conditions. A stronger concentration requires a much higher effort.

If the lipoproteins are to be used for analytical purposes in control or comparison materials, it is generally sufficient to remove about 70 to 75% of the starting proteins.

The particular advantage of the method described is that not only fresh serum or plasma, but also frozen serum can be used directly for the workup, although here the lipoproteins suffer some damage to their structure. As a result, the frozen serum collected from serum waste can be processed. Nevertheless, the solutions before and after the ultrafiltration were clearly filterable without loss of lipoproteins, and no ultrapiltration or formation of any other noticeable aggregation occurred.

Ausführungsbelsplel

20 μl of human serum are added at room temperature and with stirring to a solution of ammonium bromide solution of 20.8 mol / l, with the first 151 being added in 20 minutes and the last 51 in 2 streams. The solution is stirred for a further 1 hour, allowed to stand overnight and centrifuged. The supernatant is first concentrated by subsequent ultrafiltration with membranes dor exclusion limit 100,000 Dalton suf 151, washed with <ι (as a washing solution is used throughout the process 0.9% saline), then to 121.10 /, 81,6,71 and 5.51, washing at 41 at each stage.

It is then concentrated to 4.81 and washed with 3.01 total, maintaining the retentate volume between 4.8 and 5.21. Finally, it is concentrated to 3.51.

Depending on the initial values, the concentrate contains about 10 g of cholesterol and about 8 g of protein per liter. The ultrafiltration is completed in 5 hours (depending on the type and size of the membrane and on the pump and the pressure conditions used).

From this concentrate, 2 parts are added to 10 parts of human collecting serum having a cholesterol content of 1.8 g / l. The cholesterol content of the mixture is slightly more than 3 g / l. The spiked serum is sterile filtered, dispensed in portions and lyophilized.

Claims (1)

Process for the enrichment of lipoproteinone from plasma or serum, characterized in that proteins and immune complexes having a molecular weight of more than 1.5 x 10 ⁶ are precipitated with ammonium sulfate in a final concentration of 1.40 ± 0.05 mol / l and then the supernatant is passed through Ultrafiltration with membranes of nominal molecular weight 1 χ 10 ^s from the precipitant, low molecular weight substances of the serum and egg white zj to a molecular weight of about 6 x 10 ⁴ is released. Field of application of the invention Cardiovascular diseases are currently the most common cause of death in highly developed industrialized countries. Their main risk factors include disorders in the Lipld family, which among other things promote the occurrence of arteriosclerosis and myocardial infarction. In the prevention and treatment of these disorders, lipid analysis is of great importance in clinical laboratories. The present invention relates above all to a method for obtaining lipoproteins which are suitable for the preparation of control and calibration materials for lipid analysis. For this purpose, serum lipoproteins are used as starting material, which are largely freed from other components and concentrated by a particular ancestor. Such preparations may also serve as starting material for the recovery of apolipoproteins.