JPH023466B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for producing low density polyproteins from whole serum or plasma.
Selective extracorporeal precipitation of lipoprontein
Regarding the method.

Recent advances in the field of integrated analysis of lipoprotein systems have enabled ancient epidemiological data to link plasma cholesterin concentrations and early atherosclerosis, particularly in coronal atherosclerosis. It has been found that the close correlation between the . In human blood, approximately 70-80% of total cholesterin is normally bound to low-density or beta-lipoproteins, with the remainder being stored in other lipoprotein components (substantially VLDL, HDL, and milky lipid granules). (LDL = low-density lipoprotein, VLDL = very low-density lipoprotein,
HDL = high-density lipoprotein). In the disease process, which progresses by interfering with fat metabolism and increasing plasma lipid concentrations, leading to early atherosclerosis, LDL- or β- to total cholesterin
The proportion of cholesterin (LDL = β-lipoprotein) increases further. That is, hypercholestinaemia is usually caused by hyperβ-lipoproteinemia. Therefore, attempts have been made to selectively measure low-density lipoproteins and remove them from the blood circulation as selectively as possible.

These attempts have not yet been fully resolved considering the technical situation. The conventional methods for quantifying low-density lipoproteins are based on the separation of lipoprotein spectra into density classes by ultracentrifugation or the separation of lipoprotein spectra in electric fields used in so-called lipoprotein electrophoresis. . Furthermore, it can also be determined by precipitation methods based on the separation of apo-B-containing lipoproteins (VLDL and LDL) from non-apo-B-containing lipoproteins by precipitation with polyanions and divalent cations. In the case of apo-B-proteins, the main protein component of the problem is VLDL and pre-β-lipoproteins and lower density than the milk fat granules or β-lipoproteins. Precipitation method is VLDL
Conventionally, it was unsuitable for separation from LDL. The ultracentrifugation method described above [Havel RJ, Eder HA and Bragdon JH, "Distribution and chemical composition of lipoproteins in human serum separated by ultracentrifugation", J.Clin.Invest. No. 34 Vol. 1345, 1955]
and electrophoretic methods [Wieland H. and Seidel D., "Advances in the comprehensive analysis of lipoprotein samples",
Inn.Med., Volume 5, Pages 290-300, 1978]
are all part of a large number of routine series (Routine-
The disadvantages of this method are that it is expensive, time-consuming, and cannot be automated. Furthermore, linear measurements of low-density cholesterin are usually only possible after isolation of the components using an ultracentrifuge. Calculated evaluation of low-density cholesterin content by electrophoresis assumes that the protein-lipid composition is constant. The same applies to the methods used in precipitation techniques. Conventional precipitation techniques further reduce the lipoprotein spectrum to only two major components (apo-B-containing and non-apo-B-containing).
It has the disadvantage that it cannot distinguish or separate VLDL and LDL.

All therapeutic attempts to effectively precipitate low density lipoproteins have been inadequate. Influencing low-density lipoprotein levels with drugs, especially in genetic forms of fat metabolism disorders, is very difficult and usually insufficient. Previous results achieved with drugs do not reliably demonstrate a therapeutic effect in reducing the risk of atherosclerosis. Surgical procedures essentially based on the identification of abnormal blood circulation conditions or the relocation of a significant portion of the intestines have clear therapeutic effects, but are generally considered an alternative method due to the expected strong side effects. I'm not used to it. Indeed, such drastic therapeutic measures, if low-density lipoproteins are reduced sufficiently, will not only halt the progression of atherosclerosis but also reverse existing atherosclerotic disease. Vascular degeneration is regressed.

Research into the "mechanical" removal of low-density lipoproteins from blood has traditionally followed essentially two paths. One method is to treat sick patients with so-called plasmapheres to completely convert the total serum. According to this method,
Low-density lipoproteins can be reduced in sick patients, but at the same time high-density lipoproteins, which inhibit atherosclerosis, are also removed from the blood, resulting in an overall undesirable therapeutic effect. Furthermore, during plasmapheresis, all other proteins of the plasma, contained clotting factors, globulins and hormones are also removed. However, attempts to selectively remove low-density lipoproteins from plasma remain superior, although this method has only proven useful in the traditional and specific cases of hyperβ-lipoproteinemia. Ta. The first attempt to remove low-density lipoproteins from the blood was through the use of specialized antibodies bound to matrices. Antibodies were obtained by immunization of sheep or rabbits. Such systems (Habenicht A., Doctoral thesis (1978, Heidelberg University, Faculty of Medicine); Stoffel W. and
Demant Th. paper "Selective removal of apolipoprotein B-containing serum lipoproteins from plasma", Proc.
Nat.Acad.Sci.USA, Volume 78, Pages 611-615
A significant reduction in all apo-B-containing lipoproteins was obtained by using the method [Page et al., 1981], but this method does not allow antibodies produced in the animal's body to enter the circulating blood of the patient during treatment. It has the disadvantage that it cannot be avoided, albeit in a small amount. This must be followed over a long period of time and must be resolved as an immunological problem for treated patients. This major barrier to such methods becomes even more critical when the treatment of fat metabolic disorders is life-threatening, and the treatment should be effective and long-lasting. In the case of familial forms, treatment in adolescence is necessary to prevent or measurably delay the progression of early-onset atherosclerotic vascular disease. Another drawback of the method of introducing immobilized antibodies against apo-B is the inability to specifically remove all apo-B-containing lipoproteins. This method is also unsuitable for selectively removing low density lipoproteins as the only atherosclerotic lipoproteins. The major disadvantage of simultaneous removal of VLDL is that it stimulates the synthesis of triglycerides and cholesterin in the liver.

A third method for removing low-density lipoproteins from blood is to add beta from whole blood to a sulfated polysaccharide, such as heparin or dextran sulfate (U.S. Pat. No. 4,103,685), bound on agarose microparticles.
- and pri-β-lipoproteins are selectively absorbed. A clear drawback of the US patent is that this method has a low ability to remove lipoproteins and removes not only low density lipoproteins but also very low density lipoproteins. The US patent only describes the reduction of cholesterin in an in vitro test. The reduction in cholesterin is thought to be caused by dilution of the patient's blood rather than by selective removal, such that phospholipids and triglycerides are concomitantly reduced.

The method described in U.S. Pat. No. 4,215,993 in which lipoproteins are generally precipitated at their isoelectric point comprises:
This method differs from the method of the present invention in that sodium phosphotungstate is used. This polyanion is non-physiological and, unlike the method of the present invention, is unsuitable for therapeutic use. Since low density lipoproteins cannot be selectively removed or quantified using phosphotungstate, the object of the present invention cannot be achieved. The failure of this method is shown in U.S. Pat. No. 4,215,993 and elsewhere. That is, it is recommended that low-density lipoprotein be calculated from so-called HDL-cholesterin by a calculation formula (Friedewald formula), taking into account total cholesterin and triglycerides. Essentially, isoelectric focusing using phosphotungstic acid as disclosed in US Pat. No. 4,215,993 differs in its results from conventional polyanionic precipitation using divalent cations. Complete precipitation of VLDL and LDL in this method is only achieved when a polymer of precisely defined molecular weight, such as polyvinylpyrrolidone or polyethylene glycol, is added to the precipitation solution. There is no question that this method cannot be carried out in vitro.

Furthermore, in US Pat. No. 4,215,993, it is shown that the sum of β-lipoprotein, pre-β-lipoprotein and milky fat granules can be misleadingly interpreted as low-density lipoprotein. Since the purpose described in U.S. Pat. No. 4,215,993 is to determine high-density lipoproteins in serum, its value is that VLDL, milk fat granules and LDL present together with high-density lipoproteins are removed by this precipitation method. It is to be done. Effective low-density lipoprotein or beta
- Specific precipitation of lipoproteins is neither intended nor achieved.

The object of the present invention is to extract low density lipoproteins or β from blood, i.e. from blood components such as whole serum or plasma.
- By providing a method and device for the selective and highly efficient removal of lipoproteins, this principle can be applied for both therapeutic and diagnostic purposes.

This problem was solved by using suitable methods and devices.

That is, the present invention is characterized in that heparin in a buffer solution is added to whole serum or plasma, and the generated β-lipoprotein-heparin complex is precipitated at an isoelectric point of PH 5.05 to 5.25, and then separated. The present invention relates to a method for specifically in vitro precipitating low-density lipoproteins or β-lipoproteins from whole serum or plasma.

This method is based on the completely specific precipitation of β-lipoproteins after binding with heparin at a pH of 5.05 to 5.25, in particular at the isoelectric point of the 5.13 complex. Besides specificity, another feature of this method is the addition of a substance produced in the body (heparin) as a precipitant for low-density lipoproteins. The precipitate at the isoelectric point by lowering the PH using a buffer is subjected to appropriate filtration, and the uniform PH after the isoelectric point precipitation is easily corrected to physiological conditions. In particular, in the case of the method of the present invention, a complex of heparin and β-lipoprotein is precipitated at a pH of 5.13.

Heparin is dissolved in a buffer and mixed with whole serum or plasma. Buffers are used for adjusting and stabilizing a predetermined pH value. In principle, all buffer mixtures that guarantee stabilization in the given PH range can be used in this method. Buffers suitable for therapeutic use include phosphate buffer, phosphate-citrate-
buffers, lactate buffers, acetate buffers or mixtures thereof.

The ratio of buffer to serum, plasma or blood volume is 1:5 to 5:1. The most effective concentration range is a 1:1 ratio (these volume ratios also apply for therapeutic removal). The most effective volume ratio for selectively removing low density lipoproteins from blood for diagnostic purposes is a buffer to serum ratio of 2 to 10:1.

Heparin concentration is not particularly critical. However, it is useful to adjust the heparin concentration to almost completely precipitate β-lipoproteins. For example, in the most effective case, a predetermined amount of serum is mixed with approximately 10% by volume heparin-buffer (500-5000E/
ml).

For example, when using a mixture of equal volumes of whole serum or plasma and a phosphate-citrate buffer in which heparin has been dissolved and the pH value adjusted to approximately 5.13, precipitation occurs immediately and completely. It is specific. This means that only β-lipoproteins or low density lipoproteins are precipitated. The precipitated β-lipoproteins are e.g.
It can be separated from residual serum by simple centrifugation through a 0.8 μm membrane filter or in a continuous flow process.

The method of the invention is also suitable for therapy and diagnosis.

In the case of therapy, for example, plasma is separated from blood taken from a patient and mixed with a heparin solution in a buffer according to the invention. As a result, β-lipoprotein is precipitated as a heparin-β-lipoprotein complex at a pH of 5.05 to 5.25, especially 5.13. Residual serum is separated from the precipitate by filtration and returned to the circulation.

In the case of diagnosis, the serum residue separated from the .beta.-lipoproteins and the .beta.-lipoprotein-heparin complex is subjected to subsequent analysis.

The specificity of the method of the present invention is that quantitative immunological techniques,
It was re-examined by ultracentrifugation and quantitative lipoprotein electrophoresis. The specific PH-precipitation method of low density lipoproteins described here leads to complete precipitation of the β-lipoprotein class. Only HDL and VLDL are present as lipoproteins in the residual filtrate.

The specificity of the method of the invention and the use of only physiological substances firstly enable extracorporeal blood circulation in patients suffering from hyperlipoproteinemia or lipoproteinemic disorders, and secondly, from plasma of
Selective removal of LDL allows quantification of this component by calculation of the difference between total cholesterin and residual filtrate, or by determining the linear cholesterin for the precipitate or by measuring the turbidity of the precipitate that produces it. .
This method for direct determination of LDL- or β-cholesterin is novel, and no competing method has hitherto existed. This method is also effective for the selective removal of LDL- or β-cholesterin from whole plasma for therapeutic purposes.

The method of the present invention is a preparative ultracentrifugation method (die
0.98 for both methods for serum series of more than 100 patients.
The above correlation coefficients are shown. The specificity of the method of the invention clearly exceeds that of ultracentrifugation. The method of the present invention has an advantage over electrophoresis in that cholesterin in β-lipoprotein can be directly determined. The method according to the invention can be used both in discrete automation and in continuous flow automation.
It can also be easily implemented in a mechanized manner within a flow-automation. The daily precision (Prazision) for this series is less than 2%.

The invention further comprises a reservoir to which a blood component, such as the patient's blood or plasma, is supplied via a first pump, and a filter connected to the reservoir and a filtrate outlet connected to a conduit leading back to the patient. The present invention relates to an extracorporeal precipitation device for blood components.

The invention provides a device of this type that can be operated continuously without costly flow regulation in the working phase, in order to place as little stress on the patient as possible and to avoid expensive and failure-prone regulation systems. The task is to do so.

To solve this problem, in the device according to the invention, the filter has a second outlet for the sediment, which is connected to a return conduit leading to the reservoir, and the reservoir receives the treatment agent via a second pump. is connected to the outlet of the first container containing the pump, and the transport output is connected to the first pump and the second pump.
A filter filtrate outlet is connected to a third pump that substantially matches the total pump delivery output.

In this case, the reservoir forms a closed circuit with the filter and the return conduit, within which the fluid circulates. External fluids enter the circuit via a first pump that pumps blood or blood components within a reservoir and a second pump that pumps a processing agent (in this case heparin) within the reservoir. A third pump is used to remove fluid from this closed circuit through the filtrate outlet of the filter. By matching the transport power of the third pump with the sum of the transport powers of the first and second pumps, fluid balance in the circuit is substantially maintained, so that the level of fluid in the reservoir is always remains almost constant. Therefore, it is not necessary to frequently open and close the switch of the first pump, so that blood can be uniformly collected from the patient. In this case, the above 3
For two pumps, volumetric pumps (volumetrischen pumpen) whose transport capacity is proportional to the driving speed
is important. Hose pumps (Schlauch-pumpen) to avoid contamination of the pumped fluid
It is effective to use

The transport output of the first pump is typically determined by the patient's physical condition, since it is necessary to avoid drawing too much blood from the patient too quickly. Therefore, it is effective to configure the system so that the transport output of the first pump can be changed without causing the circulation system to become unbalanced. Furthermore, in a preferred embodiment of the present invention,
The transport output of the first pump changes depending on the rate of blood collection from the patient, and the relationship between the transport outputs of the first, second, and third pumps is maintained constant. Drive each other.

The advantage of this case is that the system can be operated while changing the transport amount over time without losing the fluid balance. The pumps are mechanically or electrically connected via their transmissions, with the first pump taking the lead and the speeds of the other two pumps being automatically adapted to this.

Despite the mutual adjustment of the transport outputs of the three pumps, the liquid level in the reservoir is gradually increased when the device is operated for a considerable length of time. To limit this rise, the reservoir is equipped with at least one fill level detector which reduces or stops the delivery output of the first pump when the fluid reaches the upper fill level. let

On the other hand, in order to avoid an excessive drop in the fluid level in the reservoir, the transport power of the third pump is reduced by the filling level detector when the fluid reaches the lower filling level; Or stop. This limiting adjustment of the filling level to the region between the upper and lower limits serves to limit the influence of ever-increasing deviations in the fluid balance of the circulation circuit.

Since filters become clogged over time, a preferred embodiment of the invention includes a cleaning phase. This device activates a second pump when the flow resistance reaches an upper limit, transports the solvent from a second container containing the solvent to the reservoir, and provides communication between the outlet and the outlet. Equipped with a resistance measuring device. In this way, the fluid circulation circuit is interrupted or closed behind the filter, and the reservoir as well as the filter are flushed with cleaning solvent, which is led together with the filter residue to the outlet.

An embodiment of the device according to the invention for carrying out plasmapheresis with precipitation of low-density lipoproteins will be explained in more detail below with reference to the drawings.

FIG. 1 is a schematic diagram of the system in which the conduits through which flow flows in the operating state are shown in bold lines.

FIG. 2 is a schematic diagram of the system of FIG. 1, with the conduits through which water flows during filter cleaning shown in bold lines.

In these figures, the portion above the dashed line shows the well-known blood collection system and blood return system on the patient side. Blood is collected from the patient via the patient connection 1 while being constantly monitored using a pressure meter 3. The collected blood is directed to a plasma filter 6 via a pump 4 and then mixed with a blood anticoagulant from a container 5. The blood, depleted of approximately a certain amount of plasma, is directed from the filter 6 via an air trap 7 and a magnetic valve 9 to a return connection 2 to the patient. The return pressure is monitored by a pressure meter 8.

The separated plasma is sent to the filtrate outlet 61 of the filter.
It is discharged from the filter 6 after passing through. In the filter 6, for example, a capillary filter, through which the blood flows, is important, in which case the fine blood components leak laterally through the membrane wall, and the coarse blood components are conveyed further into the air trap 7.

Pump selectable amount of plasma (first pump) 1
1 from the plasma filter 6. In the case of the first pump, as in the case of the other pumps used in this system, a hose pump or peristaltic pump (peristaltische pump) that automatically aspirates and capacitively transports plasma in an amount proportional to the drive speed is used. )is important. Plasma filter 6 and first pump 11
A blood leak detector 10 and a pressure measuring meter 12 are provided between them. The outlet of the first pump 11 is connected to the inlet of the reservoir 16 .

Another inlet of the reservoir 16 is connected to the outlet of a second pump 13, the inlet of which is connected via a valve 14 to a container 15 containing a heparin-containing acetate buffer solution and via a valve 30. and connect it to a container 29 containing cleaning liquid. The reservoir 16 further comprises a fill level detector 17, which responds when a certain level is reached.

The outlet provided at the lower end of the reservoir 16 is connected to the valve 3.
1 is connected to a further pump 18 which is connected in parallel with 1. The outlet of the pump 18 is the filter 21
The outlet of the filter is connected to another inlet of the reservoir 16 via a valve 22 and a return conduit 221. The pressure at the inlet and outlet of filter 21 is monitored by pressure gauges 19 and 20.
The filtrate outlet 211 of the filter 21 is connected via a third pump 23 and a valve 25 to the inlet of a dialyzer 26, through which the dialysis cleaning fluid from the dialysis device 27 flows through the cleaning fluid side of the dialyzer. Inside the dialyzer 26,
The acetate buffer and residual heparin from reservoir 15 are removed, while the plasma is flushed via pump 28 to air trap 7 and thus to the extracorporeal blood circulation. Pump 28 rotates at the same speed as pump 11, thereby maintaining fluid balance in the patient being treated. Due to the dialysis washing fluid from the dialyzer 27, the PH value and temperature of the returned plasma within the filter 26 increase to the body value at the same time. Instead of a dialyzer, the fluid can be directed to the dialyzer 26 via an open circuit.

In the case of the drive mode shown in Fig. 1, first of all,
Pump 11 transports plasma into reservoir 16 and at the same time pump 13 transports heparin-containing acetate buffer with valve 14 open. In this state, the third pump 23 is stopped. Reservoir 16
When a predetermined filling level is reached within the pump, the filling level detector 17 responds by activating the pump 23. Pump 23 rotates at a speed corresponding to the sum of the rotational speeds of pumps 11 and 13. The third pump 23 drains the plasma from the filter 21 while the precipitate is left within the filter 21 depending on the selected pore size. The further pump 18, which maintains the fluid circulation in the circuit, has a relatively large transport power, so that the low-density lipoprotein-containing precipitate is returned back into the tidal reservoir 16 via the return conduit 221.

The plasma from which low-density lipoproteins have been removed is guided to the dialyzer 26 via the third pump 23 and the opened valve 25.

Since the sediment in the reservoir 16 and filter 21 constantly increases, it is necessary to keep the sediment a certain distance from the reservoir 16 and the recirculation path. This also occurs in the case of the device of the embodiment shown in FIG. The supply of plasma via the first pump 11 and the supply of heparin-containing acetate buffer via the second pump 13 is stopped by stopping these pumps. Pump 18 is stopped and bypass valve 31 is opened. In some cases, it is also possible to leave the pump 18 rotating. In this case, the bypass valve 31 is omitted. The third pump 23 is
Filter 21 to filter plasma from which low-density lipoproteins have been removed.
The plasma is transported through the dialyzer 26 to the pump 28. If the reservoir 16 leaks, the pressure difference between the inlet and outlet of the filter 21 will increase. This increase in pressure difference is confirmed by the differential, which produces a difference between the signals of pressure gauges 19 and 20. When the pressure difference exceeds a certain limit value, the pump 13 is activated, the valve 32 opens and the valve 25 closes. Furthermore, valve 14 is closed and valve 30 is opened. At this time, solvent is supplied from container 29 to reservoir 16 . Valve 22 opens and recirculation pump 18 operates. Recirculation of the solvent dissolves any precipitate remaining in the conduit and filter 21. When the inside of the reservoir 16 reaches the operating level, the third pump 23 is activated, and the solvent together with the dissolved precipitate is fed to the reservoir 33 through the opened valve 32. If the precipitate is sufficiently dissolved, a lower pressure value is detected by the differential Frenzbildner 201. Based on this, the pump 13 is stopped and the valve 30 is closed. A similar process is performed with pressure gauge 24 indicating a relatively small pressure. The third pump 23 is activated until the contents of the reservoir 16 and the loop are delivered to the reservoir 33. After this, a new recycling is initiated to precipitate low density lipoproteins.

The invention will be further illustrated by the following examples.

Example 1 Plasma was mixed with equal volumes of citrate, phosphate or acetate buffer (0.05M, PH4.15). When 1/10 the amount of heparin (5000E/ml) of the added plasma was added to this mixture, a yellowish white precipitate was formed. This precipitate consists of precipitated β-lipoproteins and especially fibrinogen.

Example 2 0.0641M Sodium Citrate (PH 5.04) and 1% Liqemin 25000 (Roche)
1 ml of the solution consisting of 100 μl of serum was mixed. The mixture was centrifuged for 10 minutes using an Eppendorf centrifuge. The cholesterin content of the supernatant was comparable to that of VLDL and HDL. LDL is precipitated and calculated from total serum cholesterin - (VLDL cholesterin + HDL cholesterin).

Furthermore, all other polyanions that can be used in lipoprotein precipitation methods in combination with divalent cations have been found to be suitable for diagnostic purposes.

Examples of polyanions include dextran sulfate and sodium phosphotungstate having a molecular weight of 10,000 to 2,000,000.

Therefore, the present invention also provides the method of adding polyanions in a buffer to whole serum or plasma and preparing the resulting β-lipoprotein-polyanion-complex at pH 5.05 to
Selectively extract low-density lipoproteins or β-lipoproteins from whole serum or plasma for diagnostic purposes by precipitating them at an isoelectric point of 5.25, then separating and optionally further analyzing the precipitate and filtrate. This invention relates to a method for extracorporeal precipitation.

In a particularly preferred embodiment of this method, the complex of β-lipoprotein with a polyanion, such as dextran sulfate or sodium phosphotungstate, is precipitated at a pH of 5.13.

Polyanions, such as dextran sulfate, are dissolved in a buffer and whole serum or plasma is mixed. Buffers help adjust and stabilize the desired pH value. In principle, all buffer mixtures that guarantee stabilization in the given PH range can be used in this method. Suitable buffers include phosphate buffer,
Examples include phosphate-citrate buffers, lactate buffers, acetate buffers, or mixtures thereof.

The ratio of buffer to serum, plasma or blood volume is between 1:5 and 1000:1. The volume ratio of serum to buffer is most suitable for selectively removing low density lipoproteins from blood for diagnosis: 2-10:
It is 1.

The polyanion concentration is not particularly limited. However, it is preferred that the polyanion concentration be such that it is almost completely precipitated with β-lipoprotein. For example, in the most preferred case, a predetermined amount of serum is
Mix with 10% dextran sulfate buffer or 40% sodium phosphotungstate buffer by volume.

Mixing equal volumes of whole serum or plasma with a phosphate-citrate buffer that has dissolved the polyanion and adjusted the pH to about 5.13 results in complete and specific precipitation immediately.

This means that only β-lipoproteins or low density lipoproteins are precipitated. The precipitated β-lipoproteins can be filtered, for example, through a membrane filter (with a pore size of approx.
0.2-2 μm) or from residual serum by simple centrifugation in a continuous flow method.

This embodiment of the method of the invention is suitable for diagnostic testing.

In diagnostic methods, the serum residue from which β-lipoproteins have been removed and the precipitated complexes of β-lipoproteins and polyanions can be further tested. The specificity of this embodiment of the method of the invention is tested by quantitative immunological techniques and ultracentrifugation and quantitative lipoprotein electrophoresis. This specific PH-precipitation method of low density lipoproteins provides a complete precipitation method for these β-lipoprotein classes. As lipoproteins in the residual filtrate,
There are only HDL and VLDL.

In vitro selective removal of LDL from plasma can be achieved by determining the amount of this component by calculating the difference between total cholesterin and residual filtrate or by direct cholesterin determination on the precipitate or by measuring the turbidity of the precipitate formed. Allows for quantification. The linear method for quantifying LDL- or β-cholesterin is new, and no competing method has been known.

This embodiment of the method of the present invention has been compared with preparative ultracentrifugation and quantitative lipoprotein electrophoresis and has demonstrated a correlation coefficient of greater than 0.98 with both methods for serum series of more than 100 patients. Indicated. The uniqueness of this embodiment of the method of the invention is clearly superior to ultracentrifugation. Compared to electrophoresis,
This embodiment of the method of the invention has the advantage that the β-lipoprotein cholesterin can be directly quantified.
This embodiment of the process of the invention can easily be carried out as a mechanized process in discontinuous or continuous automatic equipment. The daily accuracy in the above series is within 2%.

This embodiment of the invention is further illustrated by the following examples.

Example 3 Plasma was mixed with equal volumes of citrate, phosphate or acetate buffer (0.005M, PH4.15). When about 10% by volume of 6% dextran sulfate buffer was added to this mixture, a yellowish white precipitate was produced. This precipitate consists of precipitated β-lipoproteins and especially fibrinogen.

Example 4 0.0641M sodium citrate (PH5.04) and
1 ml of a solution containing 1% by volume of 40% sodium phosphotungstate was mixed with 100μ of serum. The mixture was centrifuged in an Eppendorf centrifuge. The cholesterin content of the supernatant was comparable to that of VLDL and HDL. LDL is precipitated and total serum cholesterin − (VLD cholesterin + HDL
It can be calculated from cholesterin).

[Brief explanation of drawings]

1 and 2 are schematic diagrams showing an embodiment of the apparatus of the invention for carrying out plasmapherase with precipitation of low density lipoproteins. 1 is a connection to the patient, 2 is a return connection to the patient, 6 is a plasma filter, 7 is an air trap, 1
1 is a first pump, 13 is a second pump, 15 is a treatment agent containing container, 16 is a storage device, 21 is a filter,
23 is a third pump, 26 is a dialyzer, and 29 is a container containing a solvent.

Claims (1)

[Claims] 1. Heparin in a buffer solution is added to whole serum or plasma, and the resulting β-lipoprotein-heparin complex is precipitated at an isoelectric point of PH 5.05 to 5.25, and then separated, or A method for the selective in vitro precipitation of low density lipoproteins from whole serum or plasma, characterized in that the precipitate or filtrate is further analyzed for diagnostic purposes. 2. The method according to item 1, wherein the precipitation of the β-lipoprotein-heparin complex is carried out at pH 5.13. 3. The method according to item 1 or 2, wherein a phosphate buffer, citrate buffer, lactate buffer, acetate buffer, or a mixture thereof is used as the buffer. 4. The method according to any one of paragraphs 1 to 3, wherein the ratio of buffer to serum, plasma or blood volume is from 1:5 to 5:1, in particular 1:1. 5 The volume ratio of diagnostic buffer to serum is 2 to 10:
1. The method according to any one of paragraphs 1 to 3, which is 1. 6. The method according to any one of paragraphs 1 to 5, wherein the concentration of heparin corresponds to the concentration of β-lipoprotein. 7. The method according to any one of paragraphs 1 to 6, wherein after separating the precipitated β-lipoprotein-heparin complex, the remaining serum or plasma is mixed with normal blood. 8. After separation of the precipitated β-lipoprotein-heparin complex, either the remaining filtrate is further analyzed for diagnostic purposes or the precipitate or a part thereof is quantified in any of paragraphs 1 to 6. Method described.