JPH0453548B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a separation membrane for selectively removing free hemoglobin contained in blood, plasma, serum, and red blood cell suspensions by filtration.

Free hemoglobin is a protein that is not found in the blood that flows through the body or is taken out of the body under normal conditions. However, in open-heart surgery using a heart-lung machine, a suction pump to collect blood that has spilled into the surgical field, other blood pumps, a bubble-type, membrane-type, or rotating disk type oxygenator, etc., are used to mechanically collect red blood cells. Hemoglobin is liberated due to injury, and significant amounts of hemoglobin are also liberated during other long general surgeries. Furthermore, the release of hemoglobin is also a problem in plasma separation methods in which blood cells and plasma are first separated using a membrane in order to remove harmful substances with large molecular weights. Also,
Even in the case of whole blood transfusion or red blood cell transfusion, free hemoglobin increases as storage time increases. On the other hand, there are known cases of various hemolytic anemias that cause intravascular hemolysis and hemoglobinemia.

In this way, when the amount of free hemoglobin generated during bloodshed or injected during bloodshed exceeds the processing capacity level of the living body, for example, the concentration is 100 mg/dl, or the production rate is 15 mg/dl/hr. appears in the urine, leading to hemoglobinuria, and is also known to lead to severe acute renal failure.

The molecular weight of hemoglobin is 64,000 (Knowledge of hemolysis, Shiro Miwa, p. 23, Chugai Igakusha, 1972)
In contrast, the molecular weight of albumin, which exists in large quantities in plasma, is 66,000 (Plasma Protein, Structure, Function, and Pathology, edited by Chisato Hirayama and Shunsuke Migita, Ishiyaku Publishing, 1972), and the molecular weight of both albumins is 66,000. are extremely close to each other and it is considered extremely difficult to separate them. However, as mentioned above, free hemoglobin is a harmful component and should be removed, whereas albumin is a major component that regulates plasma colloid osmotic pressure, which governs the flow of water from interstitial fluid into the vascular system. ,Also,
It plays an important role in transporting nutrients and waste products, and its loss must be avoided as much as possible.

The blood filtration method, which uses the pressure difference applied on both sides of a semipermeable membrane as the driving force, is the most common artificial kidney therapy when trying to remove uremic waste products, and uses the concentration difference as the driving force. It is used as an alternative therapy to hemodialysis, and it uses substances with a larger molecular weight than hemodialysis to eliminate waste products, such as so-called urinary toxic intermediate molecular weight substances (molecular weight 300 or more).
Its significance is recognized as a method that can overcome the removal of 5000). The membranes used in the blood filtration method include PAN15 (manufactured by Asahi Medical Co., Ltd.) and RP-610, which are made of polyacrylonitrile polymer.
(manufactured by Rhone-Poulenc), B1-L (manufactured by Toray Industries, Inc.) whose membrane material is polymethyl methacrylate polymer, Hemofilter (manufactured by Sartorius) whose membrane material is cellulose acetate polymer,
Hemofresh (manufactured by Daicel) is known, but the molecular weight cutoff of these membranes (when filtration is performed, 90% of the concentration in the liquid to be treated is removed, and 10% is the concentration in the filtrate) The molecular weight of a substance such as
40,000 and is virtually impermeable to plasma proteins.

On the other hand, the driving principle is the same as that of the blood filtration method, but in order to remove abnormal large molecular weight proteins such as immune complexes of antigens and antibodies and γ-globulin (molecular weight 160,000), plasma is extracted from blood. Plasma separation methods using membranes have also been used. The membrane used in the plasma separation method is Plasmflo, which is made of cellulose acetate polymer.
(manufactured by Asahi Medical Co., Ltd.), PVA filter (manufactured by Kuraray Co., Ltd.) whose membrane material is polyvinyl alcohol-based polymer, and Masmax whose membrane material is polymethyl methacrylate-based polymer.
The transmittance of γ-globulin (ratio of the concentration in the permeate to the concentration in the liquid to be treated) is 80% or more.

When trying to use these membranes as membranes to remove free hemoglobin, the former blood membranes have molecular weight fractionation characteristics that basically prevent plasma proteins from permeating. The permeability of hemoglobin is at most 10%, and there is almost no effect of removing free hemoglobin.
In addition, with the latter plasma separation membrane, the permeability of free hemoglobin is extremely high at over 90%, and a superfluid with a concentration equivalent to that of free hemoglobin in the blood can be obtained. A portion of plasma protein also permeates into the liquid side, and as it is, it is completely ineffective in the sense of selectively removing free hemoglobin, which is the objective of this patent.

The present inventors conducted extensive research into a method for selectively and easily removing free hemoglobin, which is a harmful component, and surprisingly found that free hemoglobin is equivalent to other hemoglobins that normally coexist in large amounts in blood. We have found that various plasma proteins with a molecular weight of 1000 can be separated by filtration using a membrane with specific and limited molecular weight fractionation characteristics.

That is, the present invention is a free hemoglobin separation membrane having a permeability of 65% or more for dextran having a weight average molecular weight of 40,000 and a permeability of 45% or less for dextran having a weight average molecular weight of 500,000. (These dextran permeabilities are based on flux 40
The value is measured in ml/min/ ^m2 . ) The membrane of the present invention having a specific molecular weight fractionation region and a limited range of molecular weight fraction sharpness was subjected to normal use conditions, i.e., a temperature of 10°C to 45°C, and a flux of 20ml/min.
min/m ² ~1000ml/min/m ² , free hemoglobin permeability is 50% or more, albumin permeability is 30%
Hereinafter, the ratio of hemoglobin permeability to albumin permeability will be 2.0 or more, and free hemoglobin can be effectively separated from various plasma proteins including albumin. For example, for blood 2 containing a hematocrit value (volume fraction of red blood cells) of 20%, free hemoglobin 70 mg/dl, and albumin 1.5 g/dl, a membrane with a free hemoglobin permeability of 60% and an albumin permeability of 20% is used. When 800 ml of liquid was obtained by passing through the module once, the hematocrit value was 33%, and the free hemoglobin
Blood with 92 mg/dl and albumin 2.6 g/dl was obtained.
The effect achieved is that 35% of the absolute amount of free hemoglobin is removed, while only 14% of the absolute amount of albumin is lost.

A separation membrane having a permeability of less than 65% for dextran having a weight average molecular weight of 40,000 has an extremely low permeability of free hemoglobin, making it difficult to remove the hemoglobin. On the other hand, when the permeability to dextran with a weight average molecular weight of 500,000 exceeds 45%, the free hemoglobin permeability increases, but beneficial components such as albumin also permeate to the liquid side, making it impossible to selectively remove free hemoglobin. be.

The permeability of the conventional blood membrane and plasma separation rate was measured using an aqueous solution of dextran, T40, and T500 (nominal weight average molecular weight: 40,000 and 500,000, respectively) manufactured by Pharmacia, which has a relatively narrow molecular weight distribution. So, the former blood membrane is 1% at T40.
or 45%, and T500 is less than 2%. on the other hand,
The latter plasma separation membrane has T40 of 95% to 100%,
T500 is 80% to 100%.

Therefore, selective removal of free hemoglobin is not possible with these conventional membranes.

It is particularly preferable that the separation membrane of the present invention has a permeability of 75% or more for dextran having a weight average molecular weight of 40,000. Further, the permeability of dextran having a weight average molecular weight of 500,000 is particularly preferably 35% or less.

The material for the membrane of the present invention is not particularly limited as long as it has the specific molecular weight cut-off characteristics of the present invention, but for example, cellulose acetate,
Consisting of polymethyl methacrylate, polyacrylonitrile, polyvinyl alcohol, polyethylene, polypropylene, polyethersulfone polymers, etc., and copolymer (e.g. ethylene vinyl alcohol copolymer) or mixtures of these materials. Films with these characteristics can be obtained from these materials.

To produce the separation membrane of the present invention, for example, when polymethyl methacrylate polymer is used as a material, it is dissolved in a solvent such as dimethyl sulfoxide so that the polymer concentration is 10 wt% to 20 wt%, and water is the main component. It can be obtained by introducing it into a coagulation bath, solidifying it, and removing the solvent.

Further, the form of the membrane is not particularly limited as long as it has the specific molecular weight cut-off characteristics of the present invention, and for example, either a hollow fiber membrane or a flat membrane can be used. Furthermore, this kind of membrane can be used not only to simply pass through already diluted blood as is commonly seen in open-heart surgery, but also to use the concentrated membrane to be processed later in a physiological manner. Methods such as diluting with a physiological fluid such as saline or diluting with a physiological fluid before filtration may be used, and the free hemoglobin and The amount and concentration of various plasma proteins will vary relative to the amount and concentration in the untreated fluid. Note that diluting the solution in advance and then filtering it is a preferred method because it is recognized to have the effect of stabilizing membrane performance and/or improving the separation efficiency of free hemoglobin.

Hereinafter, the effects of the present invention will be explained in more detail with reference to Examples.

Example 1 A spinning stock solution in which 15 parts of a polymethyl methacrylate polymer was dissolved in 85 parts of dimethyl sulfoxide was discharged from the outer discharge hole of a circular spinneret, and dry nitrogen gas was introduced inside to form hollow fibers. This yarn is introduced into an aqueous solution containing approximately 10% dimethyl sulfoxide at 30°C to coagulate, then washed with water and heat treated to reduce the inner diameter.
A hollow fiber with a thickness of 370μ and a film thickness of 85μ was obtained. For a module with an effective area of 0.8 m ² made by bundling these hollow fibers, molecular weight fractionation characteristics were measured using dextran manufactured by Pharmacia. Processed liquid flow rate is 200
ml/min, and the overflow rate was 32ml/min, that is, the overflow rate was 40ml/min/ ^m2 . The concentration of dextran was measured using high performance liquid chromatography (column: μ-Bondagel-E500/E1000, manufactured by Waters). T40, T500 (nominal weight average molecular weight and w/n; 40000, 1.5; 500000,
The transmittance for 1% aqueous solution of 2.7) is 79, respectively.
%, 20%.

Next, blood flow experiments were conducted on the module with the above characteristics. By adding hemoglobin to blood with a hematocrit value of 42% and a total protein content of 6.5 g/dl,
Blood containing 190 mg/dl of free hemoglobin was obtained. Hemoglobin was obtained by adding water to red blood cells and causing hemolysis.

Free hemoglobin was measured by the cyanmethemoglobin method. This blood was further diluted 2.1 times with physiological saline.

Dilute the blood using the module and adjust the blood flow rate.
Overtreatment was performed at 200 ml/min and 37°C.
A suction pump was used on the overflow side to regulate overflow.

Overflow: 54ml/min (overflow: 68ml/min/m ² )
Under the conditions, the permeability of free hemoglobin is 61% and the permeability of albumin is 16%, and the ratio of the permeability of free hemoglobin to the permeability of albumin is
It was 3.8. The albumin amount was calculated from the total protein amount by the Biuret method and the cellulose acetate membrane electrophoresis fraction.

Furthermore, when the diluted blood 2.5% was circulated using the module at a blood flow rate of 300 ml/min and the module outlet liquid was returned to the blood container, 950 ml of filtrate was obtained in 15 minutes.
The amount of free hemoglobin in the blood decreased to 67% of its original level.

Example 2 A film-forming solution prepared by dissolving 10 parts of diacetyl cellulose with a degree of acetylation of 42% in 90 parts of dimethylformamide was spread on a glass plate, penetrated into 4°C water, coagulated, and removed the solvent to form a film with a thickness of 150 μm. A cellulose acetate flat membrane was obtained. This membrane was set in Amicon's filter device model 52 with a stirring bar, and the following Example 1 was prepared.
conducted a similar experiment. 37℃, excess flux 40ml/
The transmittance of dextran T40 and T500 under the condition of min/m ² was 85% and 29%, respectively.

Next, an overexperiment was performed on the same blood used in Example 1, but before dilution with physiological saline, and the permeation of free hemoglobin and albumin was performed under conditions of overflow of 45 ml/min/ ^m2. The rate is each
61% and 27%, and the ratio of both transmittances was 2.3.

Comparative Example 1 Using polymethyl methacrylate material
Both B1-L (manufactured by Toray Industries, Inc.) and PAN-15 (manufactured by Asahi Medical Co., Ltd.) using polyacrylonitrile-based materials are hollow fiber membranes and are mainly used in blood filtration methods. conducted a similar experiment.

In B1-L, the permeability of dextran T40 and T500 was 10% and 1%, respectively, and the permeation rate of free hemoglobin was 2% in the blood permeation experiment, and no permeation of albumin was observed. PAN−15
In this case, the permeability of dextran T40 and T500 was 2% and 0%, respectively, and the permeation of free hemoglobin was 0.5% in the blood permeation experiment, and no permeation of albumin was observed. As above, B1-L
, PAN-15 were both largely ineffective in removing free hemoglobin.

Comparative Experimental Example 2 The same experiment as in Example 1 was conducted using Plasmaflo (manufactured by Asahi Medical Co., Ltd.), a hollow fiber module for plasma separation using a cellulose acetate-based material.

The transmittance of dextran T40 and T500 is
98% and 95%, and a free hemoglobin permeability of 99% was obtained in the blood filtration experiment, but the albumin permeability was also 94%, which was ineffective in the sense of separating free hemoglobin by filtration.

Claims (1)

[Claims] 1. Transmittance to dextran with a weight average molecular weight of 40,000 is 65% or more, and the weight average molecular weight is 40,000.
Free hemoglobin separation membrane with a permeability of 45% or less for dextran of 500,000.