CA1108129A - Method and apparatus for extravascular treatment of blood - Google Patents
Method and apparatus for extravascular treatment of bloodInfo
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- CA1108129A CA1108129A CA305,136A CA305136A CA1108129A CA 1108129 A CA1108129 A CA 1108129A CA 305136 A CA305136 A CA 305136A CA 1108129 A CA1108129 A CA 1108129A
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Abstract
ABSTRACT OF THE DISCLOSURE
There is provided an improved complex gel and appa-ratus for the extracorporeal treatment of blood of hyperlipemic and hypercholesteremic patients whereby a portion of blood is drawn from such patients and treated with a divalent metal complex of a sulphated polysaccharide coupled to a non-sulphated polysaccharide gel having its remaining sites blocked whereby a substantial amount of the lipoproteins present in the blood are bound to the gel and then filtering the blood before returning same to the patient.
There is provided an improved complex gel and appa-ratus for the extracorporeal treatment of blood of hyperlipemic and hypercholesteremic patients whereby a portion of blood is drawn from such patients and treated with a divalent metal complex of a sulphated polysaccharide coupled to a non-sulphated polysaccharide gel having its remaining sites blocked whereby a substantial amount of the lipoproteins present in the blood are bound to the gel and then filtering the blood before returning same to the patient.
Description
1~081Z9 The present invention relates to an improved complex gel for the extravascular purification of blood to remove a substantial amount of lipoproteins therefrom thereby rendering the blood suitable for return to the blood stream of the patient suffering from abnormally high levels of lipoproteins (lipids). The invention also relates to an apparatus for the extravascular or extracorporeal treatment of blood containing abnormally high levels of lipoproteins to substantially reduce said amount of lipoproteins to a more compatible level.
PRIOR ART
It is known that millions of individuals are affected by hyperlipemia and hypercholesteremia. These diseases are caused by excess lipids bound to proteins to form macromolecu-lar complexes called lipoproteins. Presently, treatment of the diseases has involved the use of lipotropic agents such as LUFA~ sold by Arlington Laboratories, Division, U.S.V. Inc. or antihyperlipidemic agents such as clofibrate sold under the trade mark ATROMID-S by Ayerst Laboratories. Also any chemi-cal treatment of these diseases must be accompanied by special diets which are restricted in all common sources of fat.
Though these procedures are relatively useful they do not pro-vide a satisfactory answer because they fail to provide the removal of the excess lipoproteins (lipids) present in the serum.
It is also known that methods and apparatus have been proposed for the extracorporeal treatment of blood containing certain undesirable substances, such as for example, drugs whereby the undesirable substances can be removed by fil-tration. For example, Yatzidis, H., in P~oc. Eu~op. Dial T~anspZant Ass. 1:83,1964, de~onstrated that the blood of indi-viduals, who had taken an overdose of drugs, could be subjected - 1 - -~
~ )8~Z9 to an extracorporeal filtration through activated charcoal to remove the excess drug. This method requires heparinisation of the patient, and an external system for causing the blood to flow from and to the body through capsules containing activated charcoal. Unfortunately, this system and others developed along the same lines subsequently cause certain potentially harmful effects, some of which are the removal of glucose, calcium, and certain formed elements (See Duvea et al., Trans Amer. Soc. Artif. Int. Organs. ZZ:Z~8, Z965) . Finally, it has also been found that such activated charcoal filters have a tendency to block.
Accordingly, it would appear highly desirable to pro- -vide a system where the blood of patients suffering from hyper-lipemia or hypercholesteremia could be withdrawn for treatment to reduce the lipoprotein content thereof and returned to the patient without danger and discomfort to the patient.
Preparations which bind lipoproteins found in blood have been described by Iverius in Biochem. J. 124, 677 (1971) and J. Biol. Chem. 247, 2607 (1972), and by Srinayasan et al.
in Arch. Biochem. Biophys. 170, 334 (1975).
THE INVENTION
In accordance with the present invention, there is provided an improved complex gel and an apparatus whereby blood of hyperlipemic or hypercholesteremic patients can be with-drawn, treated with said improved complex gel to reduce the lipoprotein (lipids) content thereof and returned to the patient thereby reducing the general average content of lipo-proteins in the thus treated patient.
The novel method disclosed below comprises with-drawing an amount of blood from a patient suffering from hyper-lipemia or hypercholesteremia, contacting said withdrawn amount ~1~)81Z9 of blood with an improved complex gel consisting of a divalent metallic ion complex of a sulfated polysaccharide coupled to an activated non-sulfated polysaccharide gel having its remaining active sites blocked (hereinafter referred to as 'the complex gel') whereby a substantial amount of the undesired ~- and pre-~-lipoproteins ar~ chemically bonded to the complex gel, and then filtering the thus treated blood to remove the complex gel thus obtaining blood wherein the original amount of ~- and pre-~-lipoproteins have been substantially reduced. Subsequently, the thus treated blood is returned to the patient in the usual manner thus providing a substantial reduction of the lipo-proteins (lipids) in the total blood volume of the patient. It can be easily appreciated that by repeating this method over a period of a few days to a few weeks will result in reducing the lipoprotein content of hyperlipemic or hypercholesteremic patients to a more acceptable level.
As can be appreciated by those skilled in the art, the novel method of the present invention provides many distinct advantages over the treatment of hyperlipemic or hypercholesteremic patients by the use of chemical agents. As a first advantage, there is no necessity to hospitalize the patient, the method being adapted for treatment of any patient in any out-patient clinic. Further, because of its simplicity, the method of the present invention can be carried out by tech-nicians such as nurses rather than highly specialized medical personnel such as doctors. The apparatus used for carrying out the process of the present invention is simple, inexpensive and can be readily assembled.
A further advantage of the improved complex gel of the present invention is that it selectively binds to a much higher extent than previously known preparations the undesira-`' 11~8129 ble ~- and pre-~-lipoproteins which carry cholesterol, tri-glycerides and phospholipids while the alpha lipoproteins are not significantly reduced thus allowing said alpha lipoproteins to be returned to the blood stream of the patients. The sig-nificant importance of this feature is that it is believed that the alpha lipoproteins play a significant beneficiary role in liberating deposited tissular cholesterol, thus possibly ren-dering more cholesterol available for removal in subsequent treatments, while the removal of the ~-lipoproteins which are known to carry large amount of cholesterol and pre-~-lipo-proteins which are known to carry large amount of triglycerides can only prove beneficial to any hyperlipemic or hyper-cholesteremic patient.
Finally, it should also be appreciated that other important factors normally found in blood are not affected by the improved complex gel of the present invention. Thus, the total proteins, urea nitrogen (BUN), glucose, total bilirubin, electrolytes such as sodium and potassium as well as calcium and magnesium and proteins such as albumin, ~l-globulins, ~2-globulins, ~-globulins, ~-globulins and the albumin:globulins (A/G) ratio remain substantially unchanged in the portion of the treated blood. Also, it has been noted that there is no significant change in the enzymes and hormones, red cells, white cells and platelets after treating the blood from hyper-lipemic or hypercholesteremic patients in accordance with the present invention.
The improved complex gel of the present invention is a divalent metallic ion complex of a sulfated polysaccharide coupled to an activated non-sulfated polysaccharide gel having its remaining sites blocked, and containing a much higher pro-portion of the sulfated polysaccharide than similar prepa-rations known from the Prior Art. This complex gel is preparedfrom non-sulfated polysaccharide gel through a series of chemi-cal reactions whereby there is obtained an improved complex gel which possesses the unique property of selectively binding ~-and pre-~-lipoproteins.
Generally speaking, the improved complex gel used in the process of the present invention is prepared by activating a non-sulfated polysaccharide gel, for example with a cyanogen halide, reacting the activated non-sulfated polysaccharide gel with a sulfated polysaccharide, blocking the remaining active sites with a chemical blocking agent and finally causing the product obtained to react with a divalent metallic ion thereby forming a divalent metallic ion complex of a sulfated poly-saccharide coupled to an activated non-sulfated polysaccharide gel having its remaining sites blocked.
As starting non-sulfated polysaccharide there may be used a polysaccharide or its derivative which is water-insoluble when in matrix form such as a gel. Such poly-saccharides are characterized by a molecular weight of at least 30,000. As an example of suitable polysaccharides there may be mentioned agarose, dextran, cellulose, and those derived from lactose and glucose. The critical feature of this starting polysaccharide is that it must be non-sulfated thereby leaving its highly reactive sites available for subsequent coupling by activation to sulfated polysaccharide units.
The activation of the non-sulfated polysaccharides is carried out by reaction with a cyanogen halide, for example, cyanogen bromide or cyanogen chloride, by techniques which are known in the art and such as are disclosed in British Patent 1,404,507, Pharmacia Fine Chemicals AB. The thus obtained activated non-sulfated polysaccharide is then reacted with a sulfated polysaccharide in proportions which are much higher than those described in the Prior Art, whereby the cyanogen activated sites of the starting polysaccharide will react favourably with the sulfated polysaccharide, thus leaving secondary reaction sites on the starting polysaccharide availa-` ble for blocking by treatment with a blocking agent such as ethanolamine. The thus obtained product may be stored for pro-longed periods of time. It is reacted immediately before using it for the purpose of removing certain lipoproteins from the blood of patients with a divalent metallic salt thereby forming the corresponding divalent metallic ion complex. As an example of the divalent metallic ions that can be used there may be mentioned calcium, magnesium or manganese and the like. It should be appreciated that when the term "gel" is used herein it is intended to cover the product in the form of beads, filaments or fibers.
As the starting agarose gel there may be used one which is manufactured and sold by Pharmacia Fine Chemicals AB, Uppsala, Sweden under the trade mark SEPHAROSE~ or by Bio-Rad Laboratories, Richmond, California under the trade mark BIO-GEL~. Agarose is a linear polysaccharide which consists of alternating residues of D-galactose and 3,6-anhydro-L-galactose units. This product is available in commerce as gel beads and is particularly recommended for use in gel filtration, also referred to as gel chromatography or molecular (particle) sieve chromatography, of high molecular weight proteins and poly-saccharides, nucleic acids and viruses. Agarose has at least the same good gelling properties as agar, the gelling being attributed to hydrogen bonding. Agarose gel is prepared from `~ 30 agarose according to a modification of the method described in Biochem. biophys. Acta. 79 (1964) 393-398, Hjerten.S. Various - ' 11`~812~
types of agarose gels are available on the market and the type suitable for exercising the process of the present invention is the agarose gel which has been treated with cyanogen bromide and the gel which is available as "CNBr-activated SEPHAROSE~
4B" from Pharmacia Fine Chemicals has been found to be suita-ble.
As an example of sulfated polysaccharides suitable for the purpose of the present invention there may be mentioned sodium heparin and sodium dextran sulfate, with sodium heparin being preferred.
The thus prepared improved complex gel, i.e. the divalent metallic ion complex of a sulfated polysaccharide coupled to a CNBr-activated agarose gel having its remaining active sites blocked is in the form of micro beads having a ; diameter varying between the range of from 40 to 300 microns, and possesses the ability to bind and to immobilize certain lipoproteins contained in abnormal amounts in the blood of hyperlipemic or hypercholesteremic patients, thus reducing the lipoprotein content of the blood after filtration of the in-solubilized lipoproteins.
We have now found that the gels described by Iverius cited above may be substantially improved and rendered much more active by using a significant modification of the method of preparation. For example, Iverius' paper in Biochem. J. 124 (1971) describes on p. 680 the preparation of a number of such gels using proportions by weight of sulfated polysaccharide, e.g. heparin, to CNBr-activated agarose (SEPHAROSE 4B which contains about 4% agarose according to manufacturers specifi-cations) of about 1:20 to 1:18.75, with the gels obtained con-taining from 0.65-1.29 mg of heparin per milliliter of wet gel.
Using the same procedure with either SEPHAROSE 4B or with .
" 11~18~29 BI0-GEL~A5m (which ~ontains about 6~ agarose according to manu-facturers specifications), both activated with CNBr as de-scribed above, but using much h;gher proportions by weight of sulfated polysaccharide, e.g. heparin, to CNBr-activated agarose of 1:6 to 1:2 we obtain improved complex gels contain-ing from 7 - 11 mg of heparin per gram or milliliter of wet gel which are about three to five times as active as the gels pre-pared by the method of Iverius in removing certain lipoproteins e.g. cholesterol, from the blood of patients.
The advantages of using a much more active prepa-ration in the treatment of hyperlipedemic and/or hyper-cholesterolemic patients are evident. Not only that it is possible to use less of the complex gel to remove a given pro-- portion of beta- and pre-beta-lipoproteins by withdrawing and treating an amount of blood which will be well tolerated by the patient, it is also possible to remove increased proportions of beta- and pre-beta-lipoproteins in a single treatment and thus . to reduce the frequency of treatments in a significant manner.
A further advantage of the improved complex gels prepared according to this inVention oVer the gels prepared by Iverius cited above is the fact that the former possess a much higher capacity of binding excess calcium ions than the latter.
We have demonstrated this in the experiment shown in Example 4, in which calcium chloride was added to human blood so that the resulting mixture had calcium levels which were about 20%
higher than physiological levels. When the latter mixtures were treated with gels prepared according to Iverius and with gels prepared according to this invention it was found that the gels prepared according to I-verius removed only less than a quarter of the excess calcium ions, while gels prepared accord-ing to this invention removed substantially all the excess --" liO8129 calcium ions so that the calcium levels of blood after treatment were again within the physiological range of un-treated human blood.
The ability of the improved complex gels prepared according to this invention to bind excess calcium ion is par-ticularly important because unphysiologically high calcium levels can cause severe toxic effects. ~e have found that the binding of beta- and pre-beta-lipoproteins, e.g. cholesterol, to the agarose-sulfated polysaccharide gels, e.g. agarose-heparin gels prepared according to this invention i5 particu-larly effective at concentrations of calcium ions of 0.02-0.04 M, i.e. at concentrations which are about 8-16 times greater than the physiological concentrations of about 0.0025 M
which are present in normal blood. In spite of that vast difference in the calcium concentrations present in the syringe or container in which the treatment with the gel is carried out and those present in the blood of the patient, the binding of excess calcium ions by the improved complex gels prepared according to this invention is so effective that the blood after treatment and as returned to the patient contains again no more than physiologically acceptable levels of calcium ions.
In practice a quantity of blood depending upon the weight of the patient is withdrawn, for example, from about 5 to about 500 ml and caused to react with a suitable amount of divalent metallic ion complex of a sulfated polysaccharide coupled to an activated gel non-sulfated polysaccharide in bead form dispersed in a heparin solution whereby a substantial portion of the ~-and pre-~-lipoproteins of the blood are selectively reacted with the divalent metal complex gel and thus rendered insoluble an~ after this reaction has taken place causing the blood to be filtered thereby obtaining blood ~"" 11~81~;9 wherein the amount of lipoprotein has been substantially reduced and thereafter returning the thus treated blood to the patient in the usual manner.
The amount of complex gel will usually vary between 20 and 60 grams per 100 cc of blood. The complex gel is dis-persed in a heparin solution as is usually done for blood transfusion to avoid coagulation of the blood. The time of contact of the complex gel and the blood is usually between 3 and 5 minutes though longer or shorter time can be used depend-- 10 ing on the original lipoprotein content.
The present invention is also concerned with the pro-vision of a novel apparatus for carrying out the above de-scribed method. Basically, the apparatus comprises, in combi-nation, blood recipient means containing therein a divalent metallic complex of sulfated polysaccharide coupled to an activated non-sulfated polysaccharide gel support, and filter means for separating the treated blood from the complex gel support to which the lipoproteins have been fixed. These filter means must be provided with a porosity such that passage of the complex gel support therethrough is prevented and only the treated blood may be returned to the patient.
In one form of the apparatus in accordance with the present invention, blood is withdrawn from a patient's vein by means of a catheter and drained into a transfusion bag contain-ing the complex gel support. After the fixing of the ~- and pre-~-lipoproteins to the complex gel, treated blood is infused back to the patient after having gone through a series of filtering surfaces having pores allowing passage of treated blood only.
In another form of the apparatus in accordance with the present invention, blood is first collected with a syringe 8~29 which is, then, connected to a bag containing therein the complex gel support and the filter. This bag is provided with outlet means for infusing the blood back to the patient after it has been treated in the presence of complex gel and passed through the filtering surfaces.
In a third form of the apparatus of the present invention, a syringe is provided with the complex gel and the filter therein. The syringe is thus used for collecting blood, treating it, filtering it and returning treated blood to the patient.
In order that the invention be more readily under-stood, several embodiments thereof are described below solely by way of non-limiting illustrative examples with reference to the accompanying drawings, wherein:
Figures la and lb are sketches illustrative of a first embodiment of the apparatus of the present invention, showing respectively the blood collection and the blood infusion stepsi Figures 2a and 2b are sketches illustrative of a second embodiment of the apparatus of the present invention, showing respectively the blood withdrawal and the blood infusion steps; and Figure 3 illustrates a third embodiment of the appa-ratus of the present invention.
The method of the present invention will be best understood with reference to the accompanying drawings which illustrate various techniques and apparatus for the extra-vascular treatment of blood containing abnormally high level of lipoproteins.
Referring to Figs. la and lb, there is shown a first embodiment of the apparatus of the present invention. A blood ``` 110~312~
transfusion bag 10 contains therein a divalent metallic complex of sulfated polysacharride coupled to an activated non-sulfated polysaccharide gel 12. Blood to be treated is drained from a patient's vein 14 by means of catheter means 16. As illustrated in Fig. la, blood withdrawal may be carried out by gravity. When a desired amount of blood is received in bag 10, a stopcock 18 cuts the blood flow to the bag. Stopcock 18 may also be connected to a heparinized saline bag 20 through its outlet tube 22. The inlet port 24 of bag 10 is then sealed off. Outlet port 26 of bag 10 is then connected to a filtering device 28 having an outlet tube 30 connected to the stopcock 18 and equipped with a flow controlling device 31. Prior to the infusion of treated blood back to the patient, stopcock 18 may be opened to allow the liquid inside bag 20 to drip slowly to prevent coagulation in the catheter 16, the drip being con-trolled by device 23. For blood re-infusion, bag 10 is raised higher than the patient; stopcock 18 is then operated to allow the flow between bag 10 and the patient's vein. The complex gel 12 now reacted with lipoproteins (lipids) as a result of the reaction inside bag 10 pass with the treated blood in the filtering device 28. One important feature of the present invention is the provision of a filter which will have a porosity such that the complex gel reacted with lipoproteins (lipids~ will remain in the filtering device 28 whereas the treated blood only will pass to the outlet tube 30. It is preferable to have a filter of a large surface area so that, as the lipoprotein complex gel accumulates around filter 32 at the bottom of filtering device 28, treated blood will still be able to pass through the upper portion of the filter and exit through outlet tube 30. Also, preferably, filter 32 should consist of a series of successive layers of filtering surfaces ---" 11q38~29 having pores of decreasing size with the layer having the lowest size of pores being the innermost layer.
Particularly satisfactory results have been obtained with a blood filtering device known under the trade mark INTERSEPT~ and disclosed in Belgium Patent No. 827,749 issued to Johnson & Johnson and Puralator Inc. October 9, 1975. Such filter device includes three filtering layers where the outside layer in contact with the complex gel has a pore size of 170 microns; the second and third layers have respectively 40 and 20 microns. As indicated above, the complex gel prepared from agarose beads may have a diameter varying between 40 and 300 microns. It is therefore believed that such filter device is sufficient to prevent passage of any lipoprotein-complex gel reaction product or any unreacted complex gel from reaching the outlet tube 30.
Referring to Figs. 2a and 2b, a second embodiment of the apparatus of the present ;nvention consists in connecting a syringe 50 to the stopcock 18 described above. In this embodi-ment, a second stopcock 54 is connected to the inlet tube 52 of the filtering device 28. In operation, a predetermined quantity of blood is collected from the patient's vein 14 into syringe 50. Stopcock 18 is then closed and the syringe is con-nected to stopcock 54. As illustrated in Fig. 2b, blood is then injected into the filtering device 28. However, in this case, the complex gel is already in the filtering device 28.
Blood from syringe 50 mixes with the complex gel and is ; treated. Opening stopcock 18 and operating on device 31, treated blood is returned to the patient's vein.
Referring to Fig. 3, for treating only a small volume of blood such as for newborns, a syringe type container 60 may be used wherein the complex gel 12 and a filter device 62 having the porosity characteristics described above are pro-vided. Filter 62 is located adjacent the end of container 60 which is connected to the outlet tube 64 which also serves as the inlet tube for blood collection. When treated blood is returned to the patient's vein, filter 62 blocks the complex gel and allows passage of treated blood only. It should be understood, however, that this embodiment should preferably be used for only a small quantity of blood since the complex gel reacted with lipoproteins forms a cheese-like structure which, for a large volume of blood would block the flow of treated blood back to the outlet tube 64.
Although the apparatus above has been described in relation to three specific forms of the invention, it will become apparent to those skilled in the art that it may be varied and refined in various ways. For example, there may be provided a blood recipient bag wherein the complex gel and the filter device are both present therein as a unit. In such device, the filter could be mounted adjacent the inlet of the bag whereby blood to be treated would first pass through the filter before reacting with the complex gel. Then, for blood re-infusion, the bag would merely be inverted and treated blood would again pass through the filter and return to the patient via the inlet of the bag. Other arrangements of filter-and-gel in a bag may easily be envisaged.
The present invention will be more fully understood by referring to the following Examples which are given to illustrate the invention rather than limit its scope.
A. PREPARATION OF HEPARIN AND DEXTRAN SULFATE-AGAROSE IMPROVED
COMPI.EX GEL
1. Activation of agarose beads Two types of activated agarose beads were used.
11~81Z9 CNBr-activated SEPHAROSE~ (diameter 40-190~) was purchased from Pharmacia Fine Chemicals. BIO-GEL~ A-5m (diameter 149-297~) was purchased from Bio-Rad Laboratories and then activated with cyanogen bromide (K and K Laboratories) according to the method of Axén et aZ (~ature 2Z~:Z302,Z967). 100 g of hydrated BIO-GEL~ A-5m was reacted with 20 g of cyanogen bromide at 20C
and pH 11.0 with constant stirring. The reaction is usually complete in approximately 10 minutes. Free cyanogen bromide which is hazardous was then removed by washing the gel on sintered-glass filter with 10 liters of sodium bicarbonate buffer. It is also possible to purchase CNBr-activated agarose beads from Pharmacia Fine Chemicals under the trade mark "CNBr-activated SEPHAROSE~ 4B".
PRIOR ART
It is known that millions of individuals are affected by hyperlipemia and hypercholesteremia. These diseases are caused by excess lipids bound to proteins to form macromolecu-lar complexes called lipoproteins. Presently, treatment of the diseases has involved the use of lipotropic agents such as LUFA~ sold by Arlington Laboratories, Division, U.S.V. Inc. or antihyperlipidemic agents such as clofibrate sold under the trade mark ATROMID-S by Ayerst Laboratories. Also any chemi-cal treatment of these diseases must be accompanied by special diets which are restricted in all common sources of fat.
Though these procedures are relatively useful they do not pro-vide a satisfactory answer because they fail to provide the removal of the excess lipoproteins (lipids) present in the serum.
It is also known that methods and apparatus have been proposed for the extracorporeal treatment of blood containing certain undesirable substances, such as for example, drugs whereby the undesirable substances can be removed by fil-tration. For example, Yatzidis, H., in P~oc. Eu~op. Dial T~anspZant Ass. 1:83,1964, de~onstrated that the blood of indi-viduals, who had taken an overdose of drugs, could be subjected - 1 - -~
~ )8~Z9 to an extracorporeal filtration through activated charcoal to remove the excess drug. This method requires heparinisation of the patient, and an external system for causing the blood to flow from and to the body through capsules containing activated charcoal. Unfortunately, this system and others developed along the same lines subsequently cause certain potentially harmful effects, some of which are the removal of glucose, calcium, and certain formed elements (See Duvea et al., Trans Amer. Soc. Artif. Int. Organs. ZZ:Z~8, Z965) . Finally, it has also been found that such activated charcoal filters have a tendency to block.
Accordingly, it would appear highly desirable to pro- -vide a system where the blood of patients suffering from hyper-lipemia or hypercholesteremia could be withdrawn for treatment to reduce the lipoprotein content thereof and returned to the patient without danger and discomfort to the patient.
Preparations which bind lipoproteins found in blood have been described by Iverius in Biochem. J. 124, 677 (1971) and J. Biol. Chem. 247, 2607 (1972), and by Srinayasan et al.
in Arch. Biochem. Biophys. 170, 334 (1975).
THE INVENTION
In accordance with the present invention, there is provided an improved complex gel and an apparatus whereby blood of hyperlipemic or hypercholesteremic patients can be with-drawn, treated with said improved complex gel to reduce the lipoprotein (lipids) content thereof and returned to the patient thereby reducing the general average content of lipo-proteins in the thus treated patient.
The novel method disclosed below comprises with-drawing an amount of blood from a patient suffering from hyper-lipemia or hypercholesteremia, contacting said withdrawn amount ~1~)81Z9 of blood with an improved complex gel consisting of a divalent metallic ion complex of a sulfated polysaccharide coupled to an activated non-sulfated polysaccharide gel having its remaining active sites blocked (hereinafter referred to as 'the complex gel') whereby a substantial amount of the undesired ~- and pre-~-lipoproteins ar~ chemically bonded to the complex gel, and then filtering the thus treated blood to remove the complex gel thus obtaining blood wherein the original amount of ~- and pre-~-lipoproteins have been substantially reduced. Subsequently, the thus treated blood is returned to the patient in the usual manner thus providing a substantial reduction of the lipo-proteins (lipids) in the total blood volume of the patient. It can be easily appreciated that by repeating this method over a period of a few days to a few weeks will result in reducing the lipoprotein content of hyperlipemic or hypercholesteremic patients to a more acceptable level.
As can be appreciated by those skilled in the art, the novel method of the present invention provides many distinct advantages over the treatment of hyperlipemic or hypercholesteremic patients by the use of chemical agents. As a first advantage, there is no necessity to hospitalize the patient, the method being adapted for treatment of any patient in any out-patient clinic. Further, because of its simplicity, the method of the present invention can be carried out by tech-nicians such as nurses rather than highly specialized medical personnel such as doctors. The apparatus used for carrying out the process of the present invention is simple, inexpensive and can be readily assembled.
A further advantage of the improved complex gel of the present invention is that it selectively binds to a much higher extent than previously known preparations the undesira-`' 11~8129 ble ~- and pre-~-lipoproteins which carry cholesterol, tri-glycerides and phospholipids while the alpha lipoproteins are not significantly reduced thus allowing said alpha lipoproteins to be returned to the blood stream of the patients. The sig-nificant importance of this feature is that it is believed that the alpha lipoproteins play a significant beneficiary role in liberating deposited tissular cholesterol, thus possibly ren-dering more cholesterol available for removal in subsequent treatments, while the removal of the ~-lipoproteins which are known to carry large amount of cholesterol and pre-~-lipo-proteins which are known to carry large amount of triglycerides can only prove beneficial to any hyperlipemic or hyper-cholesteremic patient.
Finally, it should also be appreciated that other important factors normally found in blood are not affected by the improved complex gel of the present invention. Thus, the total proteins, urea nitrogen (BUN), glucose, total bilirubin, electrolytes such as sodium and potassium as well as calcium and magnesium and proteins such as albumin, ~l-globulins, ~2-globulins, ~-globulins, ~-globulins and the albumin:globulins (A/G) ratio remain substantially unchanged in the portion of the treated blood. Also, it has been noted that there is no significant change in the enzymes and hormones, red cells, white cells and platelets after treating the blood from hyper-lipemic or hypercholesteremic patients in accordance with the present invention.
The improved complex gel of the present invention is a divalent metallic ion complex of a sulfated polysaccharide coupled to an activated non-sulfated polysaccharide gel having its remaining sites blocked, and containing a much higher pro-portion of the sulfated polysaccharide than similar prepa-rations known from the Prior Art. This complex gel is preparedfrom non-sulfated polysaccharide gel through a series of chemi-cal reactions whereby there is obtained an improved complex gel which possesses the unique property of selectively binding ~-and pre-~-lipoproteins.
Generally speaking, the improved complex gel used in the process of the present invention is prepared by activating a non-sulfated polysaccharide gel, for example with a cyanogen halide, reacting the activated non-sulfated polysaccharide gel with a sulfated polysaccharide, blocking the remaining active sites with a chemical blocking agent and finally causing the product obtained to react with a divalent metallic ion thereby forming a divalent metallic ion complex of a sulfated poly-saccharide coupled to an activated non-sulfated polysaccharide gel having its remaining sites blocked.
As starting non-sulfated polysaccharide there may be used a polysaccharide or its derivative which is water-insoluble when in matrix form such as a gel. Such poly-saccharides are characterized by a molecular weight of at least 30,000. As an example of suitable polysaccharides there may be mentioned agarose, dextran, cellulose, and those derived from lactose and glucose. The critical feature of this starting polysaccharide is that it must be non-sulfated thereby leaving its highly reactive sites available for subsequent coupling by activation to sulfated polysaccharide units.
The activation of the non-sulfated polysaccharides is carried out by reaction with a cyanogen halide, for example, cyanogen bromide or cyanogen chloride, by techniques which are known in the art and such as are disclosed in British Patent 1,404,507, Pharmacia Fine Chemicals AB. The thus obtained activated non-sulfated polysaccharide is then reacted with a sulfated polysaccharide in proportions which are much higher than those described in the Prior Art, whereby the cyanogen activated sites of the starting polysaccharide will react favourably with the sulfated polysaccharide, thus leaving secondary reaction sites on the starting polysaccharide availa-` ble for blocking by treatment with a blocking agent such as ethanolamine. The thus obtained product may be stored for pro-longed periods of time. It is reacted immediately before using it for the purpose of removing certain lipoproteins from the blood of patients with a divalent metallic salt thereby forming the corresponding divalent metallic ion complex. As an example of the divalent metallic ions that can be used there may be mentioned calcium, magnesium or manganese and the like. It should be appreciated that when the term "gel" is used herein it is intended to cover the product in the form of beads, filaments or fibers.
As the starting agarose gel there may be used one which is manufactured and sold by Pharmacia Fine Chemicals AB, Uppsala, Sweden under the trade mark SEPHAROSE~ or by Bio-Rad Laboratories, Richmond, California under the trade mark BIO-GEL~. Agarose is a linear polysaccharide which consists of alternating residues of D-galactose and 3,6-anhydro-L-galactose units. This product is available in commerce as gel beads and is particularly recommended for use in gel filtration, also referred to as gel chromatography or molecular (particle) sieve chromatography, of high molecular weight proteins and poly-saccharides, nucleic acids and viruses. Agarose has at least the same good gelling properties as agar, the gelling being attributed to hydrogen bonding. Agarose gel is prepared from `~ 30 agarose according to a modification of the method described in Biochem. biophys. Acta. 79 (1964) 393-398, Hjerten.S. Various - ' 11`~812~
types of agarose gels are available on the market and the type suitable for exercising the process of the present invention is the agarose gel which has been treated with cyanogen bromide and the gel which is available as "CNBr-activated SEPHAROSE~
4B" from Pharmacia Fine Chemicals has been found to be suita-ble.
As an example of sulfated polysaccharides suitable for the purpose of the present invention there may be mentioned sodium heparin and sodium dextran sulfate, with sodium heparin being preferred.
The thus prepared improved complex gel, i.e. the divalent metallic ion complex of a sulfated polysaccharide coupled to a CNBr-activated agarose gel having its remaining active sites blocked is in the form of micro beads having a ; diameter varying between the range of from 40 to 300 microns, and possesses the ability to bind and to immobilize certain lipoproteins contained in abnormal amounts in the blood of hyperlipemic or hypercholesteremic patients, thus reducing the lipoprotein content of the blood after filtration of the in-solubilized lipoproteins.
We have now found that the gels described by Iverius cited above may be substantially improved and rendered much more active by using a significant modification of the method of preparation. For example, Iverius' paper in Biochem. J. 124 (1971) describes on p. 680 the preparation of a number of such gels using proportions by weight of sulfated polysaccharide, e.g. heparin, to CNBr-activated agarose (SEPHAROSE 4B which contains about 4% agarose according to manufacturers specifi-cations) of about 1:20 to 1:18.75, with the gels obtained con-taining from 0.65-1.29 mg of heparin per milliliter of wet gel.
Using the same procedure with either SEPHAROSE 4B or with .
" 11~18~29 BI0-GEL~A5m (which ~ontains about 6~ agarose according to manu-facturers specifications), both activated with CNBr as de-scribed above, but using much h;gher proportions by weight of sulfated polysaccharide, e.g. heparin, to CNBr-activated agarose of 1:6 to 1:2 we obtain improved complex gels contain-ing from 7 - 11 mg of heparin per gram or milliliter of wet gel which are about three to five times as active as the gels pre-pared by the method of Iverius in removing certain lipoproteins e.g. cholesterol, from the blood of patients.
The advantages of using a much more active prepa-ration in the treatment of hyperlipedemic and/or hyper-cholesterolemic patients are evident. Not only that it is possible to use less of the complex gel to remove a given pro-- portion of beta- and pre-beta-lipoproteins by withdrawing and treating an amount of blood which will be well tolerated by the patient, it is also possible to remove increased proportions of beta- and pre-beta-lipoproteins in a single treatment and thus . to reduce the frequency of treatments in a significant manner.
A further advantage of the improved complex gels prepared according to this inVention oVer the gels prepared by Iverius cited above is the fact that the former possess a much higher capacity of binding excess calcium ions than the latter.
We have demonstrated this in the experiment shown in Example 4, in which calcium chloride was added to human blood so that the resulting mixture had calcium levels which were about 20%
higher than physiological levels. When the latter mixtures were treated with gels prepared according to Iverius and with gels prepared according to this invention it was found that the gels prepared according to I-verius removed only less than a quarter of the excess calcium ions, while gels prepared accord-ing to this invention removed substantially all the excess --" liO8129 calcium ions so that the calcium levels of blood after treatment were again within the physiological range of un-treated human blood.
The ability of the improved complex gels prepared according to this invention to bind excess calcium ion is par-ticularly important because unphysiologically high calcium levels can cause severe toxic effects. ~e have found that the binding of beta- and pre-beta-lipoproteins, e.g. cholesterol, to the agarose-sulfated polysaccharide gels, e.g. agarose-heparin gels prepared according to this invention i5 particu-larly effective at concentrations of calcium ions of 0.02-0.04 M, i.e. at concentrations which are about 8-16 times greater than the physiological concentrations of about 0.0025 M
which are present in normal blood. In spite of that vast difference in the calcium concentrations present in the syringe or container in which the treatment with the gel is carried out and those present in the blood of the patient, the binding of excess calcium ions by the improved complex gels prepared according to this invention is so effective that the blood after treatment and as returned to the patient contains again no more than physiologically acceptable levels of calcium ions.
In practice a quantity of blood depending upon the weight of the patient is withdrawn, for example, from about 5 to about 500 ml and caused to react with a suitable amount of divalent metallic ion complex of a sulfated polysaccharide coupled to an activated gel non-sulfated polysaccharide in bead form dispersed in a heparin solution whereby a substantial portion of the ~-and pre-~-lipoproteins of the blood are selectively reacted with the divalent metal complex gel and thus rendered insoluble an~ after this reaction has taken place causing the blood to be filtered thereby obtaining blood ~"" 11~81~;9 wherein the amount of lipoprotein has been substantially reduced and thereafter returning the thus treated blood to the patient in the usual manner.
The amount of complex gel will usually vary between 20 and 60 grams per 100 cc of blood. The complex gel is dis-persed in a heparin solution as is usually done for blood transfusion to avoid coagulation of the blood. The time of contact of the complex gel and the blood is usually between 3 and 5 minutes though longer or shorter time can be used depend-- 10 ing on the original lipoprotein content.
The present invention is also concerned with the pro-vision of a novel apparatus for carrying out the above de-scribed method. Basically, the apparatus comprises, in combi-nation, blood recipient means containing therein a divalent metallic complex of sulfated polysaccharide coupled to an activated non-sulfated polysaccharide gel support, and filter means for separating the treated blood from the complex gel support to which the lipoproteins have been fixed. These filter means must be provided with a porosity such that passage of the complex gel support therethrough is prevented and only the treated blood may be returned to the patient.
In one form of the apparatus in accordance with the present invention, blood is withdrawn from a patient's vein by means of a catheter and drained into a transfusion bag contain-ing the complex gel support. After the fixing of the ~- and pre-~-lipoproteins to the complex gel, treated blood is infused back to the patient after having gone through a series of filtering surfaces having pores allowing passage of treated blood only.
In another form of the apparatus in accordance with the present invention, blood is first collected with a syringe 8~29 which is, then, connected to a bag containing therein the complex gel support and the filter. This bag is provided with outlet means for infusing the blood back to the patient after it has been treated in the presence of complex gel and passed through the filtering surfaces.
In a third form of the apparatus of the present invention, a syringe is provided with the complex gel and the filter therein. The syringe is thus used for collecting blood, treating it, filtering it and returning treated blood to the patient.
In order that the invention be more readily under-stood, several embodiments thereof are described below solely by way of non-limiting illustrative examples with reference to the accompanying drawings, wherein:
Figures la and lb are sketches illustrative of a first embodiment of the apparatus of the present invention, showing respectively the blood collection and the blood infusion stepsi Figures 2a and 2b are sketches illustrative of a second embodiment of the apparatus of the present invention, showing respectively the blood withdrawal and the blood infusion steps; and Figure 3 illustrates a third embodiment of the appa-ratus of the present invention.
The method of the present invention will be best understood with reference to the accompanying drawings which illustrate various techniques and apparatus for the extra-vascular treatment of blood containing abnormally high level of lipoproteins.
Referring to Figs. la and lb, there is shown a first embodiment of the apparatus of the present invention. A blood ``` 110~312~
transfusion bag 10 contains therein a divalent metallic complex of sulfated polysacharride coupled to an activated non-sulfated polysaccharide gel 12. Blood to be treated is drained from a patient's vein 14 by means of catheter means 16. As illustrated in Fig. la, blood withdrawal may be carried out by gravity. When a desired amount of blood is received in bag 10, a stopcock 18 cuts the blood flow to the bag. Stopcock 18 may also be connected to a heparinized saline bag 20 through its outlet tube 22. The inlet port 24 of bag 10 is then sealed off. Outlet port 26 of bag 10 is then connected to a filtering device 28 having an outlet tube 30 connected to the stopcock 18 and equipped with a flow controlling device 31. Prior to the infusion of treated blood back to the patient, stopcock 18 may be opened to allow the liquid inside bag 20 to drip slowly to prevent coagulation in the catheter 16, the drip being con-trolled by device 23. For blood re-infusion, bag 10 is raised higher than the patient; stopcock 18 is then operated to allow the flow between bag 10 and the patient's vein. The complex gel 12 now reacted with lipoproteins (lipids) as a result of the reaction inside bag 10 pass with the treated blood in the filtering device 28. One important feature of the present invention is the provision of a filter which will have a porosity such that the complex gel reacted with lipoproteins (lipids~ will remain in the filtering device 28 whereas the treated blood only will pass to the outlet tube 30. It is preferable to have a filter of a large surface area so that, as the lipoprotein complex gel accumulates around filter 32 at the bottom of filtering device 28, treated blood will still be able to pass through the upper portion of the filter and exit through outlet tube 30. Also, preferably, filter 32 should consist of a series of successive layers of filtering surfaces ---" 11q38~29 having pores of decreasing size with the layer having the lowest size of pores being the innermost layer.
Particularly satisfactory results have been obtained with a blood filtering device known under the trade mark INTERSEPT~ and disclosed in Belgium Patent No. 827,749 issued to Johnson & Johnson and Puralator Inc. October 9, 1975. Such filter device includes three filtering layers where the outside layer in contact with the complex gel has a pore size of 170 microns; the second and third layers have respectively 40 and 20 microns. As indicated above, the complex gel prepared from agarose beads may have a diameter varying between 40 and 300 microns. It is therefore believed that such filter device is sufficient to prevent passage of any lipoprotein-complex gel reaction product or any unreacted complex gel from reaching the outlet tube 30.
Referring to Figs. 2a and 2b, a second embodiment of the apparatus of the present ;nvention consists in connecting a syringe 50 to the stopcock 18 described above. In this embodi-ment, a second stopcock 54 is connected to the inlet tube 52 of the filtering device 28. In operation, a predetermined quantity of blood is collected from the patient's vein 14 into syringe 50. Stopcock 18 is then closed and the syringe is con-nected to stopcock 54. As illustrated in Fig. 2b, blood is then injected into the filtering device 28. However, in this case, the complex gel is already in the filtering device 28.
Blood from syringe 50 mixes with the complex gel and is ; treated. Opening stopcock 18 and operating on device 31, treated blood is returned to the patient's vein.
Referring to Fig. 3, for treating only a small volume of blood such as for newborns, a syringe type container 60 may be used wherein the complex gel 12 and a filter device 62 having the porosity characteristics described above are pro-vided. Filter 62 is located adjacent the end of container 60 which is connected to the outlet tube 64 which also serves as the inlet tube for blood collection. When treated blood is returned to the patient's vein, filter 62 blocks the complex gel and allows passage of treated blood only. It should be understood, however, that this embodiment should preferably be used for only a small quantity of blood since the complex gel reacted with lipoproteins forms a cheese-like structure which, for a large volume of blood would block the flow of treated blood back to the outlet tube 64.
Although the apparatus above has been described in relation to three specific forms of the invention, it will become apparent to those skilled in the art that it may be varied and refined in various ways. For example, there may be provided a blood recipient bag wherein the complex gel and the filter device are both present therein as a unit. In such device, the filter could be mounted adjacent the inlet of the bag whereby blood to be treated would first pass through the filter before reacting with the complex gel. Then, for blood re-infusion, the bag would merely be inverted and treated blood would again pass through the filter and return to the patient via the inlet of the bag. Other arrangements of filter-and-gel in a bag may easily be envisaged.
The present invention will be more fully understood by referring to the following Examples which are given to illustrate the invention rather than limit its scope.
A. PREPARATION OF HEPARIN AND DEXTRAN SULFATE-AGAROSE IMPROVED
COMPI.EX GEL
1. Activation of agarose beads Two types of activated agarose beads were used.
11~81Z9 CNBr-activated SEPHAROSE~ (diameter 40-190~) was purchased from Pharmacia Fine Chemicals. BIO-GEL~ A-5m (diameter 149-297~) was purchased from Bio-Rad Laboratories and then activated with cyanogen bromide (K and K Laboratories) according to the method of Axén et aZ (~ature 2Z~:Z302,Z967). 100 g of hydrated BIO-GEL~ A-5m was reacted with 20 g of cyanogen bromide at 20C
and pH 11.0 with constant stirring. The reaction is usually complete in approximately 10 minutes. Free cyanogen bromide which is hazardous was then removed by washing the gel on sintered-glass filter with 10 liters of sodium bicarbonate buffer. It is also possible to purchase CNBr-activated agarose beads from Pharmacia Fine Chemicals under the trade mark "CNBr-activated SEPHAROSE~ 4B".
2. Coupling of heparin and dextran sulfate to CNBr-activated agarose beads The following sulfated polysaccharides were used:
(1) Sodium heparin obtained from K and K Laboratories and (2) Sodium dextran sulfate, (M.W. 500,000) purchased from Pharmacia Fine Chemicals. Both substances were coupled to CNBr-activated agarose beads according to the method of Iverius (Bioc~em. J., Z2~:677,Z97Z). One to two grams of each of sodium heparin or sodium dextran sulfate are respectively dissolved in bicarbon-ate buffer and stirred at 4C with 100 g of hydrated CNBr-activated agarose beads for 16 hours. The remaining active groups on the gel were blocked by stirring for 4 hours with 12 ml of ethanolamine. The gel was finally transferred to sintered-glass filter and washed consecutively with 2 litres of distilled water, 1 litre of 0.5 M sodium chloride. The gel was then transferred to another sintered-glass filter (previously autoclaved to remove pyrogens) and then washed with 10 litres of pyrogen free water. The gel was then stored in autoclaved bottles with rubber sealed-caps at 4C. This preparation was sterilized with 500 rads of gamma radiation before using for the treatment of human blood.
In a similar manner, agarose (BI0-GEL~ A5m, 500 9) was diluted with water (150 ml) in a 2-liter beaker equipped with a stirrer, a pH electrode, and a thermometer. CNBr (100 g) was added all at once and sodium hydroxide (250 ml 4N
followed by lC0 ml 3N) was added in portions with stirring so as to maintain pH 10 - 11, and ice was added as required to keep the temperature of the mixture at 18 - 20C. When the mixture did no longer consume any base it was filtered on TERYLENE~ cloth and the CNBr-activated agarose beads thus obtained were washed with successive portions of water (3.5 1), 0.5 M NaCl (3.5 1), and 0.15 M NaCl (3.5 1). The washed solids were suspended in ice-water (600 9), cooled to about 4C, com-mercial heparin sodium (Sigma Grade II, 140 u/mg, 6.81 9) was added, and the mixture was stirred at 4C over night (21.5 hrs). Ethanolamine (30 ml) was added and the mixture was ; stirred at 4C for 4 hrs, filtered on TERYLENE~ cloth, washed with 0.15 M NaCl (12 1) at 4C, and the solids were suspended in about 100 ml of 0.15 M NaCl, to obtain the product which was stable when stored in this form at 4C. The same product was also obtained when using Canada Packers USP Grade heparin sodium (170 u/mg, 6.81 9) instead of the heparin sodium obtained from Sigma used above.
Again in a similar manner, 200 ml of agarose (BI0-GEL~ A5m, 78-149 ~, Bio-Rad Labs, Richmond, Calif.) was weighed and then washed on a glass sintered funnel with dis-tilled water. The gel was then transferred to a beaker with equ~l weight of distilled water. A stirring bar, a thermometer and a pH electrode were added and the beaker was placed in a 11~81Z9 hood on a magnetic stirring table. Cyanogen bromide (Eastman, Rochester, N.Y.), 50 9, was added and the pH brought to 10.5 with 2.5 N NaOH. For the next 10 minutes, 2.5 N NaOH and small amounts of ice were added to the reaction mixture to keep the pH at 10.5 and the temperature at 18 - 22C. Then a large quantity of ice was added, the activated gel was rapidly filtered and washed with 25 volumes of ice cold 0.1 M sodium bicarbonate, and then transferred to a bottle containing 6 g of heparin in sodium bicarbonate (30 mg/ml). The bottle was rotated gently overnight at 4C. On the following day, the remaining active groups on the gel were blocked by stirring the gel for four hours with 20 ml of ethanolamine. The gel was finally washed on a coarse sintered funnel with phosphate-buffered-saline, 0.5 M NaCl and 0.15 M NaCl. The washed gel was stored at 4C in 0.15 M NaCl. The amount of heparin coupled was 7 - 11 mg per g of gel.
B. PRECIPITATION AND FILTRATION OF LIPOPROTEINS
20 grams of each of the products obtained in Step A.2 was introduced into a blood transfusion bag (Blood Pack Unit) and then 3 ml of 10% CaC12 solution (depending upon the amount of blood to be withdrawn the final concentration of CaC12 should be between 0.02 to 0.04 M) was injected into the bag to obtain the improved complex gel of this invention. 100 ml of blood was withdrawn into the bag (by vein-puncture and col-lected by gravity as shown in Fig. la). Blood was mixed with gentle agitation of the bag. Under these conditions both beta-and pre-beta-lipoproteins are selectively complexed with each complex gel.
C. RE-INFUSION OF BLOOD
The treated blood is re-infused into the patient as shown in Fig. lb. The blood transfusion bag 10 is raised ~1~8129 higher than the patient as for usual transfusion procedure and the blood is allowed to flow through filter 28 which retains the insoluble lipoproteins bound to the complex gel obtained in Step B and allows the blood to flow into the patient through the same vein puncture. The withdrawal of blood, captation of lipoproteins and re-infusion of blood takes about 10 to 20 minutes.
Human blood treated in accordance with Part B of Example 1 was submitted to various tests and the results obtained are reported in Tables I to V.
TABLE I
IN VITRO PRECIPITATION OF CHOLESTEROL IN HUMAN BLOOD OF NORMO-CHOLESTEROLEMIC PATIENT (Heparin-agarose improved complex gel as described in Example 1) Cholesterol (mg %) 206 184 Triglycerides (mg %) 246 219 Phospholipids (mg %) 174 155 Cholesterol (mg %) 216 69 Triglycerides (mg %) 255 114 Phospholipids (mg %) 260 116 It will be noted that there is a significant decrease in the blood cholesterol and triglyceride levels.
Human blood treated in the manner described in Example 1 and was submitted to biochemical and hematological tests and the results for such test are reported in Tables II, III, IV and V, with the results reported in Tables III - V
having been obtained using heparin-agarose improved complex gels as described in Example 1.
)8~29 TABLE II
IN VITRO PRECIPITATION OF CHOLESTEROL IN HUMAN BLOOD OF A
HYPERCHOLESTEROLEMIC PATIENT (Dextran Sulfate-agarose improved complex gels as described in Example 1) BEFORE AFTER
Cholesterol (mg %)339 129 Triglycerides (mg %) 172 127 Phospholipids (mg %) 240 153 TABLE III - .
ENZYMES AND HORMONES
BEFORE AFTER ~-SGOT (Serum glutamic : oxalacetic transaminase) 17 16 SGPT ~Serum glutamic pyruvic transaminase) 21 17 SLDH (Serum Lactic Dehydrogenase) 181 150 Cortisol (mcg %) 19 16 TABLE IV
BIOCHEMICAL PROFILE
BEFORE AFTER
Total proteins (9 %) 7.8 6.7 Nitrogen urea (mg %) 14.0 12.8 Glucose (mg %) 94.0 102 Total bilirubin (mg %) 0.8 0.46 :;
Electrolytes Sodium (meq/L) 138 142 Potassium (meq/L) 4.1 4.7 1 9 _ 8~29 TABLE IV (Cont'd) Protein electrophoresis Albumins 55.2 58.7 al-globulins 3.7 2.8 -globulins 9.0 8.3 ~-globulins 15.7 10.9 a-globulins 14.6 18.4 Ratio A/G 1.23 1.42 TABLE V
HEMATOLOGY
BEFORE AFTER
R.B.C's (x106) 5.41 5.46 Hb (9 %) 16.5 16.60 Hct (%) 47-3 45-4 By proceeding in the same manner as in Example 1 but with animal blood and using the heparin-agarose improved complex gel described in Example 1, the following analytical results are obtained.
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(1) Sodium heparin obtained from K and K Laboratories and (2) Sodium dextran sulfate, (M.W. 500,000) purchased from Pharmacia Fine Chemicals. Both substances were coupled to CNBr-activated agarose beads according to the method of Iverius (Bioc~em. J., Z2~:677,Z97Z). One to two grams of each of sodium heparin or sodium dextran sulfate are respectively dissolved in bicarbon-ate buffer and stirred at 4C with 100 g of hydrated CNBr-activated agarose beads for 16 hours. The remaining active groups on the gel were blocked by stirring for 4 hours with 12 ml of ethanolamine. The gel was finally transferred to sintered-glass filter and washed consecutively with 2 litres of distilled water, 1 litre of 0.5 M sodium chloride. The gel was then transferred to another sintered-glass filter (previously autoclaved to remove pyrogens) and then washed with 10 litres of pyrogen free water. The gel was then stored in autoclaved bottles with rubber sealed-caps at 4C. This preparation was sterilized with 500 rads of gamma radiation before using for the treatment of human blood.
In a similar manner, agarose (BI0-GEL~ A5m, 500 9) was diluted with water (150 ml) in a 2-liter beaker equipped with a stirrer, a pH electrode, and a thermometer. CNBr (100 g) was added all at once and sodium hydroxide (250 ml 4N
followed by lC0 ml 3N) was added in portions with stirring so as to maintain pH 10 - 11, and ice was added as required to keep the temperature of the mixture at 18 - 20C. When the mixture did no longer consume any base it was filtered on TERYLENE~ cloth and the CNBr-activated agarose beads thus obtained were washed with successive portions of water (3.5 1), 0.5 M NaCl (3.5 1), and 0.15 M NaCl (3.5 1). The washed solids were suspended in ice-water (600 9), cooled to about 4C, com-mercial heparin sodium (Sigma Grade II, 140 u/mg, 6.81 9) was added, and the mixture was stirred at 4C over night (21.5 hrs). Ethanolamine (30 ml) was added and the mixture was ; stirred at 4C for 4 hrs, filtered on TERYLENE~ cloth, washed with 0.15 M NaCl (12 1) at 4C, and the solids were suspended in about 100 ml of 0.15 M NaCl, to obtain the product which was stable when stored in this form at 4C. The same product was also obtained when using Canada Packers USP Grade heparin sodium (170 u/mg, 6.81 9) instead of the heparin sodium obtained from Sigma used above.
Again in a similar manner, 200 ml of agarose (BI0-GEL~ A5m, 78-149 ~, Bio-Rad Labs, Richmond, Calif.) was weighed and then washed on a glass sintered funnel with dis-tilled water. The gel was then transferred to a beaker with equ~l weight of distilled water. A stirring bar, a thermometer and a pH electrode were added and the beaker was placed in a 11~81Z9 hood on a magnetic stirring table. Cyanogen bromide (Eastman, Rochester, N.Y.), 50 9, was added and the pH brought to 10.5 with 2.5 N NaOH. For the next 10 minutes, 2.5 N NaOH and small amounts of ice were added to the reaction mixture to keep the pH at 10.5 and the temperature at 18 - 22C. Then a large quantity of ice was added, the activated gel was rapidly filtered and washed with 25 volumes of ice cold 0.1 M sodium bicarbonate, and then transferred to a bottle containing 6 g of heparin in sodium bicarbonate (30 mg/ml). The bottle was rotated gently overnight at 4C. On the following day, the remaining active groups on the gel were blocked by stirring the gel for four hours with 20 ml of ethanolamine. The gel was finally washed on a coarse sintered funnel with phosphate-buffered-saline, 0.5 M NaCl and 0.15 M NaCl. The washed gel was stored at 4C in 0.15 M NaCl. The amount of heparin coupled was 7 - 11 mg per g of gel.
B. PRECIPITATION AND FILTRATION OF LIPOPROTEINS
20 grams of each of the products obtained in Step A.2 was introduced into a blood transfusion bag (Blood Pack Unit) and then 3 ml of 10% CaC12 solution (depending upon the amount of blood to be withdrawn the final concentration of CaC12 should be between 0.02 to 0.04 M) was injected into the bag to obtain the improved complex gel of this invention. 100 ml of blood was withdrawn into the bag (by vein-puncture and col-lected by gravity as shown in Fig. la). Blood was mixed with gentle agitation of the bag. Under these conditions both beta-and pre-beta-lipoproteins are selectively complexed with each complex gel.
C. RE-INFUSION OF BLOOD
The treated blood is re-infused into the patient as shown in Fig. lb. The blood transfusion bag 10 is raised ~1~8129 higher than the patient as for usual transfusion procedure and the blood is allowed to flow through filter 28 which retains the insoluble lipoproteins bound to the complex gel obtained in Step B and allows the blood to flow into the patient through the same vein puncture. The withdrawal of blood, captation of lipoproteins and re-infusion of blood takes about 10 to 20 minutes.
Human blood treated in accordance with Part B of Example 1 was submitted to various tests and the results obtained are reported in Tables I to V.
TABLE I
IN VITRO PRECIPITATION OF CHOLESTEROL IN HUMAN BLOOD OF NORMO-CHOLESTEROLEMIC PATIENT (Heparin-agarose improved complex gel as described in Example 1) Cholesterol (mg %) 206 184 Triglycerides (mg %) 246 219 Phospholipids (mg %) 174 155 Cholesterol (mg %) 216 69 Triglycerides (mg %) 255 114 Phospholipids (mg %) 260 116 It will be noted that there is a significant decrease in the blood cholesterol and triglyceride levels.
Human blood treated in the manner described in Example 1 and was submitted to biochemical and hematological tests and the results for such test are reported in Tables II, III, IV and V, with the results reported in Tables III - V
having been obtained using heparin-agarose improved complex gels as described in Example 1.
)8~29 TABLE II
IN VITRO PRECIPITATION OF CHOLESTEROL IN HUMAN BLOOD OF A
HYPERCHOLESTEROLEMIC PATIENT (Dextran Sulfate-agarose improved complex gels as described in Example 1) BEFORE AFTER
Cholesterol (mg %)339 129 Triglycerides (mg %) 172 127 Phospholipids (mg %) 240 153 TABLE III - .
ENZYMES AND HORMONES
BEFORE AFTER ~-SGOT (Serum glutamic : oxalacetic transaminase) 17 16 SGPT ~Serum glutamic pyruvic transaminase) 21 17 SLDH (Serum Lactic Dehydrogenase) 181 150 Cortisol (mcg %) 19 16 TABLE IV
BIOCHEMICAL PROFILE
BEFORE AFTER
Total proteins (9 %) 7.8 6.7 Nitrogen urea (mg %) 14.0 12.8 Glucose (mg %) 94.0 102 Total bilirubin (mg %) 0.8 0.46 :;
Electrolytes Sodium (meq/L) 138 142 Potassium (meq/L) 4.1 4.7 1 9 _ 8~29 TABLE IV (Cont'd) Protein electrophoresis Albumins 55.2 58.7 al-globulins 3.7 2.8 -globulins 9.0 8.3 ~-globulins 15.7 10.9 a-globulins 14.6 18.4 Ratio A/G 1.23 1.42 TABLE V
HEMATOLOGY
BEFORE AFTER
R.B.C's (x106) 5.41 5.46 Hb (9 %) 16.5 16.60 Hct (%) 47-3 45-4 By proceeding in the same manner as in Example 1 but with animal blood and using the heparin-agarose improved complex gel described in Example 1, the following analytical results are obtained.
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o ~ . . . . d-~ t ~ ~ ~ I~ ~ 0~ Ln O O
: ~ ~ ~ r--r -. .
n a~ ~ ~ ~ c (n ~ c v ~ ~ -- -- ~ c -- E
o~n . ~ ._ ._ ~ ,_ ~
C-- C ~ ~ ~ _. . ~ _ ~ _ o o ~~ ~
E _ o O o -- o o 5: 1 1 v~ ~ ~, O_ _ _ N I I rl 5 O
., U~
+, ,~ , ~ ., o o C~ ~ , o ~ , . . .
a) ~
., a~ ~ ~ I I I I
, ., ~ .
o o ~, ~ ., C ~
- ~ o ~ o .~ ~ , , , , , z a~
o ,_ o _ C~
.~ ~ Z ~_ Cl O
~ ., Cl L~ a ~ a~
S ~ ~ ~ ~,_ Z C~
1~ C~J 4-~5 ~
O O
O r~ O O~I~ O I
aJ
E
d ~n ~, , ^
l1~ E ~ E a~ o v~
s J) E~
o ~,a s E n~ E v~ E ~ E
C ~ ~, ~ s xa.l ~ ~ r~ ' ' U~ o U~ Cl o ~ :
, V o a t~
~ ~ ~ I O
-` ~1081Z9 o ~
~ ~ . :
C~
o C~
aJ ., ~ ~ o o ~ o O ~ ~ C~l 4~ N
D ,_ .
O
., d . C~l . .
~ ~) ~ O O
4- ~ ,. .
a ~, C~
O C
C~ O
~ a~ _ _ ~ C ~ ~~ CS~ O~ O
._ ~ O ~~ .,_ .
~ ~ ~ ~~t ,~
:
, 1~1 ~ , .~ _ i-- 4-C~ ~
~: ILI
. ~
O
, C_.~ , r a ,~..
C~l O C
C:~ O
a.~ _ ~ ~ ~ O O
O r8 d' r--._ E E E
cr _, _ ~ _ Ll~ E
E ~:: ~ ~ E
" ~ ' a) , ,, ._ ~ E~--E U~ _ ~v~
~ a o ~ c o o ~ ~ ~
11~81Z9 C ~
a) ~ ~> o , ,. .,_ ~ . ~ o ~ ., C~
o C
o o ~ oo o C . ,. . o aJ ~ ~ oCO
r-O
c ~ , C~
q~ta I ~
5-- ~ a~~) r-~ ~ ~ .
C~
O r-C~ O
X C~ C ~ ~ 00 0 l_ O O ~ I
~ 4- ~ COC~lr 1~ O ~ ~ ~U'~
~ l_ : cn cc ,-Cl ~ L~
.
O
.~
C ~ 00 0 a~a . .c~, .1~ t I I ~ O
r d-I--., C~J
C~
O r~
C:~ O
al , ~ O O
O~15 O
4- 1 Ir_oooo a) ~ .--~ r : ::
. ~
O
D X
O O
X X ~
~ Ul ~ r-
3 ~ I I ~
8:1Z9 TABLE X
DOG CONTROL
before after before after Total proteins (g %) 6.1 5.5 5.3 4.9 Globulins (g %) 3.6 3.5 3.4 3.3 The other biochemical parameters are unchanged.
A solution of calcium chloride (10%) was added to a sample of human blood previously determined as having a molar concentration of plasma calcium of 0.00260 M. The resulting mixture had a plasma calcium concentration of 0.00313 M, i.e.
` the resulting increase in plasma calcium concentration was 19.4%. Samples of the above mixture were treated with heparin-agarose gel prepared according to the method of Iverius cited above, and with heparin-agarose gel prepared as described in Example 1. Plasma calcium concentrations were determined after filtration of the heparin-agarose gels, and it was found that the sample of the mixture treated with heparin-agarose gel pre-pared according to Iverius had a plasma calcium concentration of 0.00300 M, i.e. an increase in the molar concentration of plasma calcium over the untreated blood of 15.5%, while the sample of the mixture treated with heparin-agarose gel prepared as described in Example 1 had a plasma calcium concentration of 0.00263 M, corresponding to an increase in the molar concen-tration of plasma calcium of 1.9% over the untreated blood, as shown in the following Table.
, lZ9 Plasma calcium % Increase Treatment (Molar Concentration) in Molarity Blood 0.00260 Blood + CaC12 0.00313 19.4%
Blood + CaC12 + H-A (1) 0.00300 15.5%
Blood + CaC12 + H-A (2) 0.00263 1.9%
(1) Heparin-agarose gel prepared according to Iverius cited above.
(2) Heparin-agarose gel prepared as described in Example 1.
For the purpose of comparing the relative efficien-cies of gels prepared according to the method described by Iverius cited above and the improved complex gels prepared as described in Example 1 the following procedure was used.
A sample of the respective gel (1.0 9. wet weight) was equilibrated three times with tris(hydroxymethyl)amino-methane hydrochloride buffer (3 mM, pH 7.4) containing 0.15 M
NaCl, centrifuging each time at 2000 rpm for 2 minutes and decanting the supernatant. Whole blood (3 ml, i.e. 3 parts per part of gel) was added with gentle agitation to obtain uniform distribution of the gel in the mixture.
It should be noted that Iverius had stated on p. 2512 of his paper in J. Biol. Chem. 247,2607 (1972) cited above that the presence of divalent cations (e.g. calcium) was not neces-sary for the interaction between lipoproteins and the gels pre-pared by himself. For that reason we conducted one set of experiments in which the gels prepared according to Iverius' method were tested for their ability to bind beta- and pre-beta-lipoproteins in the absence of calcium ions, by adding to the mixture of blood and gel obtained above 0.15 ml of the tris(hydroxymethyl)aminomethane hydrochloride buffer described 111)81~9 above (3 mM, pH 7.4) containing O.lS M NaCl only. In another set of experiments there were added 0.15 ml of the above buffer containing 0.42 M CaC12 so as to obtain a final concentration of 0.02 M CaC12 in the gel-blood mixture.
In both sets of experiments the resulting mixture was gently agitated at room temperature for 20 minutes, centrifuged at 2000 rpm for 5 minutes, and cholesterol was determined in the supernatant plasma (i.e. after treatment) and in the plasma obtained from the sample of whole blood before treatment using 0 Liebermann-Burchardt reagent in a Technicon Autoanalyser Model II. The results are shown below, with duplicate values given in brackets.
When conducting the above experiment with a sample a ~- whole blood containing 225(228) mg/100 ml cholesterol, treatment with heparin-agarose gel prepared according to the method of Iverius cited above in the absence of calcium ions gave plasma cholesterol levels of 150(150) mg/100 ml, and in the presence of 0.02 M CaC12 gave plasma cholesterol levels of ; 125(120) mg/100 ml. On the other hand, two different lots of the improved heparin-agarose complex gel prepared according to the method described in Example 1 gave plasma cholesterol levels of 39(39) and 24(24) mg/100 ml, respectively, i.e.
plasma cholesterol levels which were 3-5 times lower than those Obtained with a gel prepared according to the method of Iverius cited above, even if the latter was used in the presence of calcium ions.
As a matter of fact, the plasma cholesterol levels obtained in the above experiments after treatment with the improved complex gels of this invention were so low that we were inclined to believe that all the beta- and pre-beta-lipo-proteins had been removed and that the cholesterol levels found after treatment could perhaps be attributed to the alpha-lipo-protein fraction which could not have been affected by the above treatment. It was therefore though that the use of a larger sample of blood and of diminished amounts of gel, e.g.
a ratio of 16 parts of blood per part of gel, would result in the removal of even greater amounts of beta- and pre-beta-lipo-proteins than shown above. The procedure described above was therefore modified by using 4 ml samples of blood and 0.25 9 of gel (wet weight) in the presence of 0.02 M CaC12, all other 10 conditions remaining unchanged. Cholesterol in the supernatant plasma was determined as above, and the portion bound to the respective gel after treatment was calculated by difference and expressed in mg/100 ml, as a percentage of the tGtal cholesterol initially present, and as milligrams bound per gram of gel. The following results show that the improved complex gels of this invention are 4-5 times more effective than the gels prepared according to the method of Iverius cited above.
Plasma Cholesterol mg/100 ml % mg bound per g Unbound Bound Bound of gel per 4 ml blood Blood before treatment 206 After treatment with H-A(l) 194 12 5.9 1.15 After treatment with H-A(2) 156 50 24.2 4.80 After treatment with H-A(3) 147 59 28.6 5.66 (1) Heparin-agarose gel prepared according to Iverius' method (2) and (3) Heparin-agarose improved complex gels prepared as described in Example 1.
8:1Z9 TABLE X
DOG CONTROL
before after before after Total proteins (g %) 6.1 5.5 5.3 4.9 Globulins (g %) 3.6 3.5 3.4 3.3 The other biochemical parameters are unchanged.
A solution of calcium chloride (10%) was added to a sample of human blood previously determined as having a molar concentration of plasma calcium of 0.00260 M. The resulting mixture had a plasma calcium concentration of 0.00313 M, i.e.
` the resulting increase in plasma calcium concentration was 19.4%. Samples of the above mixture were treated with heparin-agarose gel prepared according to the method of Iverius cited above, and with heparin-agarose gel prepared as described in Example 1. Plasma calcium concentrations were determined after filtration of the heparin-agarose gels, and it was found that the sample of the mixture treated with heparin-agarose gel pre-pared according to Iverius had a plasma calcium concentration of 0.00300 M, i.e. an increase in the molar concentration of plasma calcium over the untreated blood of 15.5%, while the sample of the mixture treated with heparin-agarose gel prepared as described in Example 1 had a plasma calcium concentration of 0.00263 M, corresponding to an increase in the molar concen-tration of plasma calcium of 1.9% over the untreated blood, as shown in the following Table.
, lZ9 Plasma calcium % Increase Treatment (Molar Concentration) in Molarity Blood 0.00260 Blood + CaC12 0.00313 19.4%
Blood + CaC12 + H-A (1) 0.00300 15.5%
Blood + CaC12 + H-A (2) 0.00263 1.9%
(1) Heparin-agarose gel prepared according to Iverius cited above.
(2) Heparin-agarose gel prepared as described in Example 1.
For the purpose of comparing the relative efficien-cies of gels prepared according to the method described by Iverius cited above and the improved complex gels prepared as described in Example 1 the following procedure was used.
A sample of the respective gel (1.0 9. wet weight) was equilibrated three times with tris(hydroxymethyl)amino-methane hydrochloride buffer (3 mM, pH 7.4) containing 0.15 M
NaCl, centrifuging each time at 2000 rpm for 2 minutes and decanting the supernatant. Whole blood (3 ml, i.e. 3 parts per part of gel) was added with gentle agitation to obtain uniform distribution of the gel in the mixture.
It should be noted that Iverius had stated on p. 2512 of his paper in J. Biol. Chem. 247,2607 (1972) cited above that the presence of divalent cations (e.g. calcium) was not neces-sary for the interaction between lipoproteins and the gels pre-pared by himself. For that reason we conducted one set of experiments in which the gels prepared according to Iverius' method were tested for their ability to bind beta- and pre-beta-lipoproteins in the absence of calcium ions, by adding to the mixture of blood and gel obtained above 0.15 ml of the tris(hydroxymethyl)aminomethane hydrochloride buffer described 111)81~9 above (3 mM, pH 7.4) containing O.lS M NaCl only. In another set of experiments there were added 0.15 ml of the above buffer containing 0.42 M CaC12 so as to obtain a final concentration of 0.02 M CaC12 in the gel-blood mixture.
In both sets of experiments the resulting mixture was gently agitated at room temperature for 20 minutes, centrifuged at 2000 rpm for 5 minutes, and cholesterol was determined in the supernatant plasma (i.e. after treatment) and in the plasma obtained from the sample of whole blood before treatment using 0 Liebermann-Burchardt reagent in a Technicon Autoanalyser Model II. The results are shown below, with duplicate values given in brackets.
When conducting the above experiment with a sample a ~- whole blood containing 225(228) mg/100 ml cholesterol, treatment with heparin-agarose gel prepared according to the method of Iverius cited above in the absence of calcium ions gave plasma cholesterol levels of 150(150) mg/100 ml, and in the presence of 0.02 M CaC12 gave plasma cholesterol levels of ; 125(120) mg/100 ml. On the other hand, two different lots of the improved heparin-agarose complex gel prepared according to the method described in Example 1 gave plasma cholesterol levels of 39(39) and 24(24) mg/100 ml, respectively, i.e.
plasma cholesterol levels which were 3-5 times lower than those Obtained with a gel prepared according to the method of Iverius cited above, even if the latter was used in the presence of calcium ions.
As a matter of fact, the plasma cholesterol levels obtained in the above experiments after treatment with the improved complex gels of this invention were so low that we were inclined to believe that all the beta- and pre-beta-lipo-proteins had been removed and that the cholesterol levels found after treatment could perhaps be attributed to the alpha-lipo-protein fraction which could not have been affected by the above treatment. It was therefore though that the use of a larger sample of blood and of diminished amounts of gel, e.g.
a ratio of 16 parts of blood per part of gel, would result in the removal of even greater amounts of beta- and pre-beta-lipo-proteins than shown above. The procedure described above was therefore modified by using 4 ml samples of blood and 0.25 9 of gel (wet weight) in the presence of 0.02 M CaC12, all other 10 conditions remaining unchanged. Cholesterol in the supernatant plasma was determined as above, and the portion bound to the respective gel after treatment was calculated by difference and expressed in mg/100 ml, as a percentage of the tGtal cholesterol initially present, and as milligrams bound per gram of gel. The following results show that the improved complex gels of this invention are 4-5 times more effective than the gels prepared according to the method of Iverius cited above.
Plasma Cholesterol mg/100 ml % mg bound per g Unbound Bound Bound of gel per 4 ml blood Blood before treatment 206 After treatment with H-A(l) 194 12 5.9 1.15 After treatment with H-A(2) 156 50 24.2 4.80 After treatment with H-A(3) 147 59 28.6 5.66 (1) Heparin-agarose gel prepared according to Iverius' method (2) and (3) Heparin-agarose improved complex gels prepared as described in Example 1.
Claims (6)
1. A process for preparing an improved complex gel for effecting a reduction of the levels of beta- and pre-beta-lipoproteins in the blood of patients suffering from hyper-lipemia and/or hypercholesterolemia by extravascular treatment of said blood with said improved complex gel, which comprises activating a non-sulfated polysaccharide gel by treatment with a cyanogen halide selected from cyanogen chloride and cyanogen bromide, treating said activated non-sulfated polysaccharide gel with a sulfated polysaccharide in proportions by weight ranging from 1 part of sulfated polysaccharide to 2-6 parts of activated non-sulfated polysaccharide gel, blocking the remain-ing active sites by treatment with a blocking agent, treating the resulting product with a divalent metallic ion, and isolating said improved complex gel.
2. A process as claimed in Claim 1 in which the non-sulfated polysaccharide is agarose, the cyanogen halide is cyanogen bromide, the sulfated polysaccharide is dextran sulfate, the blocking agent is ethanolamine, and the divalent metallic ion is calcium.
3. A process as claimed in Claim 1 in which the non-sulfated polysaccharide is agarose, the cyanogen halide is cyanogen bromide, the sulfated polysaccharide is heparin, the blocking agent is ethanolamine, and the divalent metallic ion is calcium.
4. An improved complex gel consisting of a divalent metallic ion complex of a non-sulfated polysaccharide activated with a cyanogen halide selected from cyanogen chloride and cyanogen bromide and coupled to a sulfated polysaccharide in proportions by weight ranging from 1 part of sulfated poly-saccharide to 2-6 parts of activated non-sulfated poly-saccharide, in which the remaining active sites are blocked with a chemical blocking agent, when prepared by the process defined in Claim 1 or by an obvious chemical equivalent.
5. An improved complex gel consisting of the calcium complex of agarose activated with cyanogen bromide and coupled to dextran sulfate, in which the remaining active sites are blocked with ethanolamine, when prepared by the process defined in Claim 2 or by an obvious chemical equiva-lent.
6. An improved complex gel consisting of the calcium complex of agarose activated with cyanogen bromide and coupled to heparin, in which the remaining active sites are blocked with ethanolamine, when prepared by the process defined in Claim 3 or by an obvious chemical equivalent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA305,136A CA1108129A (en) | 1978-06-09 | 1978-06-09 | Method and apparatus for extravascular treatment of blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA305,136A CA1108129A (en) | 1978-06-09 | 1978-06-09 | Method and apparatus for extravascular treatment of blood |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1108129A true CA1108129A (en) | 1981-09-01 |
Family
ID=4111662
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA305,136A Expired CA1108129A (en) | 1978-06-09 | 1978-06-09 | Method and apparatus for extravascular treatment of blood |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1108129A (en) |
-
1978
- 1978-06-09 CA CA305,136A patent/CA1108129A/en not_active Expired
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