Note: Descriptions are shown in the official language in which they were submitted.
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-
8129
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
-- 5
8129
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
-- 6
- ' 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
-- 1 0
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
1 1
``` 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
- 13 -
29
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.
EXAMPLE 1
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.
- 14 -
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
81Z9
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
- 16 -
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.
EXAMPLE 2
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)
PATIENT 1 BEFORE AFTER
Cholesterol (mg %) 206 184
Triglycerides (mg %) 246 219
Phospholipids (mg %) 174 155
PATIENT 2
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.
- 18 -
)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
EXAMPLE 3
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.
- 20 -
11~J8129
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- 23 -
11~81Z9
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- 24 -
8:1Z9
TABLE X
DOG CONTROL
DOG C-111 DOG C-222
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.
EXAMPLE 4
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.
,
- 25 -
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.
EXAMPLE 5
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
- 27 -
129
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.
- 28 -
