 |
Description  |
|
|
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to standards useful in assaying for
cholesterol concentrations, and to the preparation thereof.
2. Description of the Prior Art
The use of serum standards or references in biochemistry analysis is well
known. It is frequently advantageous, for example, to determine the level
of certain constituents in the blood of a patient as a diagnostic aid.
Serum standards are used in analytic procedures to provide a reference to
which the patient's serum may be compared. Serum standards therefore may
be required to have varying concentrations to certain components. For
convenience, serum standards are commonly stored as a dry powder after
lyophilization to be reconstituted at the time of use, or they are frozen
and subsequently thawed for use. It is desirable that the serum standard
be stable and have substantial optical clarity to minimize interference
with the analytical measurement of serum constituents.
The prior art is believed to be best set forth in U.S. Pat. No. 3,853,465,
issued to Rush et al. on Dec. 10, 1974, which is hereby incorporated by
reference. This patent generally sets forth the facts of difficulties in
photometric analysis which result from turbidity in serum and plasma
samples. This is believed to be due primarily to the presence in the serum
standard of certain serum proteins, particularly the low-density,
triglyceride-rich lipoproteins. The problem of turbidity correspondingly
increases when a serum standard is required to have increased triglyceride
or cholesterol levels due to the consequent increase in the concentration
of the turbiditycausing components.
The solution to the turbidity problem which Rush et al. set forth has
generally been to add a surfactant to the serum or plasma to reduce the
turbidity. Rush et al. disclose a surfactant of polyoxyethylated lauric
acid having from 9 to 20 ethoxy groups. Compounds of this general class
which have from 10 to about 20 ethoxy groups are disclosed in U.S. Pat.
No. 3,260,648, issued to Fox on July 12, 1966, for use as an emulsifier
for cholesterol in serum. This latter patent sets forth the use of a lower
alkylphenoxypolyethoxyethanol having from about 10 to 20 ethoxy groups.
While the use of such surfactants does result in a reduction in the
turbidity of the serum, the products produced thereby tend to be
metastable and separate after a period of time. Additionally, large
amounts of surfactant are generally required and the use of such large
amounts of surfactant may interfere with biological assays of serum
ingredients. Moreover, even with the surfactants of this type,
reconstitution of lyophilized serum having elevated cholesterol levels
produces significant, measureable turbidity notwithstanding the use of the
surfactant.
Aside from the problem of turbidity, it is recognized, as mentioned
previously, that it is sometimes desirable to have a serum standard which
has increased levels of triglycerides or cholesterol. In U.S. Pat. No.
3,764,556, issued to Kuchmak et al. on Oct. 9, 1973, there is disclosed a
procedure for obtaining a cholesterol-rich protein fraction from outdated
human plasma. The use of this protein fraction, however, entails several
disadvantages. First, there are certain dangers involved in that the
procedure by which the cholesterol-rich fraction is obtained will also
result in collection and concentration of any hepatitis virus which may be
present in the plasma. Additionally, it has found that substantial serum
turbidity will result upon lyophilization and reconstitution of such a
prepared cholesterol standard. Also, a process which utilizes human blood
as a source of cholesterol to be added in preparing a serum standard will
generally be expensive.
In an article entitled "Steroid-Protein Conjugates," by Erlanger et al.,
Journal of Biological Chemistry, Volume 228, September, 1957, there is
disclosed the preparation of water soluble conjugates of bovine serum
albumin with testosterone and with cortisone. The steroids are linked by
amide bonds to the lysine residues of the albumin. The conjugates
disclosed in the Erlanger et al. article were proposed as antigenic
compounds which could elicit antibodies having antihormonal properties.
These conjugates were proposed to be useful in enabling the development
and study of antihormonal principles.
SUMMARY OF THE INVENTION
One embodiment of the present invention is a compound useful in assay
procedures for measuring serum cholesterol which comprises an ester of
cholesterol having the formula
R.sub.1 --R.sub.2 --[Cholesterol Base]
in which R.sub.1 is a water soluble, nonionic surfactant, and R.sub.2 is a
dicarboxyl group bonded by ester linkages to R.sub.1 and to the
Cholesterol Base, R.sub.1 and R.sub.2 together having a molecular weight
of from about 200 to about 3000. In an alternate embodiment the compound
comprises a pregnenolone derivative having the general formula R.sub.3
--[Pregnenolone Base]--R.sub.4, R.sub.3 being a hydrogen or a carboxyl
group having from one to about 17 carbons, R.sub.4 being selected from the
group consisting of
##STR1##
R.sub.5 being an alkane having from about 1 to about 8 carbons and R.sub.6
being chloride or serum albumin. In other embodiments of the present
invention, a compound of the described type is combined with serum or is
used to reconstitute lyophilized serum to provide a standard or reference
material useful in cholesteral assay procedures.
It is an object of the present invention to provide a stable and easily
prepared compound useful in assay procedures for measuring serum
cholesterol.
Another object of the present invention is to provide a method for
producing the above-described compound.
It is a further object of the present invention to provide a compound of
the described type which does not display turbidity upon lyophilization
and reconstitution.
Another object of the present invention is to provide a substantially human
serum which measures in assay procedures as having normal or elevated
cholesterol levels, and which does not display significant turbidity upon
lyophilization and reconstitution.
It is a further object of the present invention to provide a method for
preparing a substantially human serum which is useful as a standard or
reference material and which fulfills the above-described objectives.
Another object of the present invention is to provide a compound which
combined with serum produces a standard or reference material useful in
cholesterol assay procedures.
It is another object of the present invention to provide a compound useful
as a diluent for lyophilized serum to produce a cholesterol standard.
Further objects and advantages of the present invention will be apparent
from the description of the preferred embodiments which follows.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Standard or reference materials are generally required in the performance
of assay procedures directed toward determining the levels of cholesterol
in serum or plasma. In order to retain the reference material for use over
a period of time it is desirable to store the material either in the
frozen or lyophilized state. The material is subsequently thawed or
reconstituted with aqueous media for use. As previously noted, however,
standard or reference materials heretofore have displayed significant
turbidity, especially upon thawing or reconstitution. This turbidity
interferes with the use of standard colorimimetric or turbidimetric
measuring devices. It is therefore highly desirable to provide a standard
or reference material which may be stored in the frozen or lyophilized
state, but which does not display turbidity when thawed or reconstituted
for use.
Human serum or plasma contains four major classes of lipoproteins.
Lipoproteins are a complex of a protein with a lipid, and the four major
classes are chylomicrons, very low density lipoproteins (pre-beta
lipoproteins), low density lipoproteins (beta lipoproteins) and high
density lipoproteins (alpha lipoproteins). The actual proportions of the
various classes of lipoproteins present in a serum or plasma sample vary
with respect to certain physical and chemical parameters. However, the
alpha lipoproteins generally comprise between 20% and 40% of the
lipoprotein content of human serum or plasma. Most of the serum
cholesterol is associated with the beta lipoproteins.
The problem associated with the lipoproteins is that their presence in
serum tends to result in turbidity in the serum, particularly upon thawing
or reconstitution of a stored standard or reference material. It is
believed that the turbidity is a result particularly of the presence of
the lower density lipoproteins. As a result, standards or reference
materials used in assay procedures for measuring serum cholesterol
generally become turbid because of the presence of the lipoproteins.
Various techniques have been employed to attempt to reduce the turbidity
of the thawed or reconstituted serum standards. The present invention
provides a new and useful compound which may be applied in various manners
to produce a standard or reference material which is useful in assay
procedures for measuring serum cholesterol. The compound of the present
invention further does not become turbid upon freezing and thawing, or
upon lyophilization and reconstitution with an aqueous media.
In accordance with one embodiment of the present invention, a compound is
provided which is useful in assay procedures for measuring serum
cholesterol and which comprises an ester of cholesterol having the general
formula:
##STR2##
in which R is a water soluble, nonionic surfactant having a molecular
weight of from about 200 to about 3000. The attachment to the Cholesterol
Base is at the 3 beta position of the Cholesterol Base. More preferably, R
has a molecular weight of from about 300 to about 1000, and contains
between about 4 and about 20 carbon atoms. R should be relatively inert,
preferably containing several ethoxy groups, and more specifically
comprising polyethoxyethanyl. The term "Cholesterol Base" is defined as
the entire portion of the cholesterol molecule except for the hydroxyl
group normally attached at the 3 beta position, the term "Cholesterol
Base" thus being equivalent to the material known by conventional steroid
nomenclature as cholest-5-en.
In a related embodiment of the present invention, the compound is
represented by the general formula:
R.sub.1 --R.sub.2 --[Cholesterol Base]
in which R.sub.1 is a water soluble, nonionic surfactant, and R.sub.2 is a
dicarboxyl group bonded by ester linkages to R.sub.1 and to the
Cholesterol Base. The R.sub.2 group is attached at the 3 beta position of
the Cholesterol Base. R.sub.1 and R.sub.2 together shouuld have a
molecular weight of from about 200 to about 3000, and more preferably have
a combined molecular weight of from about 300 to about 1000. R.sub.1 and
R.sub.2 preferably have a total of between about 5 to about 21 carbon
atoms, with R.sub.2 preferably having between about 3 and about 8 carbon
atoms. R.sub.1 should be relatively inert or non-reactive to be useful
with common assay procedures, and R.sub.1 preferably includes several
ethoxy groups. A particular compound of the described class which has been
found to be well suited for use in conjunction with cholesterol assay
procedures is one in which R.sub.1 comprises polyethoxyethanyl and R.sub.2
is an adipyl group.
Compounds of the above-described classes may be readily produced in
accordance with known, chemical synthesis techniques. In the preferred
method for forming the above-described compounds, cholesterol is reacted
with a dicarboxyl compound to bond the cholesterol to one of the carboxyl
groups and to thereby form a cholesterol ester. The ester is then coupled
to a nonionic surfactant, such as polyethoxyethanol, to form the final
compound. The dicarboxyl compound preferably has betwen about 3 and about
8 carbon atoms, and the surfactant preferably has a molecular weight of
from about 300 to about 1000. It has been found that a particularly
preferred embodiment of the present invention is produced by bonding the
cholesterol to an adipyl compound, which is in turn bonded to a surfactant
containing several ethoxy groups and having from about 4 to about 20
carbon atoms.
The classes of compounds of the present invention are applied in various
manners to provide a standard or reference material useful in cholesterol
assay procedures. An aqueous solution of the water-soluble, cholesterol
compound of the present invention is used as a standard or reference
material. The cholesterol compound of the present invention is utilized to
prepare an aqueous solution which will measure as having cholesterol in
the concentration of a preselected value, such as that corresponding to a
normal or elevated level of cholesterol in human serum or plasma. The
water-soluble, cholesterol compounds of the present invention are also
combined with human or other serums to provide a standard or reference
material. The serums with which the cholesterol compounds are combined
include, for example, serum containing essentially all normal human serum
components; serum having essentially all of its normally present pre-beta
lipoproteins, beta lipoproteins, and chylomicrons removed; and, serum
having normally present lipoproteins and chylomicrons removed. Removal of
the lower density lipoproteins from the serum with which the cholesterol
compounds are combined is advantageous in that it further reduces the
possibility of turbidity upon freezing and thawing, or upon lyophilization
and reconstitution with aqueous media. Removal of all of the lipoproteins
is advantageous in that the selective removal of only the lower density
lipoproteins is not involved. In a further application of the present
invention, aqueous solutions of the cholesterol compounds are combined
with lyophilized serum which may, for example, be one of the three types
of serum previously described. Any of the standards or reference materials
of the present invention may be frozen or lyophilized for storage, and
upon thawing or reconstitution with aqueous media, do not display
significant turbidity.
The cholesterol compounds of the present invention in certain embodiments
are combined with blood plasma or serum, as previously described. These
embodiments utilize blood plasma, which is defined as the liquid part of
the blood containing fibrinogen. Normal human plasma is obtained from
pooled blood. The pooled blood includes approximately equal volumes of the
liquid portions of whole blood from not less than 8 adult humans.
Outdated, citrated whole blood, which is old whole blood to which citrate,
phosphate, and dextrose have been added, is preferable in view of its low
cost. The normal human serum is derived from the pooled blood plasma. The
serum is the clear, amber, alkaline fluid of the blood from which cellular
elements have been removed by clotting. The serum contains the salts,
soluble protein and lipoproteins. The lipoproteins are rich in
triglycerides and cholesterol.
As outlined previously, in one embodiment of the invention a cholesterol
compound of the described classes is added to the human serum. The
preferred compound is polyethoxyethanylcholesteryl-adipate, although other
compounds of the described classes may similarly be used. In another
embodiment of the invention, the human serum is processed to remove
certain constituents prior to the addition of the cholesterol compound.
Preferably, these constituents which have been found to contribute to
turbidity of the reconstituted serum are removed, as is disclosed in U.S.
Pat. No. 3,955,925, issued to Proksch and Bonderman on May 11, 1976, the
relevant parts of which are hereby incorporated by reference. The
cholesterol compound, such as the polyethoxyethanyl-cholesteryl-adipate,
is added to achieve the desired cholesterol-measuring concentration. The
resultant serum standard is stable and displays good optical clarity upon
reconstitution from the lyophilized state.
Another aspect of this invention involves the addition of the cholesterol
compound to animal serum, such as horse or bovine serum, to obtain desired
cholesterol concentrations. The resulting serum is stable in lyophilized
form and readily reconstitutes to an optically clear serum.
In still another embodiment of the present invention, the cholesterol
compounds are added as diluents to lyophilized human or animal serum to
produce an optically clear and stable standard. The lyophilized human or
animal serum have normal or reduced levels of lipids, triglyceride, or
cholesterol. The cholesterol compounds are added to the lyophilized serum
as aqueous solutions. The concentration of the lipids, triglyceride and
cholesterol in the resulting serum standards or references are controlled
by the concentration of the cholesterol compound in the aqueous diluent,
and by the amount of diluent added to the lyophilized serum.
In an alternate embodiment the compound is represented by the general
formula:
R.sub.3 --[Pregnenolone Base]--R.sub.4
R.sub.3 is attached at the 3 beta position of the Pregnenolone Base and is
a hydroxyl group or an ester group preferably having from about one to
about 17 carbon atoms. R.sub.4 is attached at the 17 position of the
Pregnenolone Base and is selected from the group consisting of
##STR3##
R.sub.5 is an alkane preferably having from about one to about 8 carbon
atoms. R.sub.6 is either a chloride atom or is serum albumin, such as
bovine serum albumin for example, attached by amide bonds with its lysine
residues. The term "Pregnenolone Base" is defined as the entire portion of
the pregnenolone molecule except for the hydroxyl group normally attached
at the 3 beta position and also except for the normally present 20 methyl
keto group, the term "Pregnenolone Base" thus being equivalent to the
material known under conventional steroid nomenclature as
.DELTA.5-androsten. The above described pregnenolone derivatives are
useful in the same fashion as the previously described cholesterol
derivatives.
Production of the above-described pregnenolone derivatives is readily
accomplished by known chemical synthesis techniques. In one procedure a
carboxylic acid is attached to the 3 beta position of the pregnenolone to
block that position from interfering with the intended reactions in
subsequent steps. The resulting product is then combined with sodium
borohydride to provide a hydroxyl group at the 20 keto position which is
subsequently reacted with a diacid chloride or with a dicarboxylic acid
and isobutyl chlorocarbonate to yield the desired product. This end
product may further be combined with serum albumin to form an alternate
compound in accordance with the present invention.
In another procedure the pregnenolone is reacted, for example with a
carboxylic acid, to provide an ester at the 3 beta position. The resulting
product is reacted with carboxymethoxylamine hemihydrochloride to produce
the desired final product. This final product may additionally be combined
with bovine serum albumin to form another form of compound in accordance
with the present invention.
In a second alternate embodiment, the compound of the present invention
comprises a cholesterol derivative having the general formula R.sub.7
--R.sub.8 --[Cholesterol Base] in which R.sub.7 is serum albumin and
R.sub.8 is a dicarboxyl group. R.sub.8 is bonded by an ester linkage to
the Cholesterol Base at the 3 beta position, and is attached to R.sub.7 by
amide bonds with the lysine residues of the serum albumin. R.sub.8
preferably has between about 1 and about 20 carbon atoms and more
preferably between about 3 and about 8 carbon atoms. This second alternate
embodiment is useful in like fashion as the previously described
embodiments.
The following examples more fully illustrate the preferred embodiments of
the present invention.
EXAMPLE 1
A cholesterol ester of the class constituting the preferred embodiment of
the present invention was prepared by the following procedure. To 150 ml
of dry toluene was added 11.6 [0.03 moles] of cholesterol, and 7.6 g [0.04
moles] of adipyl dichloride was added thereto. The mixture was incubated
at 35.degree. C. under a 5 mm of mercury vacuum for 60 minutes to form
cholesteryl-adipate.
A solution of polyethylene glycol, having an average molecular weight of
600, was separately prepared by adding 36 g [0.06 moles] of carbowax to 75
ml of toluene. The particular carbowax solution utilized is available
under the trademark PEG-600. The polyethylene glycol preparation was then
added to the cholesteryl-adipate mixture.
The resultant mixture was stirred for 45 minutes and then extracted three
times with an equal volume of water at 80.degree. C. to remove the
water-soluble impurities. The aqueous extracts were discarded. The toluene
was then removed by evaporation at 80.degree. C. The resultant material
was cooled to 70.degree. C. and was then suspended in 100 ml of methanol.
After stirring for 60 minutes, the methanol-insoluble residue was removed
by filtration, and the methanol was subsequently removed by evaporation.
The resultant viscous liquid was then twice extracted with 100 ml of
petroleum ether having a boiling point range of 30.degree.-60.degree. C.,
and the viscous liquid was placed in a vacuum oven at
50.degree.-60.degree. C. until the final traces of solvent were removed.
About 10.5 g of polyethoxyethanyl-cholesteryl-adipate (PCA) were obtained.
The viscous PCA normally contained about 300 mg of cholesterol per gram.
Analysis of the PCA by chromatographic techniques using acetone as the
solvent indicated that the preparation consisted of a series of
cholesterol-containing molecules having a mobility less than cholesterol.
Although the preparation did not represent a single unimolecular species,
but a series of homologous derivatives, the PCA was found to be useful
without further treatment as a water-soluble, cholesterol additive for
preparing standard and reference materials.
Aqueous solutions of the PCA prepared by the above method were prepared to
provide assay standards of various cholesterol concentrations and were
measured for their cholesterol activity, which as previously mentioned was
found to correspond to about 300 mg of cholesterol per gram. Various
amounts of water were then added to samples of the PCA preparation to
produce solutions with the various levels of cholesterol. These standards
were found to measure accurately in cholesterol assay procedures, and
remained stable for 6 months at 5.degree. C. The cholesterol standards
produced in this manner, when stored either in the frozen or lyophilized
condition for extended periods of time, did not show a significant effect
on the measured value of the cholesterol in the standard.
EXAMPLE 2
A variety of cholesterol esters were prepared by the method of Example 1,
except that different dicarboxyl groups were used. Dicarboxyl groups
containing from 4 to 7 carbon atoms were used to produce the succinate,
glutarate, adipate and pimelate forms of the polyethoxyethanyl-cholesteryl
compounds. Each of the compounds measured accurately in cholesterol assay
procedures, although the succinate form did not measure as accurately as
the others. These preparations also remained stable upon storage for 6
months at 5.degree. C.
EXAMPLE 3
Cholesterol esters were prepared in accordance with the methods of Examples
1 and 2, with the exception that the following surfactants were used:
polyethoxyethanyl having average molecular weights of 80, 150, 850 and
2800, and polyoxypropylene having an average molecular weight of 500. The
prepared compounds were found to measure well in cholesterol assay
procedures and remained stable upon storage for extended periods of time.
For any preparation of a cholesterol ester in accordance with the procedure
of Examples 1-3 and others it was found that the cholesterol activity of
the ester should be assayed to standardize the compound for use. Water is
then added to produce a solution of the ester having the desired level of
cholesterol. Alternatively, the ester was stored in the concentrated form,
and was subsequently diluted for use.
EXAMPLE 4
A normal 1000 ml pooled human blood plasma sample was obtained and
converted to serum by clotting. Cholesterol esters prepared in accordance
with the methods of Examples 1-3 were added to samples of the serum to
bring the cholesterol content to desired levels. The resultant,
substantially human serums provided excellent serum standards for elevated
cholesterol levels and displayed excellent optical clarity. Portions of
the prepared serums were stored for six months by freezing and by
lyophilizing, and upon thawing or reconstitution with water, respectively,
were optically clear.
EXAMPLE 5
The procedures of Example 4 were followed exactly except that the human
serum was first processed to remove the beta lipoproteins, pre-beta
lipoproteins and chylomicrons. The processing of the human serum was
performed by the method disclosed in U.S. Pat. No. 3,955,925, issued to
Proksch and Bonderman on May 11, 1976, the pertinent portions of which are
hereby incorporated by reference. The resultant, substantially human
serums provided excellent serum standards which measured accurately for
normal and elevated levels of cholesterol and which displayed excellent
optical clarity. The serum preparations remained stable upon storing for 6
months in the lyophilized or frozen state, and were optically clear upon
use.
EXAMPLE 6
The procedures of Example 4 were followed exactly except that the human
serum was first processed to remove the normally present alpha
lipoproteins, beta lipoprotens, pre-beta lipoproteins and chylomicrons.
The processing of the human serum was performed by the technique of Jonas
as described in the Journal of Biological Chemistry, Volume 247, pages
7767 et seq. Briefly, the serum was allowed to clot at room temperature
and was centrifuged at 60,000 rpm for about 24 hours. A first fraction of
primarily low density lipoproteins and chylomicrons was then removed and
discarded. The remaining solution was centrifuged under the same
conditions and a second fraction comprising primarily high density
lipoproteins was drawn off the top and discarded. The cholesterol esters
were then added in aqueous solution to portions of the processed human
serum as previously described.
The resultant substantially human serums provided excellent serum standards
which measured accurately for normal and elevated levels of cholesterol.
The serum standards displayed excellent optical clarity both originally
and after storage for 6 months in either the lyophilized or frozen state.
EXAMPLE 7
The procedures of Examples 5 and 6 were followed exactly except that the
human serum was processed by the method of Oncley et al. as disclosed in
the Journal of the American Chemical Society, Volume 79, pages 4666 et
seq. Briefly, a solution of dextran sulfate solution was added with
stirring to the serum. A lipoprotein-dextran sulfate precipitate
containing the lipoproteins was allowed to form and was separated by
centrifugation. Portions of the processed serum were then diluted with
aqueous solutions of the cholesterol esters to provide standards with the
desired levels of cholesterol. The resulting substantially human serums
provided cholesterol serum standards which measured accurately and which
displayed excellent optical clarity initially and upon storage in the
lyophilized or frozen state.
EXAMPLE 8
Substantially human serum standards were prepared by procedures similar to
those in Examples 5-7, except that the cholesterol esters were added as
diluents to lyophilized forms of the processed human serums. The human
serum was processed in accordance with the procedures detailed in Examples
5-7. The processed serums were then lyophilized. Diluents comprising
aqueous solutions of desired concentrations of the cholesterol esters were
then added in desired amounts to portions of the lyophilized, processed
human serums. The resultant, substantially human serums displayed
excellent optical clarity and measured well as cholesterol standards, even
when the human serum was stored in the lyophilized form for 6 months
before dilution with the cholesterol ester solutions.
EXAMPLE 9
The procedures of Examples 5-8 were followed exactly except that bovine and
horse serums were used. Again, excellent serum standards were obtained
which were stable, measured accurately in cholesterol assay procedures,
and displayed excellent optical clarity initially and upon storage.
EXAMPLE 10
A solution was prepared by combining 1.08 g of pregnenolone and 1.07 g of
carboxymethoxylamine hemihydrochloride in 200 ml of ethanol. This solution
was made alkaline by adding 20 ml of 5% NaOH, and the resulting solution
was refluxed for one and a half hours. The refluxed solution was diluted
with water and extracted with ether. The aqueous phase was acidified with
hydrochloric acid and the resulting precipitate was extracted with ether.
The ether extract was washed with water and dried. The resultant product
comprised pregnenolone 20-carboxymethoxyloxime and was soluble in a basic
aqueous solution. The product measured as cholesterol in standard assay
procedures and was not turbid upon lyophilization and reconstitution with
aqueous media.
EXAMPLE 11
A solution was prepared by combining 1.2 g of pregnenolone
20-carboxymethoxyloxime prepared in accordance with Example 10 and 0.80 ml
of tri-n-butylamine in 30 ml of dioxane and the solution was cooled to
10.degree. C. Added to the cooled solution was 0.40 ml of isobutyl
chlorocarbonate and the resulting reaction was allowed to proceed at
4.degree. C. for 20 minutes. The mixture was added to a stirred, cooled
solution of 4.2 g of bovine serum albumin in 220 ml of an equal mixture of
water and dioxane and 4.2 ml 1 N NaOH. An additional 2.0 ml of 1 N NaOH
was added after one hour and the mixture was continued to be stirred and
cooled for an additional three hours.
The solution was dialyzed against running water for 18 hours and
hydrochloric acid was added in an amount sufficient to achieve a pH of
4.5. The product precipitated and was collected by centrifugation after
storage for 4 days. The precipitate comprised a conjugate of the
pregnenolone 20-carboxymethoxyloxime and bovine serum albumin joined
together by amine bonds. The product was soluble in water and measured as
cholesterol in assay procedures. The product was stable and did not
produce a turbid solution upon lyophilization and reconstitution with
aqueous media.
EXAMPLE 12
A solution was prepared by adding 5.2 g of pregnenolone laurate to 200 ml
isopropyl alcohol and to this solution was added 0.125 g sodium
borohydride (NaBH.sub.4). The resulting mixture was allowed to stand at
room temperature at two days and was then combined with 40 ml of water. A
precipitate resulted and was recrystalized from 100 ml of 80% ethanol and
water. The recrystalized precipitate comprised pregnene-20-ol-3-beta
laurate, hereinafter referred to as the first precipitate. A solution was
prepared by adding 5.2 g of the first precipitate to 100 ml of the dioxane
and added to this solution were 1.55 g of succinyl chloride and 1.1 g of
triethylamine. The resulting solution was added in one portion to 12 g of
bovine serum albumin disolved in 100 ml of one to one dioxane and water
and 5 ml of 1 N soduim hydroxide to achieve a pH of 8-10. The resulting
solution was purified by dialyzing for 24 hours against distilled water
and the solution was then acidified and the resulting precipitate removed
by centrifugation.
EXAMPLE 13
A solution was prepared by adding 5.2 g of the first precipitate prepared
in accordance with example 12, together with 1.5 g of succinyl chloride
and 1.01 g of triethylamine, to 100 ml of toluene. The resulting solution
was warmed to 40.degree. C. and maintained at that temperature for 11/2
hours. The warmed solution was added in one portion to a second solution
comprising 15 g of polyethylene glycol (having an average molecular weight
of 1000) and 1.01 g triethylamine dissolved in 100 ml of toluene. The
resulting mixture was maintained for 11/2 hours at 40.degree. C. and was
extracted with 100 ml of 80% saturated sodium chloride solution, the
sodium chloride solution then being discarded. The toluene was then
removed from the resulting mixture by evaporation and the product was
dissolved in 20 ml of acetone, and 1 ml of water was added. The acetone
solution was then extracted twice with 50 ml of hexane, and the hexane was
then discarded. The acetone was removed by evaporation and the product
thereby obtained comprised 3-beta laurate pregnene-20-succinyl
polyethylene glycol. The product was found to be soluble in water and to
measure as cholesterol in common assay procedures.
EXAMPLE 14
The procedures of Examples 12 and 13 were repeated using pregnenolone
acetate and pregnenolone palmitate instead of the pregnenolone laurate,
and products displaying similar results are obtained.
EXAMPLE 15
The procedures of Examples 12-14 were repeated using sebacyl chloride and
adipyl chloride instead of the succinyl chloride and products displaying
similar results were obtained.
EXAMPLE 16
A solution was prepared by adding 4.29 g of cholesterol hemisuccinate to 50
ml of dioxane and to that solution was added 1 g of triethylamine. To the
resulting solution was added 1.37 g of isobutyl chlorocarbonate and the
resulting mixture was warmed gently for 20 minutes. The warmed solution
was then added to a second solution comprising 12 g of bovine serum
albumin added to 500 ml of one to one dioxane in water and 5 ml of 1 N
sodium hydroxide. The solution was stirred and cooled for an hour and was
then purified by dialyzing against distilled water as described in example
12. Hydrochloric acid was then added to the solution to achieve a pH of
about 4.5 and the resulting precipitate was collected by centrifugation.
The precipitated product comprised a conjugate of the cholesterol and the
bovine serum albumin with a dicarboxyl group having a first carboxyl group
attached at the 3 beta position of the cholesterol and further having a
second carboxyl attached by amide bonds to one of the lysine residues of
the bovine serum albumin. The product was found to be soluble in water and
measured as cholesterol in normal assay procedures.
EXAMPLE 17
The procedure of Example 16 was repeated using cholesterol hemisebacylate
and cholesterol hemiadipate instead of the cholesterol hemisuccinate and
products yielding similar results were obtained.
EXAMPLE 18
Performance of the procedures of Examples 12 and 14-17 using horse serum
albumin, human serum albumin and bovine serum albumin yielded products
displaying similar results.
* * * * *
|
|
 |
|
 |
Description |
|
