Note: Descriptions are shown in the official language in which they were submitted.
Field of the Invention
The present invention relates to methods for the
hydrolysis of cholesterol esters and more particularly to
methods and compositions ror the hydrolysis of protein-bound
cholesterol esters such as serum cholesterol esters.
Back~round of the Invention
In the assay of body fluids, especially blood
serum, for cholesterol concentration, the initial step requires
hydrolysis of cholesterol esters to free cholesterol.
Conventional procedures for cholesterol ester
hydrolysis use a strong base (KOH, NaOH, etc.), or for reasons
,
of simplicity and selectivity, a hydrolase enzyme (i.e., a
cholesterol esterase~. Handling of caustic materials may be
inconvenient or undesirable and, as discussed in relation to
prior publications below, while enzymatic techniques can be useful
- ~ for the hydrolysis of "free" cholesterol esters, i.e., those
not bound to protein,they are either îneffective or ver~ slow
when used to treat protein-bound cholesterol esters. The
i binding of the ester to protein apparently inhibits the action
of the esterase and thus requires some means for breaking the
protein-ester complex before the enzyme can act on the ester.
U.S. Patent No. 3~869,349 issued March 4, 1975 to
.~.
Goodhue et al describes an improved technique for hydrolyzing
serum cholesterol esters which involves the use of compositions
comprising a lipase preparation which demonstrates cholesterol
esterase activity and a protease. This patent contains no
suggestion that a surfactant can replace the protease.
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U.S. Patent No. 3,703,591 to Bucolo et al describes
the use of a combination of a lipase and a protease to achieve
serum (i.e. 5 protein-bound) triglyceride hydrolysis. No sug-
gestion is made to use a surfactant either in combination with
or as a substitute for the protease in the hydrolysis of
cholesterol esters.
U.S. Patent No. 3,759,793 to Stork et al describes
~ the hydrolysis of serum triglycerides using a lipase from
- Rhizopus arrhizus which is apparently identical to that sug-
gested by Bucolo et al, however7 with no requirement for a
protease. The reasons for this apparent anomaly are not
clear, however~ it is noted in British Patent No. 1~395,126
of the same assignee that the Stork et al hydrolysis technique
~; is very slow. This British Patent describes an improved
method for hydrolyzing triglycerides with the aforementioned
Rhizopus arrhi~us lipase comprising contacting the triglyceride
with the lipase in a buffer and in the presence of carboxyl-
esterase and an alkali metal or alkaline earth metal alkyl
sulfate, the alkyl radical of which contains 10 to 15 carbon
atoms. The pre~erred alkyl sulfate is sodium dodecyl sulfate.
There is no suggestion that the use of surfactant alone in the
absence of carboxylesterase stimulates hydrolase activity of
kriglycerides.
Helenius, Ari and Simons, Kai, Biochemistry, Vol. 10,
No. 13 (1971) describe a method for removing all major lipids
from human plasma low-density lipoprotein comprising treatment
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of the human plasma with high concentrations of ~atural and
synthetic surfactants. Lipid removal is applied for purposes
of characterizing the lipid free protein moiety of human
plasma low-density lipoprotein. There is no suggestion in
this publication that the combination o~ a surfactant and
a lipase would yield a use~ul analytical tool which would
simplify the assay of serum for cholesterol content by pro-
viding a fast and accurate hydrolysis method and a ~table
assay composition.
U.S. Patent No. 3,689,364 issued September 5, 1972
describes an assay for lipase contained in body fluids such
as blood serum using a "free" triglyceride emulsion as sub-
strate for the lipase. It is suggested that the bile salts
which stabilize the substrate emulsion of "free" triglyceride
(i.e., triglycerides not bound to protein) also concurrently
e~ert an "activating effect" on the lipase lmder assay when
it is a pancreatic lipase. The activating e~fect apparently
results in an increase in the hydrolytic activity of the
lipase on the free triglycerides of the substrate emulsion.
There is no teaching or suggestion in this patent that such
bile salts exert any effect on lipase preparations when con-
tacted with lipids bound to proteins as are found in blood
serum. In particular there is no suggestion that such lipase
can hydrolyze protein-bound cholesterol esters.
U.S. Patent No. 3,898,130 to Konatsu issued August 5
1975 describes a method for hydrolyzing triglycerides com-
; prising contacting triglyceride with a composition comprising
a mixture of a microbial lipase, particularly Candida lipase(sic),
a pancreatic lipase and a bile salt selected from sodium tauro-
3~ deoxycholate, taurocholate, taurocheno~eoxycholate and tauro-
dehydrocholate. Both the microbial and the pancreatic lipase
enzymes are critical components o~ the hydrolysis composition.
German Offenlegungsschrift No. 2,522,432 published
December 4, 1975 describes an enzymatic method for hydrolyzing
cholesterol esters using a cholesterol esterase from
Pseudomonas fluorescens. There is no suggestion of the use
of or a need for a surfactant to achieve protein-bownd
cholesterol ester hydrolysis.
French Patent No. 2,2239696 and U.S. Patent No.
3,925,164 issued December ~ 1975 describe an assay for total
cholesterol in blood serum wherein cholesterol esters ~re
~ 10 hydrolyzed with an ~nzyme preparation from Candida ru~_a,
E~ or Aspergillus in the presence of a surfactant.
The only suggested surfactant is hydroxypolyethoxy dodecane.
; As will be shown in the examples below, such surfactants are
not as effective as the materials described herein, possibly
- because of incompatibility wlth the cholesterol ester hydrolase.
German Offenlegu~gsschrift No. 2,509,156 published
September 23, 1975 describes an enzymatic method for the assay
of total cholesterol using as the cholestervl ester hydrolyzing
medium cholesterol esterase and a gallic acid or a salt of a
gallic acid. The cholesterol esterase is identified as
~C 3.1.1.13 which is derlved from Nocardia restrictus. There
is no suggestion in the foregoing publica-tion that
the synthetic surfactants described herein are useful effectors
for cholesterol esterase.
Related A~plications
Commonly-owned U. S. Patent No. 4~179,3349 issued
on December 18, 1979, entitled "Hydrolysis of Protein-Bound
Triglycerides" discloses a process and a composition for
hydrolyzing protein-bound triglycerides, featuring a com-
patible mixture of an enzyme preparation and a surfactant.
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Summary of the Invention
It has now been discovered that protein-bound
cholesterol esters can be hydrolyzed in relatively short periods
of time on the order of less than about 10 minutes (preferably
in about 5 minutes) by contacting the protein-bound cholesterol
esters with a compatible mixture of a lipase preparation which
demonstrates cholesterol ester hydrolase activity and an alkyl
phenoxy polyethoxy ethanol comprising a polyoxyethylene chain
of less than about 20 oxyethylene units.
The techniques and compositions described herein permit
the use of a much broader range of enzyme preparations
as cholesterol ester hydrolyzing agents than has been pos- ¦
sible with prior art methods. Thus, lower cost materials
can be used to attain reaction times and states of reaction
completeness at least equal to and often superior to -those
attainable with the prior art methods and materials.
Description of the Preferred mbodiments
As discussed hereinabove, there are enzyme
preparations which catalyze the hydrolysis of free cholesterol
esters. Such materials, however, catalyze the hydrolysis of
cholesterol esters bound to protein, as found in blood serum,
only at very slow rates or in an incomplete fashion. This
result is apparently due to some effe~t of the protein-lipid
complex which prevents the enzyme ~rom catalyzing the hydrolysis
in the usual fashion. The prior art has suggested the use of
what might be termed effectors3 namely, agents which increase
the rate at which lipase materials can hydrolyze protein-
bound cholesterol esters. Although the mechanism by which
- such agents act is not known, it is theorized that they disrupt
- 30 the ester-protein complex in some way to "free" the ester for
hydrolysis in a conventional mode. Protease enzymes, for
example, have been suggested ~or this purpose.
We have found that certain surfactants are effectors
and may be used as substitutes for protease to render useful,
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as hydrolyzers of protein~bound cholesterol esters~ enzyme
preparations which are normally incapable of catalyzing the
hydrolysis of protein-bound cholesterol esters or which
catalyze such hydrolysis only at undesirably slow rates.
Furthermore, since protease tends to degrade proteinaceous
binders~ such as gelatin~ used in multilaye~ elements for ;~
the detection of anaIyte as described in Belgian Patent
No. 801,742, the compositions described herein are particu~
larly useful in such elements.
The novel hydrolysis compositions of the present
invention thus comprise a compatible mixture of an enzyme
preparation which demonstrates cholesterol esterase activity
and as an effector a surfactant which is an alkyl phenoxy
, polyethoxy ethanol comprising a polyoxyethylene chain of less
than about 20 oxyethylene units.
Enzyme preparations potentially useful in the methods
and compositions described herein are those demonstrating free- `
(l.e., not protein-bound) cholesterol ester hydrolase activity. ;
~ipase preparations which demonstrate such activity are
20 specifically preferred.
; A useful screening technique for determining the
cholesterol ester hydrolase (esterase) activity of enzyme,
and particularly lipase, preparations comprises adding a
flxed amount of the enzyme preparation to a standard cholesteryl
linoleate solution at pH 7.0, incubating at 37C under N2 for
2 hours and determining the amount of ester left in the solu-
tion by the hydroxylamine method of J. Vonhoeffmyr and R. Fried,
r Z~ Klin. Chem. U. Klin. Biochem., 8, 134 (1970). By means of this
-` technique, any preparation which demonstrates a cholesterol
,.
esterase activity which releases above about 25 mg/dl cholesterol
in the-screening procedure should be considered a useful
candidate in the practice of the present invention.
~.
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Useful enzyme preparations for cholesterol ester
hydrolysis may be derived from plant or animal sources but
we prefer preparations from microbial sources such as from
Candida rugosa, Chromobacterium viscosum, variant ~
lipolyticum, crude or purified. Other useful enzyme
preparations and methods for their preparation are described
in the following U.S. Patents: 25888,385 to Grandel issued
May 26, 1959; 3,168,448 to Melcer et al issued February 2,
1965; 3,189,529 to Yamada et al issued June 15, 1965;
3,262,863 to Fukumoto et al issued July 26, 1966; and
3,513,073 to Mauvernay et al issued May 19, 1970.
Preferred commercial enzyme prepara-
tions include wheat germ Iipase from Miles Laboratorles of
Elkhart, Indiana, Lipase 3000 from Wilson Laboratories,
Steapsin from Sig~a Chemical Company (both of the latter are
pancreatic enzymes), and Lipase M (from Candida rugosa) from
Enzyme Development Company.
Certain surfactants inhibit the cholesterol esterase
activlty of certain enzyme preparations. Consequently, it
is important that before any attempt is made to combine an
enzyme preparakion and a surfactant for use as described herein
some determination of the compatibility of the two members of
the composition be made. Such a determination is preferably
made by using the test descrlbed below. An enzyme preparatlon
and sur~actant mixture which successfully meets this test is
referred to herein as a compatible mixture and each member
thereof is said to be compatible with the other.
Hydrolysis compositio~s of the present invention are
characterized by the test used in comparative Example II below.
The proposed surfactant under evaluation is added to normal
human serum. A sample of a proposed enzyme preparation is
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added and the mixture incubates at 37C for a period of
about 10 minutes, Aliquots (0.1 ml) of this solution are
then diluted to 1.9 ml with water and placed in a boiling
water bath for 10 minutes. Cholesterol is quantified in
a total volume o~ 1.2 ml by the well known cholesterol
oxidase system described below. A slmilar "control" test
is performed concurrently using only the enzyme preparation
without the surfactant. When performing the foregoing test it
is most desirable to run a blank which contains all of the
; components of the mixture but the enzyme preparation so that
~any reaction which may be due to free cholesterol or other
components of the serum can be subtracted. The preferred
compositions accomplish hydrolysis of at least 70% Or the
available cholesterol esters in less than 10 minutes and most
preferred are those which achieve substantially complete
hydrolysis, i.e., hydrolysis of at least ab~ut 90% of the
available cholesterol esters in less than about 10 minutes.
From testing of this type9 it has been discovered
that phenox~ polyethoxy ethanols are highly superior effectors,
Specifically preferred are materials available commercially
from Rohm and Haas Company under the trade~ s Triton X-114,
100, 102 and Triton n-101. Preferred alkyl phenoxy polyethoxy
ethanols comprise a polyoxyethylene chain of less than about
3 20 oxyethylene units, As will be shown in the following
éxamples, similar materials outside Qf these broad limits do
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not provide the improved hydrolysis described herein Most
preferred are those materials wherein the alkyl is either 8
or 9 carbon atoms.
As will be demonstrated in the examples below3
certain prior art surfactants discussed above do not produce
acceptably high dissociation when combined with the enzyme
preparations evaluated. This includes the hydroxypolyethoxy
dodecanes o~ French Patent No. 2,223,696 and U.S. Patent No.
3,925,164. (Exemplary useful hydrolysis compositions are
shown in Table I.)
~ he concentration of enzyme preparation and sur-
factant in the compatible mixtures useful for hydrolysis
according to the methods described herein can vary greatly
depending, for example, on such factors as the purity of the
enzyme preparation, the activity of the enzyme preparation,
.~
the nature of the bound cholesterol ester, the particular
~` surfactant used, etc. Generally, however, surfactant concen-
trations of from about 0.25 to about 10% by weight of the
analytical solution have been found useful wlth concentrations
.
of between about 0.5 and 5% bv weight of surfactant providing
optimum results. The useful range of concentrations of enzyme
preparation will vary similarly3 but concentrations of between
about 10 and 80 mg/ml of the total analytical solution have
been found quite useful when commercial preparations are used.
.:
Optimization of any such composition is, of course, within the
skill of the art.
It should be apparent that hydrolysis compositions
of the type described herein can be incorporated into any of
the single or multiple layer absorbent or other analytical
~; 30 elements (for example, test papers) described in the prior
art and that the use of the compositions and methods described
herein in such elements for the detection or determination of
protein-bound cholesterol esters is within the scope of the
invention
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In accordance with one preferred embodiment~ the
hydrolysis composi~ions described herein are incorporated
into one or more layers of multilayer analytical elements of
the type described, or example, in Belgian Patent No. 801,742.
; Such elements are intended to analyze liquids for the presence
of a predetermined analyte and they include a preferably non-
fibrous spreading layer, which delivers a uniform apparent
concentration of analysis-active components in an applied sample
to a reagent layer which contains at least some of the materials
interactive in the presence of analyte to produce a detectable
product or detectable change. Such layers are in fluid contact
under conditions of use.
Reference herein to fluid contact between layers in
; an analytical element identifies the ability of a fluid, whether
liquid or gaseous, to pass in such element between superposed
regions of the spreading layer and the reagent layer. Stated
in another manner, fluid contact refers to the ability of com-
ponents of a fluid to pass between the layers in fluid contact.
Although such layers in fluid contact can be contiguous, they
may also be separated by intervening layers as described in
detail hereinafter. However, layers in the element that
physically intervene a spreading layer and reagent layer in
mutual fluid contact will not prevent the passage of fluid
between the fluid contacting spreading and reagent layers.
Fluid contact between layers can be achieved by
preparing elements having layers that are initially contiyuous
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or effectively so for purposes of fluid passage. Alternatively,
it may be appropriate to prepare elements that have layers
initially non-contiguous, and which ~ur~her can be spaced
apart, such as by the use of interleaves as descrlbed, for
example, in U~S. Patent 3,511,608 or by the use of a resilient
absorbent material or deformable supports as described in
U.S. Patent 3,917,453 and U.S. Patent 3,933,594. As will be
appreciated, i~ the element has initially non-contiguous
layers, it may be necessary to apply compressive force or
otherwise provlde means to bring layers of the element into
fluid contact at the time of its use to provide an analytical
result.
~ According to a highly preferred embodiment of such
; an element~ the hydrolysis composition described herein is
incorporated into the spreading layer and a detection system,
for example, a cholesterol oxidase and an indicator compo-
sition sensitive to hydrogen peroxide for cholesterol detection,
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The following description of standardized procedures
and examples are presented to further illustrate the useful
scope of the present invention.
~ Standard Procedures:
`~ Quantification of Total Serum Cholesterol - Choles-
-- terol esters must first be hydrolyzed to free cholesterol.
Incubation mixtures contained in a total volume of 8 ml:2.4 units
cholesterol oxidase (N. cholesterolicum), 0.768 mg 4-amino-
__
antipyrene HCl, 0.256 mg 1,7-dihydroxynaphthalene, 0.22 mg
peroxidase (125 purpurogallin units/mg), 6.4 mg crude lipase
preparation~ and either o.48 mg protease (B subt11is (Sigma
Corporation Type VII)) or 160 mg octyl pheno~y polyethoxy
ethanol (Triton X-100). Incubation mixtures were equilibrated
at 37C for five minutes and the reaction was initiated by
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addition of 20 ~1 of human serum. A~ter 10 minutes, the
absorbance at 490 nm was measured. Blank tubes contained
all components except serum. Tot~] cholesterol concentra-
tions were obtained from a standard curve which was con-
structed~by substituting aliquots of Fermco-Test Aqueous,...
Cholesterol Standard (available from Fermco Laboratories,
, Chicago, Illinois~ for the serum substrate.
The reference method was the Liebermann-Burchard
method as described'in "Hawk's Physiological Chemistry", B. L.
Oser (Editor) 14th Edition, McGraw-Hill Book Company, New York,
(1965~ pp. 1052-1064. This method involves extrac~ion of the
cholesterol and cholesterol esters from serum prior to
quantitation.
Example 1 ~ Enzyme Catalyzed Hydrolysis of Serum Cholesterol
Esters in the Presence of Alkyl Phenoxy
Pol ethox Ethanol Surfactant
Y Y - :.
Human serum (20 ~1) was added to 8 ml of buffer
~, reagent (equilibrated---at--37) which contained---either enzyme
~' preparation and a protease or the enzyme preparation and surfactant
:::
~, to-~effect ~ydro-lysis of the cholestero~ esters. After 10
; 20 mlnutes, the absorbance at 490 nm was measured, and total
~ serum cholesterol calculated as described above.
;;~ Serum cholesterol was quantified using the
cholesterol oxidase, peroxidase system. In this system
~ cholesterol esters are first hydrolyzed to free cholesterol
'; which is subsequently oxidized to cholestenone with con-
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'~, comitant production of H202. The H2O2 is then coupled to
dye formation via a peroxidase reaction. It has been reported
, that a crude lipase preparation catalyzes hydrolysis of serum
; cholesterol esters if a protease is added to the incubation ,,
,~, 3O mixture. The data in Table I show that in the presence of
~' S-l (an octylphenoxy polyethoxy ethanol having about lO ethoxy
~ units and an HL3 number of 13.5) complete hydrolysis of
,- -13-
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cholesterol esters was observed. The surfactant efficiently
replaced the protease and thus eliminated the need for this
ex~raneous protein which may undesirably
(1) hydroly~e protein components o~ the cholesterol detection
system or (2) alter the pH of the system. Quantitation of
serum cholesterol with a lipase preparation and S-1 as the
hydrolytic system gave results (see Table I~ ~hich compared
very favorably with the reference method.
TABLE I
Total Serum Cholesterol Concentration
Lipase
Protease Lipase Referenae
Sample (Gontrol) 2% S-l Method
1 225 225 232
2 150 1~5 190 .
3 150 180 176
4 Z40 230 220
aThe reference method was a semi-automated
:` Liebermann-Burchard method
,
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~ 20 An S-l concentration of 2~ gave complete hydrolysis
-. and the surfactant produced no harmful effects at concentra-
:
tions as high as 4~. Although final color densities were
measured after 10 minutes7 reactions were essentially complete
in as little as five minutes at 37C.
.~
Comparative Example I
Repetition of the test described in Example I above
using alkylphenoxy polyethoxy ethanols having polyoxyethylene
chains above about 20 yields results which indicate that such
` 30 surfactants somehow inhibit the cholesterol esterase activity
of the enzyme~
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Comparative Example II
Direct comparative tests were conducted with a
cholesterol esterase from Candida rugosa and (a) surfactant
as described in French Patent No~ 2~223~696 and (b)
representative of` the surf'actants described herein.
Incubation mixtures were prepared containing in a
total volume of` o.6 ml:
O,5 ml normal human serum
mg cholesterol esterase (Lipase M commercial
preparation f'rom Candida rugosa)
~ moles potassium phosphate buff'er (pH 7.0)
mg ef'fector
Reactions were allowed to proceed for 10 minutes
at 37~C and then 0.1 ml aliquots were added to 1.9 ml of
water and placed in a boiling water bath for 10 minutes.
Cholesterol was then quantitated via the cholesterol oxidase-
peroxidase system using aqueous cholesterol standards to
prepare a standard curve. The results of these tests are
shown in Table II below.
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From the foregoing, it is apparent that the poly-
oxyethylene lauryl ethers (i.e.~ the dodecane materials of
French Patent No. 2~223,696) are not useful as effectors in
accordance with the invention described herein.
The invention has been described in detail with
particular reference to certain pre~erred embodiments thereof,
but it will be understood that variations and modifications can
be effected within the spirit and scope of the inventionO
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