U.S. patent number 3,925,164 [Application Number 05/454,522] was granted by the patent office on 1975-12-09 for method for the determination of cholesterol.
This patent grant is currently assigned to Boehringer Mannheim G.m.b.H.. Invention is credited to Klaus Beaucamp, Wolfgang Gruber, Gunter Lang, Hans Mollering, Peter Roeschlau.
United States Patent |
3,925,164 |
Beaucamp , et al. |
December 9, 1975 |
Method for the determination of cholesterol
Abstract
Total cholesterol or bound cholesterol in a sample is determined
by treating the sample with cholesterol esterase, thereby releasing
the bound cholesterol, and then determining the resulting total
cholesterol by known methods; specifically preferred are
cholesterol esterases from Candida rugosa ATCC 14830, Rhizopus
spec. WS 90027 and Aspergillus spec. WS 90030.
Inventors: |
Beaucamp; Klaus (Tutzing, Upper
Bavaria, DT), Mollering; Hans (Tutzing, Upper
Bavaria, DT), Lang; Gunter (Tutzing, Upper Bavaria,
DT), Gruber; Wolfgang (Tutzing, Upper Bavaria,
DT), Roeschlau; Peter (Tutzing, Upper Bavaria,
DT) |
Assignee: |
Boehringer Mannheim G.m.b.H.
(Mannheim-Waldhof, DT)
|
Family
ID: |
25764885 |
Appl.
No.: |
05/454,522 |
Filed: |
March 25, 1974 |
Foreign Application Priority Data
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|
|
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Mar 28, 1973 [DT] |
|
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2315501 |
Apr 3, 1973 [DT] |
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2316637 |
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Current U.S.
Class: |
435/11; 435/25;
435/27; 435/28; 435/197; 435/827; 435/886; 435/893; 435/911;
435/913; 435/921; 435/922; 435/933; 435/19 |
Current CPC
Class: |
C12N
9/18 (20130101); C12Q 1/60 (20130101); Y10S
435/921 (20130101); Y10S 435/886 (20130101); Y10S
435/827 (20130101); Y10S 435/922 (20130101); Y10S
435/893 (20130101); Y10S 435/933 (20130101); Y10S
435/913 (20130101); Y10S 435/911 (20130101) |
Current International
Class: |
C12Q
1/60 (20060101); C12N 9/18 (20060101); C12K
001/04 () |
Field of
Search: |
;195/13.5R |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
J Hyun et al., "The J. of Biol. Chem.," 244, No. 7, pp. 1937-1945,
1969..
|
Primary Examiner: Monacell; A. Louis
Assistant Examiner: Fan; C. A.
Attorney, Agent or Firm: Burgess, Dinklage & Sprung
Claims
What is claimed is:
1. Method of determining total cholesterol or bound cholesterol in
a sample, which method comprises treating said sample with
cholesterol esterase obtained from a micro-organism, thereby
releasing the bound cholesterol, and then determining the resulting
cholesterol content of said sample using a standard
determinatiion.
2. Method as claimed in claim 1 wherein said sample contains only
bound cholesterol and the said determination determines the amount
of said bound cholesterol.
3. Method as claimed in claim 1 wherein said sample contains free
cholesterol and bound cholesterol and the said determination
determines total cholesterol.
4. Method as claimed in claim 1 wherein said microorganism is
Candida rugosa ATCC 14830.
5. Method as claimed in claim 1 wherein said microorganism is
Rhizopus spec. WS 90027.
6. Method as claimed in claim 1 wherein said microorganism is
Aspergillus spec. WS 90030.
7. Method as claimed in claim 1 wherein said microorganism is
8. Method as claimed in claim 1 wherein said determination is an
enzymatic determination.
9. Method as claimed in claim 8 wherein the enzyme used for said
enzymatic determination is cholesterol oxidase.
10. Method as claimed in claim 9 wherein said cholesterol oxidase
is from Nocardia erythropolis ATCC 17895, Nocardia erythropolis
ATCC 4277, Nocardia formica 14811 or Proactinomyces erythropolis
NCIB 9158.
11. Method as claimed in claim 1 wherein said microorganism has
been cultivated on a cholesterol ester containing nutrient
medium.
12. Reagent composition for the determination of cholesterol in a
sample, with composition comprises cholesterol esterase obtained
from a microorganism and a system for the determination of free
cholesterol.
13. Reagent composition as claimed in claim 12 wherein said
composition comprises
a cholesterol esterase from a microorganism,
cholesterol oxidase,
a system for the determination of hydrogen peroxide.
14. Reagent composition as claimed in claim 13 wherein said
microorganism is Candida rugoas ATCC 14830, Rhizopus spec. WS 90027
or Aspergillus spec. WS 90030.
15. Reagent composition as claimed in claim 12 comprising
cholesterol esterase,
cholesterol oxidase,
catalase,
acetyl acetone,
methanol,
and a buffer containing ammonium ions.
16. Reagent composition as claimed in claim 14 containing
peroxidase,
a chromogen,
a buffer,
a system for the determination of H.sub.2 Oh.sub.2.
17. Reagent composition as claimed in claim 12 wherein said
composition comprises
a cholesterol esterase from a microorganism,
cholesterol oxidase,
a system for the determination of cholestenone.
18. Reagent composition as claimed in claim 17 wherein said system
for the determination of cholestenone is a hydrazine derivative
reacting with keto groups, to result in the formation of hydrazone.
Description
The present invention relates to a method of determining
cholesterol, and more specifically to a method of determining
either total cholesterol or bound cholesterol.
Cholesterol is present in biological matter, such as serum or the
like, partially in free form and partially in bound form as ester.
For the determination of either bound cholesterol or total
cholesterol it is necessary first to release the cholesterol that
is present in bound form. This has been done hitherto through
saponification under alkaline conditions, using alcoholic potash
lye, for example. After the saponification, the released
cholesterol can then be determined either chemically or
enzymatically by one of the known methods. The chemical
determination may be performed, for example, by the
Liebermann-Burchard method, and enzymatic determination may be
performed by means of cholesterol oxidase, cholesterol
dehydrogenase or cholesterol dehydrase. Since the individual
cholesterol esters as well as the free cholesterol are known to
have different extinction coefficients in the chemical methods of
determination, it is necessary to transform the cholesterol esters
to free cholesterol by alkaline hydrolysis.
In any case, however, the alkaline saponification of the bound
cholesterol is a troublesome and time-consuming step in the
procedure. Furthermore, the relatively agressive reagents used may
lead to a decomposition of the cholesterol. In order to prevent
such decomposition and thus forestall falsification of the results
of the analysis, a hydrolysis must be performed under relatively
mild conditions, and this in turn undesirably increases the length
of time required for the determination.
The alkaline liberation of the cholesterol is especially
disadvantageous when the determination of the cholesterol is
afterwards to be performed by the preferred enzymatic methods.
Since the enzymes are inactivated, as it is known, in the strongly
alkaline medium, the hydrolyzate must be acidified by the addition
of acid to pH 5 to 8 before the enzymatic determination can be
started. All this results in the fact that the determination of the
total cholesterol or of the bound cholesterol still takes an
undesirably long time and requires too much work.
The present invention provides a process for the determination of
total cholesterol or bound cholesterol which substantially obviates
the above-mentioned disadvantages.
Essentially, the process of the invention comprises the
determination of total cholesterol or of bound cholesterol by
releasing the bound cholesterol using cholesterol esterase,
followed by determination of the released cholesterol by known
methods.
It has been found that, using cholesterol esterase, a rapid and
quantitative saponification of bound cholesterol can be performed.
This process is especially advantageous whenever the subsequent
determination of the released cholesterol is performed
enzymatically, using cholesterol oxidase or cholesterol dehydrase,
for example. In this case, the process of the invention makes
possible the all-enzymatic determination of cholesterol and
therefore a decided improvement of routine medical diagnosis plus
an easy adaptation of the process for performance in automatic
analysis apparatus.
It is already known that in the pancreas and liver a cholesterol
esterase is present. It could not be concluded from this knowledge,
however, that such an enzyme would be suitable for the rapid,
complete saponification of cholesterol esters in the framework of a
quantitative analysis process, because the cleavage rates
determined were not quantitative, amounting to only 80% maximum
(Biochimica et Biophysica Acta 270, (1972), 156-166). Furthermore,
bound cholesterol is present in biological matter in the form of
esters of widely different acid. For an enzymatic process to be
useful in the framework of a process of analysis, it is required
that all of the esters that may occur be cleaved quantitatively
with approximately the same speed and with the same reliability. On
account of the known properties of these enzymes it is surprising
that the cholesterol esterases are capable of cleaving
quantitatively, within a very short time, all of the cholesterol
esters that occur. This is especially surprising also because in
the known cholesterol esterases there are considerable differences
with regard to their activity against various cholesterol
esters.
Cholesterol esterases from microorganisms have proven to be
especially suited for the process of the invention. They have
proven superior to cholesterol esterases of other origin in regard
to speed of cleavage and effectiveness and they are therefore
preferred within the scope of the invention.
It has also been found that a number of microorganisms contain
particularly active cholesterol esterases and can therefore be used
directly within the scope of the invention without separation and
purification of the cholesterol esterases. This is advantageous in
avoiding the separation of certain esterases from the normally used
mixture of a plurality of cholesterol esterases specific for
various cholesterol esters. Such separation would greatly
complicate the quantitative determination of all of the bound
cholesterol.
In addition, the purification of the bound cholesterol esterases in
lipoid membranes is difficult, and therefore results in a
preparation which is less suitable, on account of its price, for
use in routine diagnosis than a microorganism preparation which can
be used without any enzyme purification.
Particularly advantageous results have been obtained in the process
of the invention by the use of a cholesterol esterase derived from
Candida rugosa (also referred to as Cylindracea) ATCC 14830 and WS
90031, respectively, and from Aspergillus spec. WS 90030. These two
microorganisms may be used directly as such or in processed form,
e.g., in the form of an acetone dry powder, within the scope of the
invention. It is also, of course, possible to use a concentrated
cholesterol esterase preparation made from these microorganisms,
there being a special advantage in the fact that a certain
concentration may in this case be achieved very simply. Candida
rugosa is a microorganism that is produced on a large technical
scale and is available commercially. The customary commercial form
is an acetone dry powder stabilized with lactose, which has proven
to be outstandingly suited for the invention. Similarly attractive
properties have been found in: Actinomyces aureoverticillium WS
90002 Actinomyces cyaneofuscatus WS 90003 Actinomyces griseomycini
WS 90004 Actinomyces longisporus-fl. WS 90005 Actinomyces
malachiticus WS 90006 Actinomyces roseolus WS 90007 Actinomyces
toxytricini WS 90008 Actinomyces variabilis WS 90009 Streptomyces
spec. WS 90010 Streptomyces autotrophicus WS 90011 Streptomyces
canescens WS 90012 Streptomyces chartreusis WS 90013 Streptomyces
michiganensis WS 90014 Streptomyces murinus WS 90015 Streptomyces
hachijoensis WS 90016 Streptomyces caelestes WS 90017 Streptomyces
tendae WS 90018 Nocardia rubra WS 90019 Candida mycoderma WS 90020
Candida albicans WS 90021 Candida albicans WS 90022 Candida
albicans WS 90023 Candida spec. WS 90024 Cunninghamella elegans WS
90025 Mucor mucedo WS 90026 Rhizopus spec. WS 90027 Penicillium
spec. WS 90028 Aspergillus spec. WS 90029
In addition to the preferred cholesterol esterases of
microbiological origin, however, cholesterol esterases of other
origin may also be used in many cases.
As previously mentioned, an especially important advantage of the
process of the invention consists in the fact that it makes
possible an all-enzymatic determination of total cholesterol. It is
important in this case that, with the preferred cholesterol
esterase preparations made from microorganisms, a rapid and
quantitative release of the cholesterol from its esters is
possible. Especially with the preferred microorganisms mentioned
above, it is possible by the direct addition of same in a very
small quantity, with the maintenance of the pH values and
temperatures which are desirable in the subsequent enzymatic
determination of cholesterol, to achieve within a few minutes a
quantitative release of the cholesterol, it having been found that
the common carbohydrate-based stabilizing agents which are used for
such microorganisms do not interfere with the cholesterol
determination performed within the framework of the allenzymatic
process.
As mentioned, a separated and concentrated cholesterol esterase,
preferably one obtained from microorganism, may also be used for
the process of the invention. A suitable concentration may be
achieved by setting out from an acetone dry powder of the
microorganism or other biological material and subjecting it to a
dialysis, a treatment with weakly basic anion exchanger and to an
ammonium sulfate fractionation. In this manner, it is easy to
achieve a 20-fold to 30-fold concentration of the cholesterol
esterase. A preparation on a carbohydrate basis, modified with
diethylaminoethanol groups, has proven to be an especially suitable
weakly basic anion exchanger. In the ammonium sulfate
fractionation, the fraction between 1.8 and 2.4 moles of ammonium
sulfate is preferably obtained. The enzyme fraction thus obtained
is then chromatographed, preferably on the above-named exchanger
material.
Particularly good results are obtained within the scope of the
invention with microorganisms which have been cultivated in a
nutrient medium containing cholesterol ester. In this case, the
cholesterol ester or a mixture of cholesterol esters may be added
during the cultivation as the sole source of carbon, or may be used
together with another carbon source. Especially preferred is the
use of microorganisms which are obtained in a multi-stage
cultivating process, in which they are cultivated in the first
stage on a suitable carbon supplier, such as glycerin, and in the
second stage on a cholesterol ester. A suitable cultivation process
is described, for example, in German Published Specifications
("Offenlegungsschriften") Nos. 2,224,133 and 2,307,518.
The cholesterol esterase from Candida rugosa ATCC 14830 used
preferentially in accordance with the invention has very good
stability in the weakly acid region between pH 5 and 6.5. The
optimum pH for the enzyme is 7.5. One peculiarity of the enzymes is
that the catalytic reaction takes place especially well when the
salt content of the reaction medium is relatively high. Preferably,
therefore, the process is performed in an 0.2 to 0.8 molar buffer
solution. The pH may range between 4.5 and 7.5, and will preferably
range, as stated above, between pH 5 and 6.5. The effectiveness of
the cholesterol esterase is preferably increased by the addition of
surface active substances. Especially preferred is the addition of
hydroxypolyethoxydodecane.
As previously mentioned, it is especially preferred that the
process of the invention be performed all-enzymatically, i.e., the
cholesterol determination that follows is also performed
enzymatically, preferably with the use of cholesterol oxidase.
However, cholesterol dehydrase or dehydrogenase may also be
used.
Determination with cholesterol oxidase is described, for example,
in German Offenlegungsschrift No. 2,224,132. The process therein
described may advantageously be combined with the process of the
invention. In this case, it is possible in principle to measure the
oxygen consumption, the H.sub.2 O.sub.2 formation or the formation
of cholestenone. The determination of the oxygen consumption may be
performed, for example, by gas chromatography or polarometry, or by
the polarization method. These methods of determination are in the
prior art. The hydrogen peroxide that forms may be determined
titrimetrically, potentiometrically, and colorimetrically, as well
as enzymatically. Enzymatic determination is preferred, with the
use of catalase or peroxidase, especially determination by catalase
in the presence of beta diketones such as acetylacetone, low
alcohols and buffer containing ammonium ions, or determination by
peroxidase in the presence of a chromogen such as
2,2'-aminobenzothiazolinesulfonic acid. Cholestenone is determined
by means of keto reagents such as 2,4-dinitrophenylhydrazine, or by
photometry at 240 nm.
If the all-enzymatic determination of the total or bound
cholesterol is performed with cholesterol oxidase, a cholesterol
oxidase obtained from Nocardia erythropolis ATCC 17895, Nocardia
erythropolis ATCC 4277, Nocardia formica ATCC 14811 or
Proactinomyces erythropolis NCIB 9158 is preferably used.
Additional subject matter of the invention is a reagent for the
determination of cholesterol which consists of cholesterol esterase
and a reagent for the determination of free cholesterol.
Preferably, a reagent of this sort consists of a cholesterol
esterase of microbiological origin, cholesterol, oxidase, a system
for the determination of hydrogen peroxide, or a system for the
determination of cholestenone. Quite especially preferred in this
case is a reagent in which the cholesterol esterase is one of the
microorganisms mentioned further above, especially in the form of
an acetone dry powder or a protein fraction obtained therefrom
having cholesterol esterase activity.
In a special and preferred embodiment, such a reagent consists of
cholesterol oxidase, a cholesterol esterase preparation made from
one of the above-mentioned microorganisms, catalase, acetyl
acetone, methanol and aluminum ion-containing buffer, individually
or mixed. In still another preferred embodiment, the reagent
consists of cholesterol oxidase, a preparation made of the
above-mentioned microorganisms having cholesterol esterase
activity, peroxidase, chromogen and buffer, individually or mixed.
2,2'-aminobenzothiazolinesulfonic acid is preferred as the
chromogen.
In still another preferred embodiment, the reagent of the invention
consists of cholesterol oxidase, a cholesterol esterase preparation
made from one of the named microorganisms, and a hydrazine
derivative reacting with keto groups with the formation of
hydrazone, and in some cases a buffer. 2,4-dinitrophenylhydrazine
is preferred as the hydrazine derivative.
The above-mentioned preferred reagent combinations may contain, in
addition to the specified essential components, commonly used
solvents, stabilizers and/or suface active substances. All these
additive substances are known to persons skilled in the art and
commonly used in detection systems for hydrogen peroxide and
cholestenone.
Preferably, the above-mentioned reagent combinations will contain
the essential components in the following rations:
1. 13 to 150 U of cholesterol oxidase, 0.05 to 0.5 mg of
microorganism cholesterol esterase, 2 .times. 10.sup.4 to 5 .times.
10.sup.5 units of catalase, 0.05 to 0.2 ml of acetyl acetone and 2
to 10 ml of methanol in 100 ml of a pH 5 to 7 buffer containing
ammonium ions, plus, if desired, 0.02 to 0.3 ml of a surface active
agent, preferably hydroxypolyethoxydodecane.
2. 3 to 40 U of cholesterol oxidase, 0.05 to 0.5 mg of
microorganism cholesterol esterase, and 2 .times. .sup.2 to 1
.times. 10.sup.4 U of peroxidase, 50 to 200 mg. of
2,2'amino-benzothiasolinesulfonic acid, and, if desired, 0.05 to
0.5 ml of surface active agent, preferably
hydroxypolyethoxydodecane, in 100 ml of pH 6 to 8 buffer.
3. 0.1 to 1 U of cholesterol oxidase, 0.05 to 0.5 mg of
microorganism cholesterol esterase, 1 to 5 ml of a 1 mM solution of
2,4-dinitrophenylhydrazine, and, if desired, 0.005 to 0.1 ml of
surface active agent in 10 ml of pH 6 to 8 buffer.
4. 2 to 100 U of cholesterol oxidase, 0.05 to 0.5 mg of
microorganism cholesterol esterase, and, if desired, 0.1 to 2.0 ml
of surface active agent (preferably hydroxypolethoxydodecane), in
50 ml of pH 5 to 9 buffer, preferably 0.5 m of sodium phosphate pH
7.5 buffer.
With the process and reagent of the invention, an extremely rapid
and complete saponification of bound cholesterol determination with
cholesterol oxidase in accordance with the invention, a
quantitative cleavage of bound cholesterol is accomplished within
one to three minutes with the addition of Candida rugosa ATCC 14830
or Aspergillus sp. WS 90030 acetone dry powder in a quantity
between 0.1 to 0.3 mg.
The following examples are illustrative.
EXAMPLE 1
Using the method described in Example 1 of German
Offenlegungsschrift No. 2,224,132, the content of free cholesterol
in serum was found to be 63 mg% (63 mg in 100 ml). For the
determination of bound cholesterol, a specimen of the same serum
was treated for 30 minutes with alcoholic potash lye at
70.degree.C. After neutralization and measurement of the
cholesterol present, a total content of 181 mg% of cholesterol was
found. From this appears that 118 mg of cholesterol were present in
bound form for every 100 ml.
The process was repeated with untreated serum, but at the beginning
of the determination 0.3 mg% (with reference to the protein) of an
acetone dry powder of Candida rugosa ATCC 14830 in commercial form
was added. After 3 minutes, polarographic determination showed a
content of 183 mg% total cholesterol.
EXAMPLE 2
To concentrate the cholesterol esterase activity, commercial
acetone dry powder of Candida rugosa ATCC 14830 was dissolved in
potassium phosphate buffer pH 6.0 and dialyzed against the same
buffer. After removal of the lactose contained in the solution as
stabilizer, the specific cholesterol esterase activity was 0.3 U
per mg of protein in the dialyzed solution.
The solution thus obtained was stirred together with an ion
exchanger on a dextran basis modified with diethylaminoethanol
groups; the exchanger was separated and eluted with 0.2 M of pH 6.0
phosphate buffer. A specific cholesterol esterase activity of 1.2
U/mg was found in the eluate.
The solution thus obtained was subjected to an ammonium sulfate
fractionation. The protein fraction that precipitated between 1.8
and 2.4 M of ammonium sulfate was separated, and had a specific
cholesterol esterase activity of 2.5 U/mg.
The product obtained was again dissolved in pH 6.0 phosphate
buffer, dialyzed against the same buffer until salt-free, and then
chromatographed on a column filled with the same anion exchanger as
above. Elution was again performed with 0.2 M of pH 6.0 phosphate
buffer. A specific activity of 7 U per mg of protein was found in
the fraction having cholesterol esterase activity.
The concentrated cholesterol esterase preparation thus obtained was
used in the cholesterol determination as described in Example 1,
except that the amount used was only 0.001 mg with reference to
protein. The results were the same as in Example 1.
The cholesterol esterase from Candida rugosa can be further
purified by the conventional methods of enzyme refinement. Instead
of the concentration procedures cited above, other conventional
biochemical refinement procedures may be used, such as
precipitation or fractionation with polyethyleneimine, organic
solvents or salts, by chromatography through molecular sieve
materials or weak anion exchanger with functional groups other than
diethylaminoethanol groups, by protamine sulfate precipitation and
the like.
EXAMPLE 3
To 0.5 ml of serum in the one case and cholesterol standard in the
other, 1.0 ml of 0.5 M potassium phosphate pH 7.5 buffer containing
0.4% hydroxypolethoxydodecane, and 2.5 U of cholesterol esterase
from Example 2 were added. This reaction mixture was incubated for
40 minutes at 37.degree.C. Then 0.25 ml of this solution was added
to 3 ml of cholesterol reagent containing two parts acetic acid,
three parts acetic acid anhydride and one part sulfuric acid
(Liebermann-Burchardt reagent).
By using a standard as a reference magnitude, 170 mg% total
cholesterol was found in a typical specimen. Comparative
determination after saponification of the cholesterol ester with
alcoholic potash lye gave 165 mg%.
EXAMPLE 4
10 ml of 0.5 M potassium phosphate buffer containing 0.4%
hydroxypolyethoxydodecane, and 0.2 U of the cholesterol esterase of
Example 2, were added to 0.02 ml of serum. The reaction solution
was incubated for 60 minutes 37.degree.C. Then the extinction
(E.sub.1) at 240 nm was read in a suitable spectral photometer and
the reaction was started with 0.1 U of sterol dehydrase obtained
from Brevibacterium sterolicum. After fifteen minutes, the
extinction (E.sub.2) was again read. The concentration of the
.DELTA..sup.4 cholestenone and hence of the cholesterol was found
from the difference between the first and second reading on the
basis of the molar extinction coefficient for .DELTA..sup.4
cholestenone at 240 nm. Measurement of a typical specimen gave 183
mg% total cholesterol.
Comparative determination with a cholesterol oxidase from Nocardia
erythropolis instead of sterol dehydrase gave 181 mg%
cholesterol.
EXAMPLE 5
10 g of diammonium hydrogen phosphate was dissolved in 100 ml of
water and adjusted to pH 7.0 with 85% phosphoric acid. Then
10.sup.5 units of catalase were added. The solution thus obtained
was added to a mixture of 0.2 ml of acetyl acetone, 10 ml of
methanol and 0.1 g of hydroxypolyethoxydodecane to produce a volume
of 100 ml. To this solution, 2.5 units of cholesterol esterase from
Rhizopus spec. (WS 90027) were added. 5.0 ml of the solution thus
obtained was mixed with 0.02 ml of serum in the one case and 0.02
ml of a cholesterol standard solution containing 200 mg%
cholesterol in the other. To aliquots of the serum-containing
specimen and of the cholesterol standard-containing specimen 0.1
unit of cholesterol oxidase was added and the mixtures were
incubated for 60 minutes at 37.degree.C. Then the dye that was
formed was measured photometrically at 405 nm on the basis of the
specimen zero value.
Using a standard as a reference magnitude, the cholesterol content
of the serum-containing specimen amounted to 154 mg% total
cholesterol. The control determination performed with cholesterol
esterase from Candida rugosa ATCC 14830 instead of cholesterol
esterase from Rhizopus spec. (WS 90027) gave the same value.
It will be understood that the specification and examples are
illustrative but not limitative of the present invention and that
other embodiments within the spirit and scope of the invention will
suggest themselves to those skilled in the art.
* * * * *