Note: Descriptions are shown in the official language in which they were submitted.
~V4458Z
The present invention is concerned with a process
for the activation of cholesterol oxidase and with a diagnostic
agent for the detection and determination of cholesterol and
cholesterol esters in body fluids.
German Patent Specification No. 2,224,132 describes
a process for the determination of cholesterol in which
cholesterol is incubated in an aqueous medium with
cholesterol oxidase, followed by the determination either
of the oxygen consumption or of the amount of hydrogen
peroxide or of cholestenone formed. This German Patent
Specification also describes a reagent for the determination
of cholesterol which comprises cholesterol oxidase and a
system for the determination of hydrogen peroxide or a
system for the determination of cholestenone.
We have now found that cholesterol oxidase has an
insufficient storage stability and is inactivated relatively
quickly. In the course of the investigation of this
instability, we have found that the inactivation is brought
about by ~mall amounts of detergent which are present as an
accompanying sUbstance and originate from the process used
for the preparation of the enzyme. This preparation of the c
enzyme can be carried out, for example, by the process
described in German Patent Specification ~o. 2,224,131 in
which a micro-organism which metabolises cholesterol is
digested by destruction of the cell walls with a non-ionic,
surface-active agent present in a buffer solution and then
extracted, whereafter the extract is centrifuged, the
precipitate obtained is discarded and the supernatant ~-
liquid is applied to an anion exchanger, the enzyme then
being eluted with a buffer solution containing the non-
ionic surface-active agent and thereafter isolated from
the eluate.
- 2 -
.. ---, .
.,'`~- , .
:. .
.~ .
il~4~58Z
We have also found that, upon removal of the~e
residual traces of detergent, a completely ~atisfactory
storage stability of the enzyme can be achieved. ~owever,
we have also ascertained that the enzyme freed from
detergent ha~ a remarkably reduced acitivity. If, for
example, the enzyme is used for the determination of
cholesterol, then it is necessary either to use a compara-
tively freshly prepared preparation or considerably larger
amounts of enzyme must be employed for the test or the time
- 10 required for-carrying out the determination must be con-
siderably increased.
Consequently, it is an object of the present
invention to provide a proce~s for the activation of
cholesterol oxidase. More particularly, it is an object
of the present invention to provide such a process which,
on the one hand, permits the use of cholesterol oxidase
~ which is completely free from traces of detergent and,
"J therefore, is sufficiently storage-stable and, on the other~ hand, provide9 an activity of the enzyme during use thereof;~ 20 which corresponds to that of the freshly prepared enzyme
but which has not been subjected to the removal of small
~- amounts of detergents.
Thus, according to the present invention, there i8
provided a proce~s for the activation of cholesterol
oxidase, wherein at least one ~urface-active compound with
~;~ lipophilic and hydrophilic propertieY i~ added to the
x cholesterol oxidase before the u~e thereof.
~; .
As surface-active compounds with lipophilic and
hydrophilic properties, there are preferably used non-ionic
detergents which contain at least one hydroxyl group in the
... .
.,
.
-- 3 _
1~)4458Z
molecule. Polyoxyethylene derivativeq of alkyl, aryl and
aralkyl alcohols are preferably employed. Example~ of
such preferred surface-active compounds include polyoxy-
ethylene alkyl ethers, polyoxyethylene alkyl-carboxylic acid
ester~, polyoxyethylene-sorbitan alkyl carboxylic acid
esters, polyoxyethylene-glycerol alkyl carboxylic acid
esters, polyoxyethylene-alkylamines, polyoxyethylene-
polyoxypropylene block polymers, polyoxyethylene-alkyl thio-
ethers ànd polyoxyethylene alkyl aryl ethers, Specific
- 10 examples thereof include hydroxy-polyethoxydodecane,
ethyleneoxy adducts of alkyl-phenols, polyethoxy-ethylene
derivative~ of sorbitan anhydrides and the like. Polyoxy-
ethylene derivatives modified with mercaptans are also
equally suitable.
According to the present invention, the above-
mentioned surface-active compounds are preferably used in
an amount of between about 0.005 to 0.1 wt.%, referred to
the aqueous enzyme solution.
Considerable number~ of the above-mentioned surface-
; 20 active materials are commercially available and differ
from one another by the number of oxyethylene groups
.. . .
~' present therein, The most useful ones are those which are
water-soluble and which contain, on average, 5 to 20 oxy-
ethylene groups per molecule. Lower oxyethylated types of
compounds are usually only dispersible and are, therefore,
less useful. Higher ethoxylated compounds (greater than 25)
r~ .
are admittedly water-soluble but are ~o hydrophilic that
-their effectiveness is reduced.
Similarly good results are also obtained with
physiological surface-active substances which satisfy
. . .
.` ,
- 4 -
:: ~
58Z
the above-given de~inition, for example, desoxycholates.
In addition, other ~urface-active substances with
lipophilic and hydrophilic propertie~ which contain at
least one hydroxyl group, can al~o be used. Examples of
such compounds include ethanol, butyl-diglycol and hexylene-
glycol. However, lower mono- and dialcohols of this type
must be added in relatively large amounts of 10 to 20 vol. %
for the achievement of the desired activation and the
suitability thereof must be ascertained experimentally in
each case. Thus, for example, no activation is achieved
with methanol, ethylene glycol, polyethylene glycol,
cyclohexanol, glycerol, lecithin and saponin. Surfacé-
active compound~ which have lipophilic and hydrophilic
properties but which do not contain a hydr~xyl group in
the molecule can a}so be used.
Of the anionic wetting agents, the salts of bile
acids, such as cholic acid, taurocholic acid and desoxy-
cholic acid, are espejcially preferred, as well as fatty
acid salts and fatty acid sarcoside~. Of especial importance
are the sulphuric acid derivatives which, a~ is known, have
the additional property of stabilising the coloured radical
oxidation products of some indicators, such as o-tolidine
~ and heterocyclic azines. These include, for example, the
; following classes of compounds: sulphosuccinic acid esters,
alkyl aryl ~ulphonate~, alkyl sulphates and alkyl polyoxy-
ethylene sulphate~.
Of the cationic wetting agents, there can be used,
`~ for example, alkyl pyridinium and trimethyl ammonium salt~,
as well as more complex compounds, for example, benzethonium
chloride.
- - 5 -
~0~58Z
Of the amphoteric wetting agents, there can be used,
for example, the imidazolium betaines.
< Particular example~ of surface-active compounds which
can be u~ed according to the present invention include
sodium (2-ethyl-hexyl)-sulphosuccinate, sodium dodecyl
sulphate, sodium oleate, benzalkonium chloride and cetyl-
pyridinium chloride.
The ionic or amphoteric detergents are preferably
used in admixture with the above-mentioned non-ionic
~ 10 detergents, the appropriate amounts thereof corresponding
- to those of the non-ionic detergent.
The non-ionic, surface-active detergents containing -;
at least one hydroxyl group in the molecule are preferred
becauae they provide an approximately 5 to 10 times greater
~ activation and thus a corresponding increase of the reaction
; velocity or shortening o the period of reaction in comparison
with the other ~urface-active substances which can be employed.
They are alJo effective in smaller amounts.
By alkyl radicals, there are here to be understood
those containing up to about 20 carbon atoms and especially
those containing 12 to 18 carbon atoms.
The inactivation of cholesterol oxidase in the
pre~ence of trace~ of detergent is e~pecially marked when
the enzyme i~ present in ammonium ~ulphate ~olution. The
following Table I shows the ~torage stability of the
enzyme in LM ammonium sulphate ~olution at 33C. in the
i presence of differing amounts of detergent. The experiments
`~ were carried out with an octyl-phenol-ethylene oxide adduct.
... .
.~ . .
.. .
. ~
, - _
.~, . . . .
:;, - , ,
:. . ~ . , :.
, .. . . ~ .
j., . :
lV~58'~
TQBLE I
Activitv of cholesterol oxidase in %,
referred to freshlv prepared enzyme
.
storage detergent
period _
; in day~ without 0 02%0.05% 0.1% 0.6%
89% 60Yo lO~o 7% 3%
. 22 85% 4~h 1% 1% 1%
The effective amount of surface-active compound
used in the process according to the present invention depend~
upon it~ molecular weight and upon the degree of its
j hydrophobic and hydrophilic properties. ~oo small an
amount does not bring about an activation but too large an
amount leads to the result generally known for surface-
active substances of denaturing ths enzyme. The appropriate
concentration range, as well as the optimum concentration,
can be experimentally determined for all appropriate
ourface-active agento. Such ranges are, for example, 0.15
r to 0~6 wt.% for hydroxypolyethoxydodecane, 0.03 to 0.1 wt.%
for octylphenol-ethylene oxide adducts, 0.15 to 0.6 wt.%
for polyoxyethylene derivatives of sorbitan anhydrides,
0.03 to 0.05 wt.% for sodium desoxycholate and 0.005 to
` 0.02 wt.% for mercaptan-modified alkyl-phenol ethylene
oxide adducts (for example Sterox SE*).
Of the non-ionic surface-active polyoxy-ethylene
compounds, those are especially useful which have a balance
`` ratio of hydrophobic residues to polyoxyethylene chain and
~ are water-soluble. A measurement value for the ratio of
`f hydrophobic residue and polyoxyethylene chain is the so-
*trademark
.
.''' . `:
1(~4458Z
called HLB value (cf. W. C, Griffin, J. Soc. Co~metic. Chem.,
1, 311/1950 and 5, 249/1954).
Those ~urface-active materials with HLB values
between about 10 and 17 are especially useful. These
value~ are, however, only to be regarded as being optimum
standard value~ ~ince the effectivene~s also depends upon
the nature of the hydrophobic residue.
~he present invention i8 also concerned with a
diagno~stic agent for the detection and determination of
cholesterol and of cholecsterol esters in body fluids which
can be used for carrying out the above-deocribed process.
; The determination of cholesterol is of conssiderable importance in medical diagnosi~,
In clinical chemistry, rapid tests are being used
more and more for the detection of substances in body fluids.
Although they frequently do not provide sufficiently
accurate results, nevertheless, they do permit a quicker
and cheaper indication of routine and large-scale invest-
igations. The diagnostic agent~ employed for rapid tests
are either absorbent carriers or water-stable films which
contain all of the reagents needed for the reaction. When ~ -
these diagnostic agents are brought into contact with the
body fluids to be te~ted, then colour reactions are
obtained which can be evaluated either on the basis of
compartative colours or with the u~e of simple reflection
photometers,
Experiments for the production of agents for
, cholesterol rapid tests by impregnation of absorbent paper
with cholesterol oxidase, peroxida~e, an Qxidation indicator
and a buffer do not give satisfactory results because the
.
-- 8 --
- :: - - -. . - .
1~)4~S8Z
teqt papers thu~ obtained do not react with cholesterol-
~ontaining ~erum of the usual concentration.
We have now found that useful test papers or test
films are obtained, which react in graduated stage-q with
cholesterol-containing serum when the carrier additionally
contain~ at least one of the above-described ~urface-active
agents.
~ hus, according to the present invention, there is
also provided a diagnostic agent for the detection and
determination of cholesterol and of cholesterol esterq in
body fluids which comprise~ a carrier or a synthetic resin
, film which i~ impregnated with or has embedded therein
doleqterol oxidase, a system for the detection of hydrogen
peroxide, a buffer and at lea~t one of the above-described
surface-active agents. me surface-active agent is prefer-
ably present therein in a concentration of 2 to 3~/0 and
more preferably of 10 to 20Yo~ referred to the solid reagents.
'i A preferred Jystem for the detection of hydrogen
peroxide compri~es peroxidase and an oxidation indicator
20 optionally together with a swelling agent and/or a
stabiliser.
For the detection of cholesterol in serum, test
papers are outstandingly useful which have been obtained
by the impregnation of absorbent papers with the necessary
reagent~. If, however, it is desired to detect cholesterol
in whole blood, then the test papers are preferably rendered
- hydrophobic, for example, in the manner described in German
Patent Specification No, 1,598,048 or are coated with a
~emi-permeable membrane of celluloqe esters. However, for
.. .
.
:
; _ 9 _
-: ; . .. -- . :
,
~: .
1~)4~158Z
the detection of cholesterol in whole blocsd, it iisi ei~pecially
preferred to use test films, such ais are obtainable according
to German Patent Specification No. 1,598,153 containing the
neceissary reagents.
For the preparation of such teist films, the reagents,
for example, cholesterol oxidase, peroxidase, buffer,
indicator and optionally a swelling agent, are stirred,
together wlth the surface-active materials to be used
according to the preisent invention, into an aqueous dis-
persion of a film,forming polymer. This dispersion is
then coated as a thin film and left to dry. When
cholesterol-containing blood i3 dropped onto such a
reagent-containing film and then wiped off after about one
minute, colorations are al~o obtained, the colour depth of
which depends upon the amount of cholesterol present in
the blood. The3e colorations are particularly suitable
for a quantitative evaluation with simple remission
photometers.
The diagnostic agent according to the present
invention can be used for the detection and determination
of free choleisterol. If, however, cholesterol eisters are
also present, then the choleisiterol must firist be liberated
therefrom. Thiisi can be carried out in known manner, for
example, by saponification with aqueous alkali. However,
it iis particularly advantageous to split the ester with
choleisterol eisterase, which iisi preferably isolated from
micro-organisms, since it is then possible to work under
very mild conditions.
The choleisterol esterase can be added to the body
fluid, ~ollowed by incubation. This process can be
. ~
.
-- 10 _
, ~:
1~4458Z
carried out, for example, by drawing the body fluid into
capillarie~, the inner walls of which are coated, in a
manner analogous to that described in German Patent
Specification ~o. 2,240,672, with chole~terol esterase and
optionally with adjuvant materials. After incubation in
the capillarie~, the body fluid is then applied to the
test strips in the above-described manner. If free and
esterified cholesterol were both present in the body fluid,
then, of course, the sum of the two is detected. However,
the cholesterol esterase can also be incorporated espec-
ially advantageou31y into the test strips to give diagnostic
agent~ with which, in one step, the sum of the free and
e3terified cholesterol can be detected and determined
Systems which can be used for the detection of the
hydrogen peroxide formed by the oxidation of the choleRterol,
for example, peroxidase, buffer, oxidation indicator and
optionally a swelling agent and the like, are known, for
example,from the descriptions of rapid tests for glucose.
Example~ of component8 for thi8 preferred system are given
in the following:
Of the peroxidases, that from horseradish i~
especially preferred but lactoperoxidase and the like can
also be employed.
As buffer~, there can be u~ed those which are
conventional, for example, phosphate, citrate and borate
buffers. The pH value which they give on the diagnostic
agent should be between 4 and 9 and preferably between 5
and 8.
As oxidation indicators, there can be used various
classes of compounds, namely benzidene derivatives, for
-- 11 --
~,
. ~ .. . .
:, ,
~.
.- :
i~4582
example o-tolidine and o-vanillin derivatives according to
German Patent Specification No. 1,598,133, or heterocyclic
azines according to German Patent Specification No. 1,648,840.
The formulation~ for the diagnostic agent~ and
especially tho~e for te~t film_, can alqo contain a con~
ventional _welling agent, such as 30dium alginate, carboxy-
methyl-cellulo3e or the like, a~ well aq a stabilising
agent for the enzyme, for example, dithioerythritol.
Substrate materials for test films are synthetic
re9in di9persion~ of, for example, polyvinyl propionate
or acetate. Into these are stirred all the necessary
reagents, preferably in dissolved form, whereafter the ~;-
mixture obtained is then coated into thin films and dried.
Test papers can be produced by dissolving the re-
~` agents in water or in a mixture of water and organic ~olvents
with which filter papers are impregnated and then dried.
However, the paper can first be impregnated with the
water-~oluble reagent~ and then impregnated with, for
example, the indicator~ in organic solution.
The following Examples are given for the purpo~e
~1 of illu~trating the present invention:-
-~ Exam~le 1.
0,05 ml. of serum are added to 10 ml. 0.5M potassium
`~ phosphate buffer of pH 7.5 which contains 0.4% hydroxy-
,4 polyethoxydodecane. The extinction (El) is read off at
2~0 nm in a suitable spectrophotometer and the reaction
started with 0.02 ml. (0.1 U) of storage-stable cholesterol
oxidase, freed from traces of detergent, in lM aqueous
ammonium ~ulphate ~olution. After 3 minutes, the extinction
'
- 12 -
. . , . , : -
1~)44S8'~
(E2) is read off. The concentration of the cholestenone
formed and thus of the cholesterol is given by the difference
between the first and second readings, having regard to
the molar extinction coefficient~ for cholesterol at
240 nm. The mea~urement of a typical qample gave 62 mg.%
free cholesterol and 167 mg.% total cholesterol (after
saponification).
A comparative determination but without the addition
of the surface-active agent required a reaction time of
15 minutes.
The separation of traces of detergent for the
improvement of the storage stability of the enzyme is
preferably carried out with the use of hydrophobic
adsorption resins, Especially preferred for this purpoQe
are, for example, the material which is commercially
available as "Bio-Beads"* from Biorad, as well as the
products obtainable from Rohm & Haas under the designation
XAD*-re~ins,
Example 2.
0.05 ml. serum are added to 10 ml. 0.5M potassium
phosphate buffer of pH 7.5 which contain~ 0.02% hydroxy-
polyethoxy-dodecane and 0.03% qodium desoxycholate. The
extinction (El) is read off at 240 nm in a suitable spectro-
photometer and the reaction is started with 0.02 ml.
(= 0.1 U) storage-stable cholesterol oxidase, freed from
traces of detergent, in LM aqueous ammonium sulphate
solution.
J . After 3 minutes, the extinction (E2) is again read
off, The concentration of the cholestenone formed and
` 30 thus of the chole3terol i~ given from the difference
*trademark
- 13 _
.--~, ' - ' :.- - .
58Z :-:
between the first and second readings, having regard to
the molar extinction coefficients for cholestenone at '~ ~
240 nm (~ = 15.5 cm2/~mol). ~ -
- The meaqurement of a typical sample gave 65 mg.%
free cholesterol and 170 mg.% total cholesterol (after
saponification)
Example 3.
0.05 ml. serum are added to 10 ml. 0.5M potassium
, pho~phate buffer of pH 7.5 which contains 0.02% hydroxy-
polyethoxy-dodecane and 0.1% secondary alkyl sulphate.
' The extinction (El) is measured at 240 nm in a suitable
,' spectrophotometer and the reaction is started with 0.02 ml.
;' (= 0.1 U) storage-stable cholesterol oxidaqe, freed from
" traces of detergent, in LM aqueous ammonium sulphate
j ~olution.
, After 3 minute~, the extinction (E2) is again read
" off. The concentration of the cholestenone formed and
, thuA of the cholesterol is given by the difference between
j the fir~t and second readings, having regard to the molar
¦ 20 extinction coeffici,ent9 of cholestenone at 240 nm.
The measurement of a typical sample gave 62 mg.%
free cholesterol and 167 mg.% total cholesterol (after
saponification).
Example 4.
' Filter paper (Schleicher & Schull No. 597 ~F Ind.*)
3~ is impregnated with a solution of the following composition
.3, and dried at 40C.:
~ 1 M citrate buffer, pH 5.25 20 ml.
; cholesterol oxidase (60 U/mg.) 0.1 g.
peroxidase (70 U/mg.) 0.05 g.
distilled water ad 100 ml.
*trademark
. ~ . - , .
- 14 _
., . . , .. - . .. .
. . , . , , , - ., - . .
.:., .. , :-
16~58Z
This paper i~ then impregnated with ~olutions of
0.2 g. o-tolidine in 100 ml. methylene chloride which, in
addition, each contain 1 g. of the following surface-active
materials:
a) polyoxyethylene tributyl-phenol ether
b) polyoxyethylene ~orbitan monolaurate
c) polyoxyethylene-nonyl-phenol ether
d) polyoxyethylene lauryl ether
e) polyoxyethylene cetyl ether
f) polyoxyethylene stearate
g) polyoxyethylene dodecyl thioether.
After drying, te~t papers are obtained which react
with chole~terol-containing sera with a green colour. If
the sera also contain cholesterol eætero, then stronger
green colorations are obtained if the sera have previously
been mixed with a drop of cholesterol esterase qolution.
A test paper which does not contain one of the above-
mentioned ~urface-active materials does not react with
the sera.
Example 5.
Filter paper (Schleicher & Sch~ll No. 597 NF Ind.*)
i~ impregnated with a solution of the following composïtion
and dried at 40C,:
, 1 M citrate buffer, pH 7 20 ml.
cholesterol oxidase (60 U/mg.) 0.1 g.
chole~terol e~terase (18 U/mg.) 0.25 g.
, peroxidase (70 U/mg.) 0.05 g-
distilled water 100 ml.
This paper is impregnated with solutions of 0.2 g.
o-tolidine in 100 ml. methylene chloride which, in addition,
*trademæk
.'
~ - 15 -
582
each contain 0.5 g. of one of the following surface-active
materials:
a) polyoxyethylene cocoate
b) polyoxyethylene oleate
c) polyoxyethylene polypropylene glycol
d) polyoxyethylene stearylamine
e) polyoxyethylene glycerol monolaurate.
After drying, test papers are obtained which react
with a green colour with tho~e ~era which contain
chole9terol and/or chole~terol esters. Test papers without
surface-active agent show no reaction.
Practically the same behaviour is shown by test paperQ
which contain the same amount of citrate buffer with a pH
of 5.25 or 6.
le 6.
Paper pre-impregnated in the manner described in
Example 4 iJ ~ubsequently impregnated with ~olutions of
0.3 g. o-tolidine in 100 ml. acetone which contain 1.5 g.
of one of the following surface-active material3:
20, a) dioctyl sodium Julphosuccinate
b) sodium dodecyl-benzene-sulphonate
c) ~odium lauryl polyglycol ether ~ulphate
d) sodium lauryl sarco3inate
e) ~odium laurate
f) cholic acid
g) desoxycholic acid
h) sodium taurocholate
These test papers possess practically the ~ame
properties as the test papers produced according to
' 30 Example 4 but the reaction colours are stable for a longer
period of time.
- 16 -
.
,, : ~ . .-
: .
, . .. .. .
~)4~S82
- Te~t papers with a phosphate buffer of pH 7 posseAs
similar propertie~.
Example 7.
Paper pre-impregnated in the manner described in
Example 5 is impregnated with ~olutions which contain
0.4 g. o-tolidine and 0.5 g. of one of the following
surface-active materials:
a) lauryl pyridinium chloride
b) benzethonium chloride
c) cetyl trimethyl ammonium chloride
d) l-hydroxyethyl-l-carboxymethyl-2-alkyl-imidazolinium
betaine.
The properties of these test papers correspond to
those of Example 5.
, Example 8.
Filter paper (Schleicher & Sch~ll No. 597 ~F Ind.)
i9 impregnated with a ~olution of the following compo~ition
~i and dried at 40C.:
lM pho~phate buffer, pH 6 25 ml.
cholesterol oxida~e (60 U/mg.)0.1 g.
peroxidase (70 U/mg.) 0.05 g.
cholic acid 1.0 g,
' acetone 10 ml.
distilled water ad 100 ml.
!,, This paper i~ subsequently impregnated with solutions
of oxidation indicators in 100 ml. acetone.
The amount of indicator used, the chemical name
thereof and the colour reaction with cholesterol-containing
era are summarised in the following Table II:
- 17 -
158Z
T~BLE II
. .,
amount indicator reaction
:
O.3 g. o-vanillylidene-E~vanilloyl-violet
hydrazone
0.2 g. azino-bis-(N-ethyl-benzthia- green
zolone-2-sulphonic acid-5)
diammonium salt
0,1 g. azino-bis-(N-alkylquinolone- blue-
2-~ulphonic acid-6) di- violet
ammonium salt
0,1 g. bii-(N-alkyl-quinolone-2)- violet
0,1 g. (~-methyl-benzthiazolone-2)- blue-
~-ethyl-quinolone-2)-azine green
0,3 g, (~-methyl-benzthiazolone-2)- blue
l-phenyl-3l,4-dimethyl-tri-
azolone-5)-azine
Exam~le 9,
A mixture is prepared from the following components:
polyvinyl propionate dispersions 45 g,
sodium alginate, 1,85% in O,SM 35 g.
phosphate buffer, pH 5,5
cholesterol oxidase (60 TJ/mg~ ) 0.5 g-
peroxidase (70 U/mg,) 0.25 g,
dioctyl sodium sulphosuccinate 1 g,
o-tolidine, dissolved in 6 ml. acetone0.6 g.
water 50 ml.
This mixture is either spread out to give a film
~ with a wet film thickness of about 300 nm or is painted
o~ 30 on to a solid carrier and dried at 35C. When blood
containing chole~terol is dropped on to the film and the
blood wiped off after one minute, then, depending upon
~ .
.
- 18 _
. . : ~ . -
58'~
the cholesterol concentration, green coloration~ of
varying intensity are obtained.
If, in addition, 1.0 g. cholesterol esterase is
added to the above-de~cribed formulation, then films are
obtained with which it is also possible to determine
; increased cholesterol ester content~.
, -- 19 _
