Note: Descriptions are shown in the official language in which they were submitted.
-2-
The present invent;on is concerned wi~h a process
and an agent for the activation of cholesterol esterase,
especially in the case of the enzymatic reaction-of
esters in reaction mixt~res. Such reaction mixtures are
used. inter alia, in clinical-chemical analysis and in
the chemical analysis of foodstuffs.
Known problems in the designing of tests depending
upon the use of cholesterol esterase are the saving of
the expensive enzyme, the achievement of comparatively
high reaction rates and the widest possible choice of
the nature and content of the surface-active agent~ and
buffers according to superordinate points of view.
In a buffered medium which contains no further _`
components, the cholesterol esterase develops no or only
a very slight activity towards its substrates. There-
fore, it is necessary to find activating additives for
the enzyme by means of which the ester splitting is
increased.
Some surface-active agents are already known as
activating additives (see, for example, U.S. Patent
Specification ~o. 3,884,764, Federal Republic of Germany
Patent Specifications Nos. 2,409,696; 2,512,605,
2,512,585 and 2,506,712, and Biochim. Biop}lys. Acta,
270, 156-166/1972), examples of which include bile acids,
"Triton"*X-100 and thesit. A comparatively high salt
content of the medium promotes their activating action,
* trade mark
, -3-
However, not all buffer systems are suitable in
the same manner and this also applies to the surface-
active agents. Under certain conditions, there are here
found those with a good and less good action, as well as
those which have no activating action at all. Which
surface-active agent is, ln this regard, suitable can
usually not be predicted.
A common feature of the activating surface-active
agents is that they manifest their optimum activating
action in a particular medium and in a particular range
of concentration. However, the optima can lie in un-
favourably high concentration ranges. Outside the optima,
a comparatively high amount of the expensive enzyme does
not react in an optimum manner at comparatively high
reaction rates.
Furthermore, an increase of activity by means of
optimisation of the surface-active agent content and
increase of the ionic strength can only be achieved to a
relatively limited extent.
Since,in normal cases, the reaction mixtures in question
also contain further enzymes and substrates for subsequent
and indicator reactions, there is no free choice of huffer
and of surface-active agent, as well as of the optimis-
ation of the particular amounts to be employed.
On the contrary, in the ascertainment of the
reaction conditions, account must always be taken of the
interactions of all the reaction components with one
UI
-4-
another ancl especially of possible inhibitions or dis-
advantageous influences on ~he stability of other
enzymes due to the surface-active agents and/or high
ionic strengths.
Furthermore, in the designing of a test system,
solubility limits and formulation problems must also be
taken into account.
In the latter case, the Frequently liquid to wax-
like surface-active agents can rarely be introduced, in
the amount needed for activation, into the solicl mixtures
which are to be dry and flowable. This is of particular
importance in the case of one-pot reagents.
Finally, there is also to be borne in mind that `-
most of the surface-active agents in question are not
definite compounds but rather mixtures of substances.
Consequently, batch differences arise which also manifest
themselves in the activating action. Therefore, it can
be very disadvantageous when activation depends solely
upon a surface-active agent.
Consequently, there is a considerable need for
further and better agents and processes for the activation
of cholesterol esterase.
It is an object of the present invention to over-
come the above-described disadvantages and to provide a
process and an agent of the said kind in which less
cholesterol esterase is used, higher reaction rates can
be achieved under certain concli-tions, operating can be
carried out with low ionic s-trength and surface~active
agents are only needed in the smallest possible concen-
trations which alone display only a poor or even no
activating action.
Thvs, according to the present invention, there
is provided a process for activating cholestero7 esterase
in ion-containing solution by the addition of at least
one surface-active agent, wherein there is used
a) as surface-active agent, a polyethoxy ether or ester
and/or a bile acid com~ound and/or a salt of a fatty
- acid which contains 6 to lo carbon atoms, in combin-
ation with
b~ a synergistically~acting alcohol selected from
straight-chained, branched and cyclic aliphatic
alcohols which contain 5 to 12 carbon atoms, option-
ally halogen-substituted aromatic alcohols and
aliphatic alcohols which contain up to 3 carbon atoms
and are substituted by several halogen atoms.
The present invention depends upon the surprising
ascertainment of the fact that the above-mentioned
alcohol~, which themselves do not display any activating
action on cholesterol esterase, markedly strengthen the
~er se known activating action of certain surface~active
agents and, in the case of other surface-active agents,
which alone do not activate, bring about an activation.
1101
r --5~
The result of this is that use can be made of non-
activating surface-active agents w'nich are commercially
widely available. Use can also be made of ionic surface-
active agents which possess a desirable definite molecular
structure and have a solid consistency but w~ich were
hitherto not suitable because of their poor activating
properties. Furthermore, enzyme can be saved, the maximlm
achievable reaction rate can be increased, the necessaryO
ionic strength can be lowered and, finally, due to the wide
range of possibilities of combination, the esterase-
activating system ca~ be adapted to the conditions
dictated by further components of the reaction mixture.
Examples of polyoxy ethers which can be used
according to the present invention include fatty alcohol
polyglycol ethers, such as polyoxyethylene lauryl ether
and hydroxypolyethoxydodecane, alkylaryl ethers and
arylalkyl ethers, such as polyoxyethylenenonylphenyl
ether and polyoxyethyleneoctylphenyl ether, and fatty
acid esters, such as polyoxyethylenesorbitan monolaurate
and the like. In the case of the bile acid compounds,
cholic acid and desoxycholic acid and their alkali metal
salts are preferred. In the same way, of the fatty acid
salts, the alkali metal salts and more e~specially the
sodium salts are preferred.
Typical examples of straight-chained, branched and
cyclic aliphatic alcohols which can be used according to
the present invention include pentanol, tert.-amyl alcohol,
0
methylpentanol, hexanol, cyclohexanol, octanol, iso-
octanol, decanol and dodecanol, Examples of aromatic
alcohols which can be used inc:Lude phenol and dihydroxy-
naphthol. The halogen-substitllted phenols and naphthols,
such as the mono-, di- and trichlorophenols, are pre-
ferred. As polyhalogen-substituted aliphatic alcohols,
there can be used, for example, dichloroethanol, tri-
chloroethanol, dichloromethanol, trichloromethanol, tri-
chloropropanol, tetrachloropropanol and the like. In
the case of the aromatic and aliphatic alcohols, the
halogen substituents can be chlorine, bromine and fluorine,
chlorine being preferred. The aromatic alcohols can also
contain one or two methyl or ethyl radicalsO
Which of these alcohols (hereinafter called
adjuvants) is especially suitable for a particular case
can easily be ascertained by preliminary experiments.
Conditions, such as the nature and strength of the buffer
systel~, the nature and concentration of the surface-active
agent, as well as the storage stability and desired con-
sistency of the storage form of the reaction mixture, are
mostly predetermined. These conditions can then be taken
into account by selection of the suitable adjuvant and
adaptation of the amount to be added. In general, we
have found that concentrations of from 1 to 300 mM of
adjuvant/litre give good results.
The alcohols are preferably employed in amounts of
from 0.01 to 5% by volume in the case of liquid alcohols
113111C~
-8-
and of 0.01 to 5% by weight in the case of solid alcohols.
The amount used is more preferably 0.05 to 3/0 by volume
or weight, respectively.
In the polyethoxy derivatives, the alkyl radicals
generally contain 8 to 22 carbon atoms and the aralkyl
and alkylaryl groups preferably contain the phenyl
radical. The glycol residue of these surface-active
agents generally contains 3 to 25 oxyethylene groups.
The surface-active agents are preferably used in
an amount of from 0.05 to 5% by volume in the case of
liquid substances and of 0.05 to 5% by weight in the
case of solid substances, the especially preferred amount
being from 0.1 to 3% by volume or weight, respectively.
The present invention also provides an agent for
the activation of cholesterol esterase in ion-containing
solution, comprising at least one surface-active agent
based upon a polyethoxy ether or ester and/or a bile
acid compound and/or a salt of a fatty acid containing
6 to 10 carbon atoms, together with a synergistically-
acting alcohol selected from straight-chained, branched
and cyclic aliphatic alcohols which contain 5 to 12 carbon
atoms, optionally halogen-substituted aromatic alcohols
and halogen-substituted aliphatic alcohols containing up
to 3 carbon atoms.
The present invention can be used in conjunction
with all kinds of measurements and reactions in which
cholesterol esterase is employed in the reaction mixture.
1~3~ .0
Such reactions mixtures are generally known to the expertand are,
insofar as they are intended for analytical purposes,
descri;ed, for example, in "Methoden der enzymatischen
Analyse" by ~I.U. Bergmeyer, pub. Verlag Chemie T~einheim.
I~lerefore, a more detailed description of such reaction
mixtures is here unnecessary. By means of the present
invention, it is possible to save enzyme, to achieve
higher reaction rates under given conditions, to use
buffers of lowerionic strength, to use surface-active
agents in lower concentration and also to make use of
those surface-active agents which alone show insufficient
or even no activating actionO The present invention can
be used in conjunction with cholesterol esterase from -
micro~organisms or of other orlgin, for example from
pancreas or liver.
The following Examples are given for the purpose
of illustrating the present invention, reference thereby
being made to the accompanying drawings, in which:
Fig.l is a schematic illustration of the apparatus
used for the determination of the cholesterol
esterase activity, and
Fig.2 is a gra~hic illustration of the extinction
changes per unit time occurring in the case
of the method used, which were used for the
determination of the rate of the ester
splitting.
The following gives the chemical composition of the
r
surface-active agents referred to by their trade marks
alld common names:
"Brij"*35 polyoxyethylene lauryl ether
"Genapol"*OX-100 fatty alcohol-polyglycol ether
"Tergitol"*NPX polyoxyethylenenonylphenyl ether
(about lo . 5 oxyethylene groups)
thesit hydroxypolyethoxydodecane
"~riton"*X-100 polyethoxyoctylphenyl ether (9 to 10
oxyethylene groups)
"Tween"*20 polyoxyethylene sorbitan monolaurate
For the demonstration of the activating action of
the synergistic combination of the present invention to
be investigated on cholesterol esterase, use was made of
the apparatus illustrated in Fig.l of the accompanying `~
- drawings.
From a temperat~re-controllable reaction vessel 1
(25Co), in which the ester splitting takes place, there
is continuously taken off, via a peristaltic pump 2,
through a conveyor tube 3 of definite cross section,
0.32 ml. of reaction mixture per minute. Via the same
pump, there is continuously conveyed through a second
tube 4, from a storage vessel 5, 1.0 ml. of indicator
solution per minute, which is brought together, down-
- stream of the pump 2, with a current of air 6 of con-
stantly 0.6 ml. air/minute and thu~ segmented and then
combined with the previously described reaction mixture
current. The total stream thereupon passe~ through two
glass coils 7, a mixing up of the liquids and the
* trade mark
B
c
1~31~
necessary reaction time for the indicator reaction to
the end point thereby being guaranteed. By taking off
a current of 1.2 ml./minute immediately before a cuvette
9 through a tube 8 in a vertically downward direction,
the air bubbles are again removed. The non-segmented
residual current is passed, for photometric measurement
(at 546 nm), into the flow-through cuvette 9 (1 cm. optical
pathlenght)and the measurement values recorded by the
recorder 10.
Because of the constant conditions with regard to
the indicator reaction, changes in the height and course
of the measurement signal are solely dependent upon the
processes-in the reaction vessel. If an ester splitting
there takes place~ then its kinetic course brings about
an increase of the measurement signal which is registered
by the recorder lo as a positive increase. The steepness
of the increase is a measure of the rate ~f reaction and
thus of the cholesterol esterase activity in the reaction
batch.
Because of the separate course of the ester splitt-
ing and indicator reaction, a mutual influencing is
excluded. Of course, other indicator systems, including
the oxygen electrode, can also be usedO
Indicator solution:
O.lM/l. potassium phosphate buffer, pH 7.0
1.5 mM/l. 4-aminoantipyrine
5 mM/l. phenol
113~1iO
-12-
3%o hydroxypolyethoxydodecane
8 U/ml. peroxidase
1 U/ml. cholesterol oxidase
The indicator solution first reacts with free
cholesterol to give cholestenone and hydrogen peroxide.
The resulting hydrogen peroxide is reacted with phenol
and 4-aminoantipyrine to give a red-coloured material,
the absorption of which is measured photometrically. The-
amount of resultant coloured material is proportional to
the concentration of free cholesterol. The concentration
of free cholesterol is made up of the proportion of free
cholesterol already present in the substrate, as well as
of cholesterol liberated by the cholesterol esterase.
Batch in the reaction vessel:
0.5 ml. substrate (control serum, 360 mg. total
cholesterol/dl.)
surface-active agent, depending upon the experiment
adjuvant, depending upon the experiment
buffer to make up to 5 ml.
Start with 50 ~1. of a cholesterol esterase stock
solution, whereupon 0.003 U/ml. cholesterol esterase are
present in the batch. In the case of activity graduations,
this amount is varied.
After the start and mixing have taken place, with
the pump 2 running, the tube 3 is dipped into the sol-
ution in the reaction vessel 1 for about 10 minutes.
Subse~uently, for a better separation of the signals, in,
~3~ 0
-13-
in each case, about 10 segments, air and double distilled
water are alternatingly sucked up. The next determination
can then follow.
The following Examples illustrate the saving of
enzyme (a) made possible by the present invention, the
achievement of high rates of reaction under given con-
ditions ~bl, the use of buffers of lower ionic strength (c),
the use of surface-active agents in lower concentrations
(d) and the use of those surface-active agents which,
under certain conditions, only show an insufficient or
even no activating action (e).
a) Savinq of cholesterol esterase.
The possibility of saving cholesterol esterase is
demonstrated as follows:
First, there is carried out an enzyme activity
graduation in the absence of adjuvants. The comparative
measurements then take place with the addition of
adjuvants in the case of a cholesterol esterase use of
0.003 U/ml. On the basis of the indicated relationship
between introduced activity and resulting rate, there is
then determined the increase of activity achieved.
Exam~le 1.
In 0.1 M tris tartaric acid buffer, pH 8.0, 5/~o "Triton"
X-100
-14-
introduced activity resulting rate
0 U/ml. 0 ext./10 minutes
0.0012 0.012
0-0030 0.032
0.0045 0.052
O.C060 0.058
0.0089 o.og~
1.1 Addition of 2 vol.% tert.-amyl alcohol
introduced activity 0.0030 U/ml.
rate achieved 0.046 ext./10 min.
necessary amount of enzyme for 0 0044 U/ml
this rate without adjuvant
activity achieved 147%
1.2 Addition of 0.4 vol.% 4-methYlpentan~2_ol.
introduced activity 0O003 U/mlO
rate achieved 0.065 extl/10 minO
necessary amount of enzyme for 0 0063 U/ml.
this rate without adjuvant
activity achieved 210%
1.3 Addition of 1 vol.% cyclohexanol.
introduced activity 0.0030 U/ml.
rate achieved 0.077 ext./10 min.
necessary amount of enzyme for 0O0075 U/mlO
this rate without ad~uvant
activity achieved 250%
Exam~le 2.
In 0.1 M potassium phosphate buffer, pH 7.0, 1.5%
"Tergitol" ~PX
110
-15-
introduced activ ty resulting rate
0.0012 U/ml. 0.019 ext./10 min.
0.0030 U/ml. 0.030 ext./10 min.
0.0060 U/ml. 0.055 ext./10 min.
0.0090 U/ml. 0.091 ext./10 min.
o.ol~o U/ml. 0.120 ext./10 min.
2 1 Addition of 0 4 vol~% 2,2,2-trichloroethanol.
introduced activity 0.003 U/ml.
rate achieved 0.046 ext./10 min.
necessary amount of enzyme for 0.0046 U/ml.
this rate without ad]uvant
activity achieved 153%
2.2 Addition of 2 vol.% tert.-amyl alcohol.
introduced activity 0.003 U/ml.
rate achieved 0O053 ext./10 min.
necessary amount of enzyme for 0.0053 U/ml.
this rate without ad~uvant
activity achieved 177%
2.3 Addition of 1,4 vol.% 4-methylpentan-2-ol.
introduced activity 0.003 U/ml.
rate achieved 0.059 ext./10 min.
necessary amount of enzyme for 0 0059 U/ml
this rate without adjuvant
activity achieved 197%
2.4 A~dition of 2 vol.% cyclohexanol
introduced activity 0.003 U/ml.
rate achieved 0.114 ext./10 min.
necessary amount of enzyme for 0.0114 U/ml.
this rate without ad~uvant
activity achieved 380%
1~3~1~0
--15--
Exam~le 3.
In 0.3 M potassium phosphate buffer, pH 7.0, 5/Oo
"Genapol" ox-loo/5O,/cO sodium caprylate
introduced activity resulting rate
0.0012 U/ml. 0.01 ext./10 min.
0.0030 U/ml. 0.023 ext./10 min.
0.0060 U/ml. 0.038 ext./10 min.
0.0090 U/ml. 0O055 ext./10 min.
0.0150 U/ml. 0.093 ext./10 min.
0.0240 U/ml. 0.138 ext./10 min.
3.1 Addition of 4 mM/l. 3,4-dichlorophenol (0.065 wt.%)
introduced activity 0.0030 U/ml.
rate achieved 0 058 ext./10 min. ~-
necessary amount of enzyme for 0 0093 U/ml
thi-s rat:e without adjuvant
activity achieved 3100/o
3.2 Addition of 10 mM/l. phenol (about 0.09 wt.%)
. ,.~....
introduced activity 0.003 U/ml.
rate achieved 0.064 ext./10 min.
necessary amount of enzyme for 0 0104 U/ml.
this rate without adjuvant
activity achieved 347%
3 3 Addition of 1 vol.% 4-methylpentan-2-ol.
introduced activity 0.003 U/ml.
rate achieved 0.069 ext. /lo min.
necessary amount of enzyme for 0.0112 U/ml.
this rate without adjuvant
activity achieved 373%
~131110
3.4 Addition of 2 vol.~, cyclohexanol.
introduced activity 0.003 U/ml.
rate achieved 0.080 ext./10 min.
necessary amount of enzyme for 0 0129 U/ml
this rate without adjuvant
activity achieved 430%
3.5 Addition of 0.3 vol.% 2,2,2-trichloroethanol.
introduced activity 0.003 U/ml.
rate achieved 0.039 ext~/10 min.
necessary amount of enzyme for 0 0144 U/ml
this rate without adjuvant
activity achieved 4~0%
b) Achievement of higher reaction rates under given
conditions.
In many cases, certain conditions, such as, for
example, buffer and ionic strength, as well as the nature
of the surface-active agent in the reaction mixtures,
are already present on the basis of superordinate points
of view. Such reasons can, for example, be given by the
greater sensitivity of another necessary enzyme also
present in the mixture. In this case, the maximum rate
of the ester splitting achievable in the case of a con-
stant amount of cholesterol esterase used is character-
ised and limited by the nature and concentration of the
surface-active agent. In general, this dependence dis-
plays an optimum in the low concentration range or the
surface-active agent. The rate achieved at this point
cannot, according to the above-described conditions, be
~13i~
-18-
improved upon and, in this regard, represents a l~mit of
the system.
The combinations according to the present invention,
described by way of example in the following, are suitable
for exceeding such limits.
Exam~le 4.
Present: 0.1 M tris/tartaric acid buffer, pH 8.0, "Triton"
X-100, 0.003 U/ml. cholesterol esterase
/Oo "Triton" X-100 in resulting rate
the batch
0/Oo 0 ext./10 min.
lo/oO 0.003 ext./10 min.
3/Oo 0.028 ext./10 min.
5~Oo 0.037 ext./10 min.
7/Oo 0.037 extO/10 minO
10%O 0.037 ext./10 min.
15%O 0.029 extO/10 min.
optimum limit of the system
5%o 0.037 ext./10 min.
Addition of adjuvants takes place at 5/Oo "Triton" X-100.
4.1 Addition of 2 vol.% tert.-amyl alcohol.
rate achieved 0. 046 ext./10 min.
increase to 1240/o
4.2 Addition of 0.4 vol.o,~ 4-methyl~entan-2-ol.
rate achieved 0.065 ext./10 min.
increase to 176%
113~
--19--
4 3 Addition of 1 vol.~ cyclohexanol.
.
rate achieved 0.077 ext./10 min.
increase to 208%
Example 5.
Present: 0.3 M potassium phosphate buffer, pH 7~0,
"Tergitol" NPX, 0O003 U/ml. cholesterol esterase
/Oo "Tergitol" NPX in resulting rate
the batch
0/~ 0 ext./10 min.
2%o 0 ext./10 min.
5D/oO 0.029 extO/lo min.
10%o 0.042 ext~/10 min.
15O/oo 0~049 ext./10 min.
2.0%O . 0 O 039 ext./10 min.
- opt.imum -: ~ limit of-~he system
15O/~ . 0.049 extO/10 min.
Addition of adjuvants-takes place at 15%O "Tergitol" NPX
5.1 Addition of 2 mM~ 1. 3,4-dichloro~henol and 1 vol.%
cyclohexanone.
rate achieved 0.058 ext./10 min.
increaqe to 118%
5.2 Addition o _ vol.% tert.-amy__alcohol.
rate achieved ~ -0~066 ext./10 min.
increase to 135%
5.3 hddition of 3.4 vol.% cycloheYanol.
rate achieved . 0.118 ext./10 min.
increase to 241%
113~
-20-
c) Use of buffers of lower ion strength.
As already mentioned, buffers of higher concen-
tration promote the actlvating aciion of surface-active
agents upon cholesterol esterase. In the scope of the
required freest possible selectability of the conditions,
it is, ho~ever, desirable not to have to have high salt
contents in the solution. The following Example shows
that, according to the present invention, the salt con~
tent can be substantially reduced.
There are compared:
0.3M potassium phosphate buffer, pH 7.0
O.lM potassium phosphate buffer, pH 7.0
By the addition of suitable adjuvants to the O.lM
buffer system, the rates can ba increased in such a
manner that they reach the higher comparative value of
the 0.3M buffer system without adjuvant and, in some
cases, clearly exceed it.
Example 6.
15%o "Tergitol" ~PX, 0.003 U/ml. cholesterol esterase.
6.1. Addition of 2 vol.% tert.-amyl alcohol.
system rate
o.lM buffer without addition 0~030 extO/10 min.
0.3M buffer without addition 00049 ext/10 min.
O.lM buffer with addition 0.053 ext./min.
The comparison value is exceeded.
6 2 Addition of 1.4 vol.% 4-methylpentan-2-ol.
.
system rate
O.lM buffer without addition 0.030 ext./10 min.
1.0
-21-
0.3~ buffer without addition 0 04c ext./10 min.
O.lM buffer with addition 0.059 ext./10 min.
The comparison value is exceeded.
6.3 Addition of 2 vol.k cyclohexanol.
~.
system rate
0 lM buffer without addition 0.030 ext./10 min.
0.3M buffer without addition 0.049 ext./10 min.
0 lM buffer with addition 0.11~ ext./10 min.
The comparison value is clearly exceeded.
Exam~le 7.
10~/oO "Genapol" ox~loo/lo~/Oo sodium caprylate, 0.003 U/ml.
cholesterol esterase
7 1 Addition of 40 mM phenol (about 0.35 wt.%)
. . =
system rate
OolM buffer without addition 0~017 ext~/10 min.
0.3M buffer without addition 0.053 ext./10 min.
O.lM buffer with addition 0.053 ext~/10 min.
The comparison value isAachieved.
7.2 Addition of 1 vol.% 2,2,2-trichloroethanol.
system rate
OolM buffer without additio~ 0.017 ext./10 min.
0O3M buffer without addition 0.053 ext./10 min.
O~lM buffer with addition 00063 extO/10 min.
The comparison value is exceeded.
7O3 Addition of 2 vol.% cYclohexanol.
system rate
o.lM buffer without addition 0.017 extO/10 min.
1131~
-22-
0.3M bufCer without addition 0.053 ext./10 min.
o.lM buffer with addition 0.072 ext./10 min.
The comparison value is exceeded.
d) Use of surface-active agents in lower concentration.
-
If a surface-active agent is able to activate
cholesterol esterase, then, for the complete manifestation
of this property, it must be present in the reaction
mixture in a certain concentration. This surface-active
agent optimum is essentially dependent upon the nature
of the surface-active agent and can, in many cases, be
in an unfavourably high concentration range. It can
thereby result in inhibitions in the case of other
enzymes needed for subsequent reactions and can also
result in difficulties for reasons of solubility and of
formuLation. Below the surface7active agent optimum,
only an insufficient activation is observed. In the
interest of as free a choice as possible of the con-
ditions, it is, however, desirable to be able to use
as many different surface-active agent^s as possible,
including also those with higher concentration optima.
The following Example shows that, according to
the present invention, it is possible to achieve, with
less surface-activ~ agent, the reaction rate of the
concentration optimum and even to exceed it, surface-
active agent thereby being saved.
Example 8. ~i~
0.3M potassium phosphate buffer, pH 7.0, "Tergitol" NPX,
0.003 U/ml. cholesterol esterase
1131~10
--23--
~/Oo "Tergitol" N~X in resulting rate
the batch
0,~,o 0 ext./10 min.
2/~o 0 ext./10 min.
5,~Oo 0.029 ext./10 min.
70/~o 0.042 ext./10 min.
75/OO 0.049 ext./10 min.
20%o 0.039 ext./10 min,
-optimum comparison value
15,'c7O 0.049 ext./10 min.
8.1 Addition of 0.1 vol.,'O 2,2,2-trichloroethanol.
system rate
/oO "Tergitol" without addition 0.049 ext./10 min.
5/Oo "Tergitol" without addition 0.029 extn/10 min.
-5~/Oo "Tergitoll' with addition 0.059 extO/10 min.
The comparison rate is exceededO
8~2 Addition of 2 -vol.% tert.-amyl alcohol.
system rate
15O,'oo "Tergitol" without addition 0.049 ext./10 min.
~/OO "Tergitol" without addition 0.029 ext./10 min.
5O~oO "Tergitol" with addition 0.075 ext./10 min.
The comparison value is clearly exceeded.
~3,3 Addition of l vol~/O cyclohexanol
system rate
15O/oO "Tergitol" without addition 0.049 ext./10 min.
5O/oO "Tergitol" without addition 0.029 ext./10 min.
5/Oo "Tergitol" with additlon0.124 extO/10 min~
The comparison value is clearly exceeded.
-24-
Example 9.
0.3M potassium phosphate buffer, pH 7.0, "Genapol" OX-100
sodium caprylate, 0.003 U/ml. cholesterol esterase
/OO "Genapol" OX-100/sodium rate
caprylate
0~Oo 0 ext~/10 min.
5%O 0 ~ 020 ext./l0 min.
loo~Oo 0Oo53 ext./10 minO
15O/oO 0O 062 ext./10 min
20O/oO 0 ~ 066 ext./10 min.
30%O 0 ~ 068 ext./10 minO
40/Oo 0. 068 ext./10 min.
optimum comparison value
30~/oO 0 ~ 068 ext./10 minO
901 Additlon of 1 vol./0_4~met~
system rate
30/Oo "Genapol"/caprylate without
addition 0. 068 ext./10 min.
3%O "Genapol"/caprylate without
addition 0. 020 ext./10 min,
5/Oo "Genapol"/caprylate with
addition 0. 069 ext./10 minO
The comparison value is achieved.
9 2 Addition of 2 vol.% cyclohexanol
o
system rate
30/Oo "Genapol"/caprylate without
addition 0.068 ext./10 min.
5/Oo "Genapol"/caprylate without
addition 0. 020 ext./10 minO
5/Oo "Genapol"/caprylate with
addition 0.080 ext./10 min.
The comparison ~Jalue is exceeded.
-~5~
9.3 Addition of 0.3 vol.% 2,2,~-trichloroethanol.
=
system rate
30/~ "Gena~ol"/caprylate without
addition 0.068 ext./10 min.
5~/~o "Genapol"/caprylate without
addition 0.020 ext./10 min.
5c/oO "Genapol"/caprylate with
addition 0.089 ext./10 min.
The comparison value i5 clearly exceeded~
e) Use _~ surface-active agents with, under certain
conditions, no or only insufficient activatinq action.
Of the large number of available surface active
agents, only ~ few are suitable for activating cholesterol
esterase to a sufficient extentO Inhibition of other ~.
reaction components, ~ability of the -reaction mixture,
consistency, solubility and uniformity of the compound
~r of the preparation belong to the important reasons
which make the freest possible choice of the surface-
active agents desirable. The following Example shows
that even surface-active agents with, under the given
~onditions, only a slight or even no activating action
can be used according to the present invention.
Example 10.
o~l~ Phosphate buffer,- pH 7.0, 0.003 U/ml~ cholesterol
esterase.
surface-active agent~ Oo thesit
additive: 1 vol.% n-amyl alcohol
-20-
system rate
surface-active agent without additive 0.003 ext./10 min.
additive 0 ext./10 min.
surface-active agent with additive 0.029 ext./10 min.
Example 11.
O.lM Phosphate buffer, pH 7.0, 0.003 U/mlO cholesterol
esterase
surface-active agent: 6%O thesit
additive: 10 mM/l. (about 0.16 wt.%) 1,7-
dihydroxynaphthalene
system - rate
surface-active agent without additive 0.003 ext./10 min.
additive 0.005 ext./10 min.
surface-active agent with additive 0.024 ext./10 minO
Exam~le 12
.
OolM Phosphate buffer, pH 7~0, 0O003 U/ml. cholesterol
esterase
surface~active agent: 12%o thesit
additive: 2 vol.% cyclohexanol
system rate
surface-active agent without additive 0.010 ext./10 min.
additive 0.003 ext./10 min.
surface-active agent with additive 0.105 ext./10 min~
Exam~le 13.
0,lM Phosphate ~uffer, pH 7.0, 0.003 U/ml. cholesterol
esterase
surface-active agent: 12,/oO thesit
additive: 1 vol.% 2,2,2-trichloroethanol
11;~1110
-27-
system rate
surface-active agent without additive 0o010 ext./10 min.
additive 0 ext./10 min.
surface-active agent with additive 0.081 ext./10 min.
Exam~le l~o
0~lM Tris/tartaric acid buffer, pH 8.0, 0.003 U/mlO
cholesterol esterase
surface-active agents: 2 vol.%o thesit/2 vol./Oo "Tween" ~0
additive: 0.4 volO% hexan-l-ol
system rate
-surface-active agents without additive 0.018 ext./10 min.
additive 0 ext./10 min.
surface-active agents with additive 0.028 extO/10 min.
Example 15,
.
0.~M Phosphate buffer, p~ 7.0, Q~00~ U/mlO cholesterol
esterase
surface-active agents: 5 vol.%o thesit/5 vol.,'Oo "Tween" 20
additive: 8 mM/l. 3,5-dichlorophenol
system rate
-surface-active agents without additive 0.005 ext./10 minO
additive 0 ext./10 min.
surface-active agents with additive 0.033-ext./10 min.
Example 160
0.3M Phosphate buffer, pH 7.0, 0.003 U/ml. cholesterol
esterase
surface-active agents 5 vol.%o "Genapol" ox-loo/5 wt.%o
sodium caprylate
additive: 10 mM/l. phenol
1131~lO
-28-
system rate
surface-active agents without additive 0.020 ext./10 min.
additive 0 ext./10 min.
surface-active agents with additive 0.064 ext./10 min.
Example 17.
0.3M Phosphate buffer, pH 7.0, 0.003 U/mlG cholesterol
esterase
surface-active agents: 5 vol.~Oo "Genapol" ox-loo/5 wt.~
sodium caprylate
additive: 4 mM/l. (about 0.065 wt.%) 3,4-
dichlorophenol
system rate
surface-active agents without additive 0.020 extO/10 mi,n.
additive 0 ext~/10 min.
surface-active agents with additive 00058 extO/10 min.
Exam~le 18.
O.1M Phosphate buffer, pH 7.0, 0.003 U/ml. cholesterol
esterase
surface-active agent: 10 vol.o~Oo "Brij" 35 (30% solution)
additive: 4 mM/l. 3,4-dichlorophenoL
system rate
surface~active agent without additive 0 ext./10 min.
additive 0 ext./10 minO
sur.ace active agent with additive 0.010 ext./10 min.
Example 19
.
O.1M Phosphate buffer, pH 7.0, 0.003 U/ml. cholesterol
esterase
0
-23-
surface-active agent: 10 vol~o/~o"srij" 35 (30% solution)
additive: 1 vol./~ 2,2,2-trichloroethanol
system rate
surface-active agent without additive 0 ext./10 min.
additive 0 extO/10 min.
surface-active agent with addi'ive 0.013 ext./10 min.
Exam~le 20.
0,lM Phosphate huffer, pH 7.0, 0.003 U/ml. cholesterol
esterase
surface-active agent: 2 vol.~Oo "Brij" 35 (30% solution)
additive: 2 vol.% cyclohexanol
system rate
surface-active agent without additive 0 extO/10 min.
additive 0~003 ext./10 min.
surface~active agent with additive 0u027 extO/10 min~
Exam~le 21.
O.lM Phosphate buffer, pH 7.0, 0.003 U/ml. cholesterol
esterase
sur~ace~active agent: 6 Wt.%O cholic acid
additive: 1 vol.% 2,2,2~trichloroethanol
system rate
surface-active agent without additive 0 extO/10 min.
additive 0 ext./10 minO
surface-active agent with additive 0.029 ext./10 min.
Exam~le 22.
0,lM Phosphate huffer, pH 7.0, 0.003 U/ml. cholesterol
esterase
~1311 1~1
-30-
surface-active agent: 5 wt.~o cholic acid
additive: 16 ml~/l. 3,5-dichlorophenol
system rate
surface-active agent without additive 0 ext./10 min.
additive 0 ext./10 min.
surface-active agent with additive 0.035 ext./10 min.
Example 23.
0,lM Phosphate buffer, pH 7 0, 0.003 u/ml. cholesterol
esterase
surface-active agent: 1 wt.~ cholic acid
additives: 4 mM/l. 3,5-dichlorophenol and
1 vol.% cyclohexanol
system rate
surface~active agent without additive 0 extn/10 min.
additives 0.010 extO/10 min.
surface-active agent with additives 0.103 extO/10 min.
Exam~le 24.
0,lM Tris/tartarlc acid buffer, pH 8D0~ 0.003 U/ml.
cholesterol esterase
surface-active agent~ -3 wtr/Oo sodium desoxycholate
additive: 002 vol.% hexan-l-ol
system rate
surface-active agent without additive 0 extO/10 min.
additive 0 ext./10 min.
surface-active agent with additive 0.056 ext./10 min.
Exam~le 25.
0,lM Tris/tartaric acid buffer, pH 8.0, 0.003 U/mlO
cholesterol esterase
11~1110
-31-
surface-active agent: 3 wt.%o sodium desoxycholate
additive: 1 vol.% 2,2,2-trichloroethanol
system rate
surface-active agent without additive O ext./10 min.
additive O ext./10 min.
surface-active agent with additive 0~069 ext./10 min.
Exam~le 26.
O.lM Phosphate buffer, pH 7.0, 0.003 U/ml. cholesterol
esterase
surface-active agent: 6 vol./Oo "Genapol" OX-100
additive: 0.4 vol.% 2,2,2-trichloroethanol
system rate
surface-active agent, without additive O ext./10 min.
additlve O ext~/10 min.
surface-active agent with additive 00059 ext./10 min.
Example 27~
O.lM Phosphate buffer, pH 7.0, 0~003 U/ml. cholesterol
esterase
surface-active agent: 20 vol./Oo "Tween" 20
additive~ 1 vol~% 2,2,2-trichloroethanol
system rate
surface~active agent without additive 0.005 ext./10 min.
additive O ext./10 min.
surface-active agent with additive 0.067 ext.~10 min.
The following Example illustrates a formulation oE
a reagent ~or use:
113~
-32-
Exam~le 23.
Reaqent for the determination of cholesterol in serum.
Formulation-
4.5 g./l. tartaric acid
7~5 g~/l. tris
3.7 g./l. sodium sulphate
200 mg./l. 4-aminoantipyrine
2~32 mgO/l. phenol
5000 U/l. peroxidase
430 U/l. cholesterol esterase
310 U/l. cholesterol oxidase
activating agent:
2.2 g~/1. thesit
1,3 g./l. sodium desoxycholate
0O815 gO'/l D 3,4-dichlorophenol
~ The abo~e made choice o~ the additives makes
possible the activation of the cholesterol esterase
under the conditions required by the properties of the
other enzymes. At the same time, it is possible to
- provide a storage-stable form of the reaction mixture
- as a one-pot reagent in solid form.
