DD244826A5 - Method for optical determination of the lipase - Google Patents

Abstract

The invention relates to a new method and the reagent for the optical determination of the lipase. The invention provides a method for lipase determination which is very easy to handle and readily adapted to the various automatic analyzer systems. The object of the invention is to provide a color test for the determination of the lipase, which does not have the disadvantages of the known color tests. A new lipase substrate has the general formula H2CYOAO H2CYCACX HCYR H2CZR1worin the substituents which have the meaning given in the description. This substrate is particularly suitable for the optical determination of the lipase by subjecting it to the development of the lipase-containing sample and determining the amount of the released aromatic hydroxy or thiol compound directly or, after coupling with a suitable chromogen, the color formed therefrom.

Description

H ₂ C -

where -j .j,

A is an alkylene or alkenylene group having 1 to 16 C atoms,

R and R _{1 may be} the same or different, depending on an alkyl, alkenyl or acyl group having 1 to 20 carbon atoms or an optionally alkyl-substituted aryl or aralkyl group having 1 to 8 carbon atoms in the alkyl radical, one of the radicals R and Ri is also a hydrogen atom,

X is the radical of an aromatic hydroxy or thiol compound and each Y and Z are independently -S-or -O-, Z also -CO ₂ -,

subjected to the action of the lipase-containing sample and the amount of the liberated aromatic hydroxy or thiol compound, directly or after coupling with suitable chromogen, the color formed therefrom, optically determined.

2. The method according to item 1, characterized in that one uses a lipase substrate, wherein R and / or R-i has 8 to 12 carbon atoms.

3. The method according to item!, Characterized in that one uses a lipase substrate, wherein A3 to 7 carbon atoms.

4. The method according to item 1, characterized in that one uses a lipase substrate, wherein X is an optionally substituted resorufin, a chlorophenol red, indoxyl, naphthol, thiophenols thiofluorescein or phenol radical.

5. The method according to item 1, characterized in that is used as the lipase substrate i ^ -O-dioctyl-rac-glycero-S-azelainsäureesorufinester.

6. The method according to item 1, characterized in that is used as the lipase substrate i ^ -O-didecyl-rac-glycero-S-imelinsäureuresorfinester.

7. The method according to item 1, characterized in that is used as the lipase 1,2-didecyl-rac-glycero-3-glutarsäureresorufinester.

8. The method according to item 1, characterized in that is used as lipase substrate i ^ -O-didodecyl-rac-glycero-S-glutarsäureresorufinester.

9. The method according to item!, Characterized in that one carries out the determination at a salt concentration in the test of 0.1 to 10 mg / ml.

10. A reagent for the optical determination of the lipase, characterized in that it contains at least one compound according to any one of claims 1 to 8 and buffer substance, and bile acid alkali metal salt, colipase, salt, urea and / or chromogenic coupler.

11. Reagent according to item 1, characterized in that it contains 0.05 to 10 mg / ml of substrate, 2 to 50 mg / ml of deoxycholate, 0.001 to 0.01 mg / ml of colipase, 1 to 100 mg / ml of urea, 0.1 to 10 mg / ml NaCl, 1 to 50 mg / ml buffer substance, in each case based on ready to use solution, contains in the test.

12. Reagent according to item 10 or 11, characterized in that it is impregnated on a carrier material.

Field of application of the invention

The invention relates to a method for the optical determination of the lipase and the reagent for the optical determination of the lipase.

Lipase (Triacyglycerinacylhydrolase EC 3.1.1.3) hydrolyzes emulsified triglycerides of long-chain fatty acids at the interface between oil droplets and aqueous phase. In certain diseases, such as acute pancreatitis or pancreatic carcinoma, the normally very low lipase concentration in the serum is increased and the determination of lipase activity therefore has considerable diagnostic significance. The lipase determination is therefore of considerable importance for clinical chemistry, but also for biochemistry, pharmaceutical chemistry and food chemistry.

Characteristic of the known technical solutions

There are already a number of Lipasemeßmethoden known. Thus, the liberated acid is determined by titration with lye. The method is susceptible to interference and not very specific. Furthermore, the photometric determination in UV is known. Thus, in DE-OS 3342106, the use of a mono or diglyceride of a higher fatty acid in combination with a

nonionic surfactant described as a substrate for UV determination. However, UV tests require a relatively expensive measuring apparatus and can often not detect possible disturbances such as device defects, depletion of the reagent, easily. Therefore, a need exists for a color test which can be performed with a simple apparatus and directly visually inspected. Known already is the color test of Kurooka using a Dimerkaptopropanol triester as a substrate together with a Dithiobisnitrobenzoesäure as a chromogen, an esterase inhibitor and a lipase activator. However, this method has unsatisfactory accuracy (J.CMn.Chem.Cliri. Biochem.20, 537-552 [1982]). Next is known a method with trilinolein as a substrate, lipoxygenase as an auxiliary system, which is oxidized in a subsequent color reaction by fatty acid hydroperoxide iron-Il to iron-Ill and detected as iron-lll-thiocyanate. This method gives no reliable results (J.C. Chem. Biochem., 20,745-752 [1982])

 Also known is a turbidity determination, which, however, has a relatively low sensitivity.

Object of the invention

The present invention provides a method of optical determination of the lipase which provides very accurate results with high sensitivity. It is very easy to handle and is even suitable for test strips. Since it has only a very low or no lag phase, it can be readily adapted to the various automatic analyzer systems.

Explanation of the essence of the invention

The invention is therefore based on the object to provide a color test for the determination of the lipase, which does not have the disadvantages of the known color tests, provides accurate results, easy to handle, has high sensitivity and has only a low lag phase, so that the adaptation to the various automatic analyzer systems is not difficult. This object is achieved according to the invention by a lipase substrate of the general formula

₂ C-YCACX HC-YR

H ₂ CZ

A is an alkylene or alkenylene group having 1 to 16 C atoms, R and Ri may be the same or different, each having an alkyl, alkenyl or acyl group having 1 to 20 carbon atoms or an optionally alkyl-substituted aryl or aralkyl group with 1 to 8 carbon atoms in alkyl radical, one of the radicals R and R _{1 is} also a hydrogen atom,

X is the radical of an aromatic hydroxy or thiol compound and each Y and Z are independently -S- or -O-, Z is also -CH ₂ -.

Under the action of the lipase, the lipase substrate according to the invention is cleaved to release the aromatic hydroxy or thiol compound corresponding to the radical X, which is either optically determined directly or coupled with a suitable chromophore or fluorophore and measures the product of the coupling.

R and / or Ri preferably have 6 to 18 C atoms, particularly preferably 8 to 12 C atoms. Because of their insensitivity to hydrolysis, the alkyl groups are particularly preferred over the acyl groups for R and R ₁ .

Surprisingly, the compounds with R and R, = alkyl, alkenyl or aralkyl turn out to be good lipase substrates, although the natural triglycerides bear acyl groups.

Examples of R and / or R ₁ are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl and Octadecyl radicals as alkyl groups and the corresponding acyl groups such as the acetyl, propionyl, butyryl, valeryl, copronyl, capryl, caprinyl, lauryl, myristyl, palmityl and stearyl group, the oleyl, crotonyl, linolyl group, Phenyl, benzyl or octylphenyl group.

The lipase substrate according to the invention further contains the radical of a dicarboxylic acid COOH-A-COOH in which A preferably has 3 to 7 C atoms. Examples of acids from which A is derived are malonic, succinic, glutaric, adipic, pimelic, suberic, azelaic, sebacic, nonanedicarboxylic, decanedicarboxylic and undecanedicarboxylic acids.

The acids of glutaric acid to azelaic acid, which correspond to A of 3 to 7 C atoms, are preferred as mentioned above.

X can be an aromatic hydroxy or thiol compound which represents a chromophore or can be converted into a dye only by a subsequent reaction. Typical examples are phenol, thiophenol, naphthol, thionaphthol and their derivatives as well as the per se chromogenic compounds such as the resorufin, chlorophenol red, indoxyl or thiofluoreszeinrest.

An exhaustive list of suitable hydroxy or thiol compounds is not possible because of their large number, but the directly chromophoric or convertible into chromophores aromatic hydroxy or thiol compounds are known in the art.

Preferred are chromophores which have a low polarity and are lipophilic. However, the water solubility should still be guaranteed.

The lipophilic character of o. G. Chromophores can be replaced by suitable substitution, such as. B. with alkyl groups are positively influenced. Suitable substituents for the resorf residue have been described i.a. the methyl, dimethyl and ethyl groups as well as the substitution with bromine proved to be suitable.

The compounds of the invention are new. They have an asymmetric center and are therefore optically active. The lipase substrate used can be both the racemates obtained in the customary preparation methods and the optical isomers.

The production of the lipase substrates according to the invention can be carried out by methods known per se. Thus, suitable methods for the synthesis of the 1,2-O-di-tert-and-1-diacylglycerol compounds are described, for example, in Methods in Enzymology, 98, 633 (1983) and Oleagineux, 23, 185 (1968). The synthesis of alkanediol derivatives is described, for example, in Can. J. Biochem. 46, 69 (1968).

The corresponding glycerodicarboxylic acid monoesters are then obtained from the 1.2-0-diether and 1.2-diacyl-glycine compounds by reaction with the corresponding dicarboxylic acid anhydrides in an anhydrous medium such as chloroform / pyridine. Suitable methods for the preparation of the dicarboxylic anhydrides are described in Houben-Weyl-Müller "Methods of Organic Chemistry", Volume IV / 4, page 786.

The esterification of the monoester with the hydroxy or thiol aromatic compound from which the X radical is derived can be carried out, for example, by direct reaction of the dicarboxylic acid monoester with the aromatic alcohol or thiol in the presence of a water-withdrawing agent such as dicyclohexylcarbodiimide. Alternatively, the dicarboxylic acid monoester is first converted to an activated ester, for example, the hydroxysuccinimide ester or the imidazolide, and the activated ester is then reacted with the aromatic alcohol or thiol.

It is also possible to first prepare a monoester of the dicarboxylic acid with the aromatic alcohol or thiol, for example the adipic acid mononitrophenyl ester or glutaric acid monophenylester, and then esterify it with the 1,2-0-dialkyl- or diacylglycerol, for example via intermediate formation of an acid chloride anhydride or activated ester. The preparation of the dicarboxylic acid monoesters with the aromatic alcohol or thiol can be carried out, for example, from acid anhydride and aromatic compound in the molar ratio 1: 1 or from dicarboxylic acid and aromatic compound in a molar ratio of 2: 1 or from a dicarboxylic acid monoester with a readily cleavable protective group and the aromatic compound. A suitable method is described, for example, in Arch. Pharm. 287, 514 (1954).

Alternatively, the 1.2-0-dialkyl or 1.2-0-diacylglycero-dicarboxylic acid monoester can also be prepared by first a dicarboxylic acid monoester of the dicarboxylic acid and an easily cleavable alcohol, such as. B. benzyl alcohol or 2,2,2-trichloroethyl alcohol and the resulting acid is then esterified with the mentioned dialkyl or diacyl-glycerol. Subsequently, the protecting group is removed and the reaction is carried out with the aromatic alcohol or thiol as described above.

Another method is to first react a protected glycerol such as 1,2-isopropylidene glycerol with a dicarboxylic acid monoester to form the corresponding protected glycero-3-dicarboxylic acid diester, then deprotect the glycerol and alkylate or liberate the released OH groups acylated. Finally, the originally existing monoester group (carboxyl-protecting group) is split off and reacted with the aromatic alcohol or thiol.

The preparation of the substrates according to the invention described above is not exhaustive and those skilled in the art are a number of other known methods available, which allow him to readily prepare each of the compounds of the invention. If desired, the pure optical isomers can be obtained by known separation processes from the racemic products obtained by the methods described above. Likewise, however, the isomers can also be obtained by stereospecifically proceeding synthesis according to methods which are also known per se.

The method according to the invention for the optical determination of the lipase is characterized in that a lipase substrate according to the invention is subjected to the action of the lipase-containing sample and the amount of the released aromatic hydroxy or thiol compound is determined optically or, after being coupled with a suitable chromogen, optically determines the color formed therefrom.

A special feature of this method is that it does not require auxiliary enzymes or esterase inhibitors, as are often required in the known methods. Such additives are not only expensive, but often not very stable. A particular advantage of the invention is that it also makes a simple and well-tolerated reagent for the optical determination of the lipase possible, which in addition to a lipase substrate according to the invention and buffer substance also a surface-active agent, in particular bile acid salt, colipase, a chromogenic coupler or / and contains a salt such as sodium chloride. In addition, the reagent may conveniently contain urea, preservative and / or activator. In a preferred composition, this reagent contains

0.05 to 10 mg / ml of substrate,

2 to 50 mg / ml deoxycholate,

0.001 to 0> 01 mg / ml colipase,

1 to 100 mg / ml urea,

0.1 to 10 mg / ml NaCl,

1 to 50 mg / ml buffer substance,

in each case based on ready-to-use solution in the test.

Suitable bile acids are the known surface-active bile acids, such as cholic acid, taurocholic acid, deoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid or their alkali metal salts, in particular the sodium salt. The preferred amount is 2 to 50 mg / ml.

Another essential component of the reagent according to the invention is colipase. Particularly suitable is an impurities-free colipase. The preferred amount is 0.001 to 0.01 mg / ml.

The reagent of the invention may further contain urea, preferably 1 to 100 mg / ml.

Suitable buffer substances are all known buffers which are capable of setting a pH between 6.0 and 10.5 within the scope of the reagent according to the invention. The preferred pH range is between 7.0 and 9.5. Examples of suitable buffers are diethanolamine buffer, triethanolamine buffer, Tris buffer and Good buffer, such as Hepes buffer (well suited for addition before lyophilization), Taps buffer, CHES buffer (2- (cyclohexylamino) ethanesulfonic acid) and Bicine. Particularly preferred is Tris buffer. The preferred amount of buffer substance is between 1 and 50 mg / ml.

As salts, alkali, alkaline earth and ammonium salts are suitable and are preferably used in concentrations of 0.1 to 10 mg / ml.

Preservatives used in the invention are those which do not impair the enzymatic activity of the lipase to be determined. Particularly suitable are alkali metal azides, especially sodium azide. Other preservatives, such as. B-. However, thiocide and other sulfur-containing preservatives are also suitable. The preferred amount of preservative is 0.001 to 2 mg / ml.

Suitable activators are alkaline earth ions, preferably calcium ions. Since these enter into compounds which are insoluble in deoxycholic acid, taurodeoxycholic acid is preferred in the presence of calcium as bile acid, since it allows higher calcium concentrations in the range of 1 to 5 μmol.

If the reagent according to the invention is used in dry or concentrated form which is intended for dilution to the final composition, it contains the substances mentioned in appropriate proportions, and preferably protective colloid.

Suitable protective colloids are the substances known to the chemist for this purpose, such as polyhydroxy compounds, serum albumin, polyvinylpyrrolidone, solid polyethylene oxides and the like. Preference is given to polyhydroxy compounds, in particular monomeric or polymeric pentose or hexose having 1 to 10 pentose or hexose units in the molecule and / or solid polyethylene glycol at room temperature. Preferred examples of suitable polyhydroxy compounds are mannitol and like sugar alcohols, oligosaccharide of glucose, mannose, maltoheptaose, polyethylene glycol having an average molecular weight between 3500 and 7000 μ.a. Other suitable protective colloids are for. As amino acids, such as alanine, plant gums, such as gum arabic, etc. The preferred amount of protective colloid or a mixture of protective colloids is 20 to 90 wt .-%. Particularly suitable proved to be a mixture of sugar alcohol and polyalkylene glycol.

The reagent according to the invention can also be present impregnated on a suitable carrier material. Both an absorbent carrier material and a swellable, soluble film-forming carrier material are suitable. In this form, the reagent according to the invention makes it possible to produce test strips which can be evaluated directly visually or by means of suitable measuring devices.

The color test according to the invention for the determination of the lipase gives very accurate results with high sensitivity. It is very easy to handle and is suitable even for test strips. Since it has only a very low or no lag phase, it can be easily adapted to the various automatic analyzer systems.

The determination itself can be carried out both as an end point determination and kinetically. Compared with many known processes, the kinetic feasibility has the advantage that neither stopping nor shaking out the formed reaction product has to be carried out.

The following examples further illustrate the invention.

embodiments example 1

a) i ^ -O-Dihexyl-rac-glycero-S-glutaric acid monoester

To a solution of 3.3 g (11.5 mM) of 1,2-O-dihexylglycerol in 30 ml of chloroform is added sequentially 2.5 ml of pyridine, a spatula tip of 4-dimethylaminopyridine and 2.6 g (23 mM) of glutaric anhydride. The mixture is heated under reflux for 10 hours and diluted after cooling with 200 ml of chloroform. The chloroform phase is shaken with 1 N hydrochloric acid and dried. After the desiccant has been filtered off, the solvent is stripped off and the residue is purified on a silica gel column (eluent: ethyl acetate / petroleum ether 1: 1)

TLC: R _f : 0.45 (ethyl acetate / petroleum ether 1: 2 + 1% glacial acetic acid) 1-O-dihexyl-rac-glycero-S-glutaric acid resorufin ester:

b) 1.4 g (3.7 mM) are dissolved in 20 ml of chloroform and added dropwise while cooling with ice with 2 ml (23.3 mM) of oxalyl chloride. The ice bath is removed and the solution is stirred for 12 hours at room temperature. The solvent is then stripped off, the residue taken up in toluene and evaporated again. The resulting oil is used without further purification.

c) 0.8 g (3.7 mM) of resorufin are slurried in 40 ml of dimethylformamide with the addition of .1.1 ml of pyridine and a spatula tip of 4-dimethylaminopyridine. For this purpose, a solution of 1 b) is added dropwise in 20 ml of dimethylformamide. After one to two days of stirring, filtered at room temperature and the solvent evaporated. The residue is taken up in ethyl acetate, insoluble constituents are filtered off and the filtrate is shaken with 1 N hydrochloric acid, then with water. After drying the organic phase and distilling off the solvent to obtain an oily residue, which is purified by column chromatography on silica gel (eluant: ethyl acetate / petroleum ether 1: 1).

TLC: R _f : 0.70 (ethyl acetate / hexane 1: 1).

Example 2

a) i ^ -O-dioctyl-rac-glycero-S-glutaric acid monoester

Preparation analogous to Example 1 a) from 13 g (41 mM) 1,2-0-dioctyl-glycerol, 150 ml of chloroform, 10 ml of pyridine and 6.8 g (59.5 mM) glutaric anhydride. Yield: 6.5 g (37%) TLC: R f: 0.31 (ethyl acetate / petroleum ether 1: 1)

 i ^ O-dioctyl-rac-glycero-S-glutaric acid resorufin ester:

b) Preparation analogous to Example 1 b) from 2 g (4.5 mM) 2a)

c) 0.96 g (4.5 mM) of resorufin are dissolved in 50 ml of chloroform with the addition of 0.75 ml (5 mM) of 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 0.1 g of 4- Dimethylaminopyridine dissolved. For this purpose, a solution of 2b) is added dropwise in 20 ml of chloroform. After one to two days of stirring at room temperature is filtered and the solvent removed. Workup analogously to Example 1c).

TLC: R _f : 0.66 (ethyl acetate / hexane 1: 1).

Example 3

a) i ^ -O-dioctyl-rac-glycero-S-pimelic acid monoester

Preparation analogous to Example 1 a) from 3.2 g (10 mM) 1,2-O-dioctyl-glycerol and 2.1 g (15 mM) pimelic anhydride, TLC: R _f : 0.61 (ethyl acetate / petroleum ether 1: 1) IR ( cm " ¹ ): (Film): 1 740.1 710 1 -O-dioctyl-rac-glycero-S-pimelic acid resorufin ester:

b) preparation analogously to Example 1 b) from 2.2 g (4,7 mM) 3a)

c) Preparation analogous to Example 2c) from 1 g (4.7mM) resorufin, 0.7 rhi 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 3b). TLC: Rf = 0.78 (ethyl acetate / hexane 1: 2)

Example 4

a) i ^ -O-dioctyl-rac-glycero-S-azelaic acid monoesters

Preparation analogous to Example 1 a) from 6.3 g (2OmM) 1,2-0-dioctyl-glycerol, 80 ml of chloroform, 5miPyridin and 5.2 g (3OmM) azelaic anhydride. i ^ O-dioctyl-rac-glycero-S-azelaic acid resorufin ester:

b) Preparation analogous to Example 1 b) from 3.2 g (6.5 mM) 4a) and 3 ml of oxalyl chloride.

c) Preparation analogous to Example 2c) from 1.4 g (6,5rtiM) resorufin, 65 ml chloroform 1.1 ml 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 4b).

TLC: Rf = 0.86 (ethyl acetate / hexane 1: 2) IR (cm- ¹ ): (film) 1762.1736

Example 5

a) i ^ -O-Didecyl-rac-glycero-S-glutaric acid monoester

Preparation analogous to Example 1 a) from 3.7 g (10 mM) of 1,2-O-didecylglycerol, 40 ml of chloroform, 2.5 ml of pyridine and 1.8 g (15.8 mM) of glutaric anhydride.

TLC: R _f : 0.77 (ethyl acetate / hexane 1: 2).

i ^ -O-didecyl-rac-glycero-S-glutaric acid resorufin ester:

b) Preparation analogous to Example 1 b) from 2.5 g (5 mM) 5a) 50 ml of chloroform and 2.2 ml of oxalyl chloride.

c) Preparation analogous to Example 2c) from 1.1 g (5 mM) of resorufin, 1 ml of 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 5b). TLC: Rf = 0.70 (ethyl acetate / hexane 1: 1)

Example 6

a) i ^ -O-diundecyl-rac-glycero-S-glutaric acid monoester

Preparation analogous to Example 1 a) from 2 g (5 mM) of 1,2-diundecylglycerol, 25 ml of chloroform, 1.4 ml of pyridine and 0.9 ml (7.5 mM) of glutaric anhydride.

TLC: R _f : 0.55 (ethyl acetate / petroleum ether 1: 2)

IR (cm " ¹ ): (film) 1740,1710 '

i ^ -O-Diundecyl-rac-glycero-S-glutaric acid resorufin ester:

b) Preparation analogous to Example! b) from 2 g (3.9 mM) 6a), 5 ml chloroform and 1.8 ml oxalyl chloride.

c) Preparation analogous to Example 2c) from 0.83 g (3.9 mM) of resorufin, 0.61 ml of 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 6b). TLC: Rf = 0.47 (RP 18, ethanol / acetone 2: 1) _

Example 7

a) i ^ -O-dilauryl-rac-glycero-S-glutaric acid monoester

Preparation analogous to Example 1 a) from 10.7 g (25 mM) of 1,2-dilaurylglycerol, 70 ml of chloroform, 5.5 ml of pyridine and 3.3 g (29 mM) of glutaric anhydride.

TLC: Rf: 0.33 (ethyl acetate / petroleum ether 1: 1).

IR (Cm ^-1 Ji (FiIm): 1741, 1708

1-O-Dilauryl-rac-glycero-S-glutaric acid resorufin ester

b) Preparation analogous to Example 1 b) from 5.5 g (1OmM) 7a), 50 ml of chloroform and 4.3 ml of oxalyl chloride.

c) Preparation analogous to Example 2c) from 2.2 g (1OmM) resorufin, 100 ml of chloroform and 1.5 ml of 1,8-diazabicyclo (5,4,0) undec-7-en and7b).

TLC: R _f = 0.78 (ethyl acetate / petroleum ether 1: 1) IR (cm ^-1 ): (film) 1765, 1720

Example 8

a) i-O-Dilauryl-rac-glycero-S-pimelic acid monoester

'Preparation analogous to Example 1 a) from 8.6 g (2OmM) 1,2-dilauryl-glycerol, 50 ml of chloroform, 15 ml of pyridine and 4.3 g (3OmM) pimelic anhydride

TLC: R _f : 0.5 (ethyl acetate / petroleum ether 1: 2)

IR (cm ^-1 ): (film): 1740, 1710 i ^ -O-dilauryl-rac-glycero-S-pimelic acid resorufin ester:

b) Preparation analogous to Example 1 b) from 1.66 g (3 mM) 8a) and 1.3 ml oxalyl chloride.

c) Preparation analogous to Example 2c) from 0.65 g (3 mM) resorufin, 30 ml chloroform 0.5 ml 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 8b).

TLC: Rf = 0.75 (ethyl acetate / hexane 1: 1) IR (cm- ¹ ): (film): 1786.1739

- e -

Example 9

a) i ^ -O-Ditetradecyl-rac-glycero-S-glutaric acid monoester

Preparation analogous to Example 1 a) from 14.6 g (30 mM) of i-O-ditetradecyl-glycerol, 150 ml of chloroform, 8.2 ml of pyridine and 5.1 g (45 mM) of glutaric anhydride.

TLC: R _f : 0.42 (ethyl acetate / petroleum ether 1: 2)

IR (cm " ¹ ): (KBr) 1740, 1710.

i ^ -O-ditetradecyl-rac-glycero-S-glutaric acid resorufin ester:

b) Preparation analogous to Example 1 b) from 3 g (5 mM) 9a) and 2.2 ml of oxalyl chloride.

c) Preparation analogous to Example 2c) from 1.06 g (5 mM) resorufin, 50 ml chloroform, 0.75 ml !, S-diazabicyclo-f? -Al-undec ^ -en and 9b).

TLC: Rf = 0.34 (RP18, acetonitrile / dichloromethane 1: 1) IR (cm- ¹ ): (KBr): 1763.1735

Example 10

a) i ^ -O-Ditetradecyl-rac-glycero-S-pimelic acid monoester Preparation analogous to Example 9a) from 6.4 g (45 mM) pimelic anhydride TLC: R _f : 0.45 {ethyl acetate / petroleum ether 1: 2) IR (cm ^-1 ): (Film) 1740.1708 i ^ -O-Ditetradecyl-rac-glycero-S-pimelic acid resorufin ester

b) Preparation analogous to Example 1 b) from 3.1 g (5 mM) 10a) and 2.2 ml oxalyl chloride.

c) Preparation analogous to Example 2c) from 1.06 g (5 mM) resorufin, 50 ml chloroform and 0.78 ml 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 10b).

TLC: Rf = 0.71 (ethyl acetate / petroleum ether 1: 2) ^f

IR (cm " ¹ MFilm): 1755, 1734

Example 11

a) i ^ -O-Dihexadecyl-sn-glycero-S-glutaric acid monoester

Preparation analogous to Example 1 a) from 2.7 g (5 mM) of i-O-dihexadecyl-sn-glycerol, 50 ml of chloroform, 3 ml of pyridine and 1.5 g (13 mM) of glutaric anhydride. TLC: Rf: 0.65 (ethyl acetate / petroleum ether 1: 1) • | R (cm ^-1 ): (KBr) 1740, 1710 i ^ -O-dihexadecyl-sn-glycero-S-glutaric acid resorufin ester:

b) Preparation analogous to Example 1 b) from 2.2 g (3.3 mM) 11a) and 1 ml of oxalyl chloride.

c) Preparation analogous to Example 1c) from 0.71 g (3.3mM) resorufin, 20 ml dimethylformamide 0.5m pyridine and 11 b). TLC: Rf = 0.72 (ethyl acetate / petroleum ether 1: 2).

Example 12

a) i ^ -O-Dibenzyl-rac-glycero-S-glutric acid monoester

Preparation analogous to Example 1 a) from 3 g (11 mM) 1,2-O-dibenzyl-glycerol, 30 ml (chloroform, 2.5 ml pyridine and 1.8 g (16 mM) glutaric anhydride.

- TLC: Rf: 0.39 (ethyl acetate / petroleum ether 1: 1 + 1% glacial acetic acid) i ^ -O-dibenzyl-rac-glycero-S-glutaric acid resorufin ester:

b) Preparation analogous to Example 1 b) from 2.9 g (7.5 mM) 12a), 30 ml of chloroform and 3.3 ml of oxalyl chloride.

c) Preparation analogous to Example 2c) from 1.6 g (7.5 mM) of resorufin, 75 ml of chloroform, 1.2 ml of 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 12b).

TLC: Rf = 0.48 (ethyl acetate / hexane 1: 1)

Example 13

a) 1-0-Octadecyl-2-0-benzyl-sn-glycero-3-glutaric acid monoester

Preparation analogous to Example 1a) from 2.2 g (5 mmol) of 1-octadecyl-2-O-benzyl-sn-glycerol, 50 ml of chloroform, 3 ml of pyridine and 1.5 g (13mM) of glutaric anhydride. IR (CiTT ¹ MFiIm) 1730.1700 iO-Octadecyl ^ -O-benzyl-sn-glycero-S-glutaric acid resorufin ester:

b) Preparation analogous to Example 1 b) from 1.7 g (3.1 mM) 13a) and 1.3 ml of oxalyl chloride.

c) Preparation analogous to Example 1 c) from 0.8 g (37 mM) resorufin, 20 ml dimethylformamide, 0.7 ml pyridine and 13b). TLC: Rf = 0.68 (ethyl acetate / petroleum ether 1: 1) IR (cm ^-1 ) - (KBr): 1768.1739

Example 14

a) i-dioctanoyl-sn-glycero-S-glutaric acid monoester

Preparation analogous to Example 1 a) from 6.8 g (2OmM) i ^ -Dioctanoyl-sn-glycerol, 100 ml of chloroform, 12.5 ml of pyridine and 5.8 g (5OmM) glutaric anhydride.

TLC: R _f : 0.49 (ethyl acetate / petroleum ether 1: 1).

i ^ -Dioctanoyl-sn-glycero-S-glutaric acid resorufin ester

b) Preparation analogous to Example 1 b) from 2.2 g (5 mM) 14a) and 2.2 ml oxalyl chloride

c) Preparation analogous to Example 1 c) from 1.05 g (5 mM) resorufin, 30 ml dimethylformamide, 0.75 ml pyridine and 14b). TLC: Rf = 0.86 (RP 18, acetonitrile / dichloromethane Ϊ72Τ

Example 15

a) i ^ .- dioleyl-rac-glycero-S-glutaric acid monoester

Preparation analogous to Example 1 a) from 3.1 g (5 m mol) of diolein, 40 ml of chloroform, 3 ml of pyridine and 1.5 g (13 mM) glutaric anhydride.

TLC: R _f : 0.32 (ethyl acetate / petroleum ether 1: 1)

IR (cm ^-1 ): (film): 1740, 1706

Diolein can be prepared from technical diolein by column chromatography on silica gel using ethyl acetate / petroleum ether 1: 3 as the eluent in pure form.

i ^ .- dioleoyl-rac-glycero-S-glutaric acid resorufin ester :,

b) Preparation according to Example b) from 3.5 g (4.8mM) 15a) and 1.3 ml oxalyl chloride.

c) Preparation analogous to Example 1 c) from 0.9 g (4.2mM) resorufin, 20 ml dimethylformamide, 1 ml pyridine and 15b). TLC: Rf = 0.78 (ethyl acetate / petroleum ether 1: 1)

Example 16

i ^ -O-ditetradecyl-rac-glycero-pimelic acid naphthyl

The preparation is carried out analogously to Example 10 b) and c) from 0.72 g (5 mM) of 1-naphthol. Purification by flash chromatography on silica gel with the eluent ethyl acetate / hexane 1: 3

TLC: R _f = 0.89 (ethyl acetate / petroleum ether 1: 5)

Example 17

i ^ .- dioleoyl-rac-glycero-S-glutaric acid resorufin ester

10.3 g (14.2 mM) of di-dioleyl-S-glyceroglutaric acid monoester of Example 15a), 3.2 g (15 mM) of resorufin, 6.2 g (30 mM) of dicyclohexylcarbodiimide and a spatula tip of 4-dimethylaminopyridine are stirred in 75 ml of dimethylformamide for 2 to 3 days at room temperature. Then it is diluted with ethyl acetate and the precipitate filtered off. The ethyl acetate phase is extracted by shaking with 1 N hydrochloric acid and dried over sodium sulfate. After distilling off the solvent leaves an oily residue, which is purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether 1: 1). Likewise, the corresponding chlorophenol red derivative was prepared

DC; R _f = 0.69 (RP18, isopropanol / methanol 1: 2).

Example 18

Preparation of 1,2-O-dioctyl-3-pimelic acid monoester

1st Stage Lit: J.H.Short, U. Biermacher Chim. Ther. 1966,456 Synthesis of pemelic acid monobenzyl ester

2nd stage: Analogously to Example 1 b) from 2.5 g (10 mM) pimelic acid monobenzyl ester. The resulting oil becomes a solution of 3.2 g

(10 mM) 1,2-dioctylglycerol, 7 ml pyridine are added dropwise. Workup analogously to Example 1 c).

3rd stage: The above product was dissolved in 20 ml of tetrahydrofuran and, after addition of 0.4 g of palladiumZ activated carbon. The

Crude product is purified on a silica gel column

 Eluent: ethyl acetate / petroleum ether 1: 1

TLC: Rf = 0.61 (ethyl acetate / petroleum ether 1: 1) IR (cm- ¹ ): (film): 1740.1710.

Example 19

Synthesis of 1 ^ .- dioleyl-glycero-S-glutaric acid monoester

a) i ^ -O-isopropylidene-glycero-S-glutaric acid trichloroethyl ester

Step 1: Dissolve 5.55 g (42 mM) of isopropylidene glycol and 12 g (45.3 mM) of 2,2,2-trichloroethyl hydrogen glutarate in ethylene glycol dimethyl ether and add 10.5 g (51 mM) of dicyclohexylcarbodiimide. After stirring for two days, it is filtered and distilled.

Bp: 170 ° C (0.1 torr) farbl.ÖI

DC: R _f = 0.82

(Acetone / chloroform 1: 8) Step 2: The resulting oil is dissolved in 11 ml of ether, treated with 3 ml of methanol and 3 ml of 3 N hydrochloric acid and stirred for 12 hours at room temperature. The organic phase is shaken with saturated sodium bicarbonate solution, then with saturated sodium chloride solution and dried. After removal of the solvent remains an oily residue.

TLC: Rf = 0.23 (ethyl acetate / petroleum ether 1: 1)

b) i-Dioleyl-glycero-S-glutaric acid trichloroethyl ester

Step 3: Dissolve 6.1 g (18 mM) of the product obtained above and 7.84 g (38 mM) of oleic acid in 100 ml of ethylene glycol dimethyl ether and add dropwise a solution of 10.5 g (37 mM) of dicyclohexylcarbodiimide in 50 ml of ethylene glycol dimethyl ether. After stirring at room temperature for 12 hours, it is filtered and shaken out successively with 3N hydrochloric acid, with sodium bicarbonate solution, then with water. After drying and concentration of the organic phase, the residue is chromatographed on silica gel.

Step 4 i-Dioleyl-glycero-S-glutaric acid monoester

Cleavage of the trichloroethyl protective group according to the literature: Juste, Synthesis 1976,457.

Example 20 '

a) i ^ -O-Didecyl-rac-glycero-S-pimelic acid monoester

Preparation analogous to Example 1a) from 5.6 g (15 mM) of 1,2-O-didecylglycene, 100 ml of chloroform, 3.2 ml of pyridine and 4.0 g (28 mM) of pimelic anhydride.

TLC: R _f = 0.36 (ethyl acetate / hexab 1: 1)

IR (cm ^-1 ): (film) 1735, 1710

i ^ -O-didecyl-rac-glycero-S-pimelsäure resorufin ester

b) Preparation analogous to Example 1 b) from 3 g (5.8 mM) 20a, 50 ml of chloroform and 2.2 ml of oxalyl chloride

c) Preparation analogous to Example 2c) from 1.1 g (5 mM) of resorufin, 1 m (6.4 mM), SDiazabicyclo-fOAOJ undecene and 20b. TLC: R _f = 0.71 (ethyl acetate / hexane 1: 1)

IR (cm ^-1 ): (film) 1755, 1720

Example 21

a) i ^ -O-Dilauryl-rac-glycero-S-azelaic acid monoesters

Preparation analogous to Example 1 a) from 6.7 g (15.6 mM) of 1,2-O-dilaurylglycerol, 100 ml of chloroform, 3.2 ml of pyridine and 4.0 g (23 mM) of azelaic anhydride.

TLC: Rf = 0.22 (ethyl acetate / hexane 1: 1)

IR (Cm- ¹ J = (FiIm): 1738.1710

i ^ O-dilauryl-rac-glycero-S-azelaic acid resorufin ester

b) Preparation analogous to Example 1 b) from 1.5 g (2.5 mM) 21 a, 30 ml of chloroform and 1.5 ml of oxalyl chloride

c) Preparation analogous to Example 2c) from 0.55 g (2.5 mM) resorufin, 25 ml chloroform, 0.5 ml (3.2 ml) 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 21 b

TLC: Rf = 0.78 (ethyl acetate / hexane 1: 1) IR (cm-1): (film): 1762.1740

Example 22

In 60 ml of distilled water are dissolved with stirring 1.2 g of sodium deoxycholate and 0.15 mg of colipase (from pig).

With vigorous stirring, a solution of 70 mg of lipase substrate 1-O-dioctyl-rac-glycero-S-azelaic acid resorufin ester (Example 4) in 1.7 ml of n-propanol is injected under pressure in a stream as thin as possible. A solution containing, in 200 ml of distilled water, 1.5 g of urea, 1 g of sodium deoxy cholate, 200 mg of sodium chloride, 800 mg of TRIS and 107 mg of TRIS-HCl, is well mixed with the emulsion prepared above.

2.5 ml of the solution thus prepared are mixed with 100μ, Ι sample (serum). The reaction is followed photometrically at 578 nm Hg.

When evaluated by a standard of known lipase activity, the lipase activity of the sample is calculated as follows:

Activity , " ,,,.

Activity _Ιτ> . , (Default)

(Sample) _: -

Δ E / min (standard)

A calculation of the lipase activity of the sample is also possible according to the following formula:

Activity (sample) ß] / 'ij = 1000. ^Vges

Sample. d

V _tot : total volume of the test batch [cm ³ ]

Vprobe ^ volume of the sample [cm ³ ]

ε: extinction coefficient of the chromogen at 578 nm

d: layer thickness of the cuvette [cm]

ΔΕ / min: absorbance change per minute at 578 nm

In the reaction conditions mentioned, the extinction coefficient is ε = 60.65 cm · μηιοΓ. ¹

Example 23

Blanking change, esterase sensitivity, lipase sensitivity, and correlation to a turbidimetric turbidity test were determined for various lipase substrates.

The blank value change was determined using a reagent according to Example 1 and various lipase substrates. Instead of the sample 100μΙ water were added and the change in absorbance at 578 nm photometrically followed (mE / min).

To determine the esterase sensitivity, 100% carboxyl esterase (EC 3.1.1.1, about 20000 U / l Boehringer Mannheim GmbH, order No. 104698) was added instead of the sample, and the extinction change was monitored as described above.

To determine the lipase sensitivity, 100 μl of lipase (EC 3.1.1.3, about 100 U / l, Boehringer Mannheim GmbH, order no. 414590) were added instead of the sample, and the extinction change was monitored as described above.

To determine the correlation to a turbidimetric turbidity test, a turbidity test (Boehringer Mannheim GmbH, Order No. 262358) was performed with increasing amounts of lipase (0-1000 U / l), compared with a color test according to Example 22 and the correlation coefficient was determined.

The results are shown in Table I:

Table I

example blank Esteraseempfind Lipaseempfind- correlation friendliness friendliness coefficient [AmE / min] (Activity 20 000 U / I) (Activity 100 U / l) [AmE / min] [AmE / min] 1 13.2 > 1000 - - 2 7.0 144.0 -100 not determined 3 3.9-4.1 112.5 12.28 .9639 4 0.4-0.8 33.6 18,04 .9421 5 2.4-3.0 30.2 22.18 .9815 6 2.0-2.8 23.6 6.96 .9179 7 2.4-3.0 34.8 10.9 .9636 8th 2.1-2.4 27.8 4.68 .9286 11 0.1 1.8 0.97 .9830 20 1.3-2.0 48.7 9.8 .9888 21 9.8 to 11.9 9.5 13.2 .9716

Example 24

8.5 g sodium desoxycholate, 0.05 g colipase, 20 g mannitol, 0.05 g calcium chloride, 0.82 g sodium chloride, 2.7 g TRIS and 0.4 g TRIS · HCl are dissolved in 200 ml distilled water. While stirring, a solution containing 0.35 g of 1,2-o-ditetradecyl-racglycero-3-pimelic acid naphthol ester dissolved in 7 ml of propanol is injected. The emulsion thus obtained is frozen at -40 ⁰ C and lyophilized.

70 mg of the resulting lyophilisate are dissolved in 2 ml of distilled water and admixed with 100 μl of a true red TR solution (fast red = 4-chloro-3-methyl-benzenediazonium naphthalene-1,5-disulfonate) (231 mg in 10 ml of distilled water). After addition of 100 / xl sample (serum), the reaction is monitored photometrically at 405 nm. The determination of the lipase concentration is carried out via a calibration curve analogously to Example 22.

Example 25

In a solution of 3.04 g taurodeoxycholate, 2.7 g polywax 4000.7 mg calcium chloride, 0.2 mg colipase (from pig) in 120 ml dist. Water is added with stirring to 150 mg of lipase substrate i ^ -O-didecyl-rac-glycero-S-glutaric acid resomfinester in Sjörnl n-propanol injected.

To the emulsion thus obtained, the following solution was added and mixed well:

10 g taurodeoxycholate, 6.4 g polywax, 50 g mannitol, 14 g urea, 800 mg sodium chloride and 15 g tris are distilled in 300 ml.

Water dissolved. With a hydrochloric acid solution, the pH is adjusted to pH = 7.5. Thereafter, the solution is diluted to 400 ml with dist.

Water filled up.

2.5 ml of the reaction mixture thus prepared are mixed with 100μ, Ι sample (serum). The reaction is monitored photometrically at 578 nm.

The evaluation is made as in Example 22.

Example 26

In a solution of 3.04 g taurodeoxycholate, 2.7 g polywax 4000.7 mg calcium chloride, 0.2 mg colipase (porcine) in 120 ml dist. Water is injected with stirring 150 mg Lipasesubstrati ^ -O-didecyl-rac-glycero-S-glutaric acid resofurinester in 3.5 ml of n-propanol.

To the emulsion thus obtained, the following solution was added and mixed well:

10g taurodeoxycholate, 6.4g polywax, 50g mannitol, 14g urea, 800mg sodium chloride, 28g CHES are distilled in 300ml.

Water dissolved. With a hydrochloric acid solution, the pH is adjusted to pH = 8.5. Thereafter, the solution is diluted to 400 ml with dist.

Water filled up.

2.5 ml of the reaction mixture thus prepared are mixed with 100μ, Ι (serum). The reaction is monitored photometrically at 578nm.

The evaluation is made as indicated in Example 22.

Example 27

In a solution of 4.0g sodium taurodeoxycholate, 0.06g calcium chloride, 0.2mg colipase (porcine), 5.0g mannitol and 2.0g polywax 4000 in 100ml dist. Water is injected while stirring 150 ml of i ^ -o-ditetradecyl-rac-glycero-S-pimelinsäure naphtholester, which is dissolved in 4 ml of n-propanol. With good cooling, the emulsion thus obtained is ultrasonicated for a few minutes. - _

A second solution contains in 100 ml of 2.4 g Na-Taurodesoxycholat, 2, OgTRIS, 12.0 g mannitol, 3.5 g urea and 0.5 g sodium chloride. With hydrochloric acid, the pH of this solution is adjusted to pH = 8.3.

A third solution contains Tg Fast Red TR (Fast Red TR = 4-chloro-3-methyl-benzene-diazonium-naphthalene-1,5-disulfonate) in 40 ml of dist. Water dissolved.

5 parts of the solution 1 and 6 parts of the solution 2 are mixed with a part of the solution 3. With the solution thus obtained, a suitable for test strip production absorbent paper, z. B. the variety VS 532 from Schleicher and Schüll soaked and gently dried in a convection oven at a temperature of 30 to 65 ⁰ C. Until further use, it is recommended to store in the presence of a dehumidifying agent.

To test for lipase of the sample (serum), a small amount (a few drops) of the sample material is placed on the strip. From the time course of yellowing can be concluded that the lipase activity of the sample to be examined.

Example 28

a) i-O-Dilauryl-rac-glycero-S-tetradecanedioic acid monoester:

Preparation analogous to Example 1 a) from 8.6 g (2OmM) of 1,2-dilaurylglycerol, 100 ml of chloroform, 4 ml of pyridine, 0.4 g of dimethylaminopyridine, 9.6 g (4OmM) of tetradecanedioic anhydride

TLC: R ₁ = 0.45 (ethyl acetate / petroleum ether 1: 5) 1-O-dilauryl-rac-glycero-S-tetradecanedioic acid-methylresorufin-Jester:

b) Preparation analogous to Example 1 b) from 3.35 g (5 mM) 28a) and 2.2 ml oxalyl chloride

c) Preparation analogous to Example 2c) from 1.2 g (5 mM) of 4-methylresorufin, 20 ml of chloroform, 0.75 ml of S-Diaxabicyclo-föAOl undec-7-ene, 0.1 g of dimethylaminopyridine and 28b).

TLC: R, = 0.63 (ethyl acetate / hexane 1: 4)

d) As indicated in Example 22, an emulsion is prepared. However, instead of the lipase substrate mentioned there, a solution of 70 mg of lipase substrate 1-O-dilauryl-rac-glycero-S-tetradecanedioic acid-Fe-methylresorufin-1-ester dissolved in 1.7 ml of n-propanol is used.

The following test-specific parameters were obtained (see Example 23 and Table I): Blank value: 0.2 mU / min Esterase activity: 0.7 mU / min Lipase sensitivity: 23.6 mU / min per 100 U / l Correlation coefficient: 0.09509

Example 29

i ^ -O-Dilauryl-rac-glycero-S-glutaric acid-te-methyl-resorufinJester:

a) Preparation analogous to Examples 7a-c with 2.3 g (1OmM) 4-methylresorufin. TLC: R ₁ = 0.68 (ethyl acetate / hexane 1: 2)

b) In 60 ml dist. Water is dissolved with stirring 0.9 g of sodium taurodeoxycholate and 0.3 g of colipase (from pig). With good stirring, a solution of T, -O-dilauryl-rac-glycero-S-glutaric acid, ie-methyl-resorufin-Jester, 70 mg in 1.7 ml of n-porpanol is injected therein. To this is added a solution (200 ml) containing in 200 ml of 0.5 g of urea, 1 g of sodium taurodeoxycholate, 200 mg of sodium chloride and 2.9 g of TRIS and its pH adjusted to 7.5. After mixing well, add 2.5 ml of the solution so prepared with 100 μl sample (serum). The reaction is followed photometrically at 578 nm Hg and evaluated as indicated in Example 22.

Test-specific parameters (compare Example 21 and Table I): Blank value: 0.4mE / min _f

Lipase sensitivity: 28mE / min per 100mE / min Correlation Coefficient: 0.988

Example 30

4-methylresorufin:

Preparation as described in patent DE 3411 574 from 7.5 g of 2-methyl-4-nitrosoresorcinol, 4.2 g of resorcinol, 3.6 g of manganese dioxide, 4.3 ml of sulfuric acid, 75 ml of methanol, 4 g of zinc powder and 18 ml of 25% ammonia.

TLC: R ₁ = 0.70 (ethanol / acetone 2: 1)

UV / VIS (0.1 M potassium phosphate buffer pH 8.5):

max = 579nm

The preparation of 2-methyl-nitrosoresorcin takes place according to DE 3411 574, A1.

Example 31

1,2-0-dilauryl-rac-glycero-3-glutaric acid (4-methylumbelliferyl) ester:

a) 2.75 g (5 mM) 7a and 1 g (5 mM) of 4-methylumbelliferone are dissolved in 30 ml of tetrahydrofuran and then 2 g (10 mM) of dicyclohexylcarbodiimide and 0.15 g of dimethylaminopyridine are added. After stirring for 24 hours at room temperature, it is filtered and concentrated. The crude product is purified by flash chromatography.

DCiR ₁ = 0.40 (ethyl acetate / petroleum ether 1: 3)

b) The emulsion preparation is carried out as in Example 22, but instead of the lipase substrate used therein, 35 mg of 1,2-dilauryl-rac-glycero-3-glutaric acid (4-methylumbelliferyl) ester are dissolved in 1.7 ml of n-propanol used.

The reaction is followed by fluorimetry at 25 ⁰ C and that at an excitation wavelength of 364 nm, slit width 5 nm and an emission wavelength of 448 nm and a slit width of 10nm.

Test-specific parameters (see Example 23 and Table I):

Blank value: 0.4mE / min

Lipase sensitivity: 28mE / min per 100U / l,

Correlation coefficient: 0.9942

Example 32

1,2-O-dilauryl-rac-glycero-3-glutaric acid (p-nitrothiophenyl ester: preparation of 32a-b) analogously to Examples 7a-b.

c) Preparation analogous to Example 1 c) from 1.4 g (10 mM) of p-nitrothiophenol, 100 ml of dimethyl permeant, 2.4 ml of pyridine, 0.2 g of dimethylaminopyridine and 32 b. TLC: R ₁ = 0.76 (ethyl acetate / hexane 1: 4)

-11- Z44 8ZÜ

d) In 27ml dist. Water, the following compounds are dissolved with good stirring:

4.83mg sodium desoxycholate 28.00mg CHES * 17.50g urea

2.13mg sodium chloride 0.16mg pig colipase

0.33 mg calcium chloride 152.00 mg sodium taurodeoxycholate The pH of the solution is adjusted to pH = 8.3. To this solution, with good stirring, a solution of i ^ -O-dilauryl-rac-glycero-S-glutaric acid-lp-nitrothiophenyll ester is injected. The solution was then sonicated (medium intensity) with cooling for 2 minutes.

To 1 ml of the thus-obtained emulsion solution was added 50 ml of a sample (lipase-containing human serum) and mixed. The reaction was monitored photometrically at 405 nm Hg and calculated as indicated in Example 22, test-specific parameters: Blank: 0.3 mU / min

Lipase sensitivity: 12.2mE / min / per 100T / L Correlation coefficient: 0.998

Example 33

a) 1-0- (2-methoxy-octadecyl) -2-O-methyl-rac-glycero-3-glutaric acid monoester:

Preparation analogous to Example 1a) from 3 g (7.7 mM) of 1-0- (2-methoxy-octadecyl) -2-0-methyl-glycerol, 25 ml of chloroform, 1.8 ml of pyridine, 0.1 g of dimethylaminopyridine and 1.75 (15 mM) glutaric anhydride.

Yield: 1.5g (39%)

TLC: R ₁ = 0.68 (RP18, acetone / ethanol 1: 2) 1-0- (2-methoxy-octadecyl) -2-O-methyl-rac-glycero-3-glutaric acid (6-methylresorufin) ester:

b) Preparation analogous to Example 1 b) from 1.5 g (3 mM) 33a) and 1.5 ml oxalyl chloride

c) Preparation analogous to Example 2c) from 0.68 g (3 mM) 4-methylresorufin, 0.45 ml!, S-diazabicyclo-FBAOlundec ^ en and 33b). TLC: R ₁ = 0.86 (ethyl acetate)

d) The preparation of the test emulsion is carried out as described in Example 22, but this time as lipase substrate 1-0- (2-methoxy-octadecyl) -2-0-methyl-rac-glycero-3-glutaric acid (6-methyl-resorufin ) ester used.

The determination of the lipase concentration is carried out using a calibration line of two standards of different lipase concentration, in which the absorbance change per minute is plotted against the change in absorbance. Blank: 8.4mE / min Esterase sensitivity: 25.3mE / min Lipase sensitivity: 19.0mE / min per 100U / I Correlation coefficient: 0.9483

Example 34

a) i ^ -O-Dilauryl-rac-glycero-S-succinic acid monoester:

Preparation analogous to Example 1 a) from 8.56 g (2OmM) of 1,2-dilaurylglycerol, 4 g (4OmM) succinic anhydride, 60 ml of chloroform, 4.6 ml of pyridine and 0.24 g of dimethylaminopyridine. The product crystallizes from hexane.

FP: 41-43 ⁰ C

TLC: R ₁ = 0.26 (ethyl acetate / hexane 1: 4) 1-O-dilauryl-rac-glycero-S-succinic acid resorufin ester:

b) Preparation analogous to Example 17) from 2.65 g (5 mM) 34a) 1.06 g (5 mM) resorufin, 6.18 g (30 mM) dicyclohexylcarbodiimide, 0.1 g dimethylaminopyridine and 50 ml dimethylformamide.

TLC: R ₁ = 0.47 (ethyl acetate / petroleum ether 1: 3)

c) As stated in Example 22, the emulsion is also prepared here. In this case, however, the lipase substrate used is 70 mg of i-O-dilauryl-rac-glycero-S-succinic acid resorufin ester in 1.7 ml of propanol.

The following test-specific parameters were obtained (see Example 23 and Table I): Blank value: 3.8-4.0 mU / min Esterase sensitivity: not determined Lipase sensitivity: 8.7 mU / min per 100 U / I Correlation coefficient: 0.8793

Example 35

a). 2-0-Lauryl-octadecanediol (1,2) -1-glutaric acid monoester:

Preparation analogous to Example 1 a) from 4,6 g (10 mM) 2-O-lauryl octadecanediol (T, 2), 29 ml chloroform 2,3 ml pyridine, 0,12 g dimethylaminopyridine, 2,3 g (2OmM) glutaric anhydride TLC: R ₁ = 0 , 54 (Petroleum ether / ethyl acetate 4: 1) 2-0-Lauryl-octadecanediol (1,2) -1-glutaric acid (f-methylresorufin) ester:

b) Preparation analogous to Example 1 b) from 1.15 g (2 mM) 35a) and 0.88 ml oxalyl chloride

c) Preparation analogous to Example 2c) from 0.45 g (2 mM) 4-methylresorufin, 20 ml of chloroform, 0.3 ml!, 7-ene, 40 mg of dimethylaminopyridine and 35b).

TLC: Ri = 0.37 (petroleum ether / ethyl acetate 5: 1)

CHES * = Z-icyclohexylaminol-ethane-sulfonic acid

d) As described in Example 22, an emulsion is prepared, but are used as Lipasesubstrat70mg 2-0-Lauryloctadecanedio-i ^ i-glutaric acid ie-methylresorufinl ester dissolved in 1.7 ml of n-propanol used

 According to Example 23, Table I, the following test-specific parameters were obtained: blank value: 0.3-1, OmE / min esterase sensitivity: 1, OmE / min lipase sensitivity: 25.5 mU / min per 100 U / l correlation coefficient: 0.996

Example 36

a) i-O-Dilauryl-rac-S-thioglycero-S-S-glutaric acid monoester:

Preparation analogous to Example la) from 2.5 g (4.8 mM) i ^ -O-dilauryl-rac-S-thioglycerol, 14 ml of chloroform, 1.1 ml of pyridine and 1.1 g (9.6mMLGIutarsäureanhydrid.

TLC: R ₁ = 0.5 (hexane / tetrahydrofuran 1: 4)

i ^ O-dilauryl-rac-S-S-S-thioglycero glutaric fB-methylresorufin-Jester:

b) Preparation analogous to Example 1 b) from 0.74 g (1.3 mM) 36a) and 0.6 ml of oxalyl chloride.

c) Preparation analogous to Example 2c) aus0,3g (1.3mM) 4-methylresorufin, 13ml chloroform, 0.2ml !, S-diazabicyclo-ioAOJ undec-7-ene, 27mg dimethylaminopyridine and 36b).

TLC: R ₁ = 0.38 (petroleum ether / ethyl acetate 17: 3)

The 1,2-O-dilauryl-rac-3-thioglycerol used as starting material is obtained analogously to Org. Synth III, p. 366 and p.

Reaction of 1,2-O-dilaurylglycerol with toluene sulphonic acid chloride to dilaurylglycero-3-toluenesulphonate, then reaction with thiourea to give the corresponding isothiuronium salt, followed by hydrolysis with hydrochloric acid

TLC: R ₁ = 0.52 (petroleum ether / ethyl acetate 49: 1)

d) As indicated in Example 22, an emulsion is prepared. However, instead of the lipase substrate used there, a solution of 70 mg of 1-O-dilauryl-rac-S-thioglycero-S-S-glutaric acid-Fe-methylresorufinl-ester in 1.7 ml of n-propanol is used.

Assay-specific characteristics: Blank: 0.2-0.9 mU / min Esterase sensitivity: 1.3 mU / min Lipase sensitivity: 4.1 mU / min per 100 U / l Correlation coefficient: 0.857

Example 37

a) i ^ -S-dilauryl-rac-1'-dithioglycero-S-glutaric acid monoester:

Preparation analogous to Example 1 a) from 3 g (6.5 mM) of 1,2-S-dilauryl-1,2-dithioglycerol, 30 ml of pyridine and 1.5 g (13 ml of Fl)

Glutaric.

TLC: R ₁ = 0.43 (petroleum ether / ethyl acetate 1: 1)

i ^ -S-dilauryl-rac-i ^ -dithioglycero-S-fe-glutaric acid-resorufin-methyl-Jester:

b) Preparation analogous to Example 1 b) from 1.2 g (2 mM) 37a) and 1 ml of oxalyl chloride TLC: R f = 0.37 (ethyl acetate / petroleum ether 1: 4)

c) Preparation analogous to Example 2c) from 0.46 g (2 mM) 4-methylresorufin, 20 ml chloroform, 0.3 ml !, S-diazabicyclo-1-O-O-undec-7-ene and 37b)

The starting iS-dilauryl-rac-1-dithioglycerol is prepared as follows:

To a solution of 9g (16OmM) potassium hydroxide in 250ml ethanol at room temperature 10g (8OmM) 2,3-

Dimercaptopropanol in 100 ml of ethanol. After one hour of stirring, a solution of 40 g (16OmM) dodecyl bromide in 100 ml of ethanol is added dropwise. To complete the reaction is stirred for two more days, then filtered and the filtrate is mixed with ice. After acidification with 2 N hydrochloric acid is extracted three times with ether, the organic phase is dried and concentrated. The residue is purified over a silica gel column (eluant: ethyl acetate / petroleum ether 1:10).

TLC: R ₁ = 0.54 (ethyl acetate / petroleum ether 1:10)

Blank value: 3me / min

Esterase sensitivity: 3.4me / min

Lipase sensitivity: 24me / min

Correlation coefficient: 0.9943

d) As indicated in Example 22, an emulsion is prepared. Instead of the lipase substrate used in this case but i ^ -S-dilauryl-rac-i ^ -dithioglycero-S-S-glutar-acid-methylresorufin-lester, 70mg dissolved in 1.7ml n-popranol used.

Claims (1)

Invention claim: 1. A method for the optical determination of the lipase, characterized in that a lipase substrate of the general formula O V-M. O H ₂ CYCACX
HC-YR