Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-05-08
2001-11-27
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S018000, C435S004000, C435S968000, C435S007720, C435S007710
Reexamination Certificate
active
06322993
ABSTRACT:
This is a Section 371 PCT/EP97/05051 filed Sept. 16, 1997.
The invention concerns a method and reagent for the determination of lipase in biological liquids as well as the use of a lower molecular N-substituted carboxylic acid amide derivative to eliminate unspecific reactions in the determination of enzymes. In particular it has proven to be advantageous when N-(1,2-dicarboxylethyl)-N-alkyl-sulfosuccinamide or N-(1,2-dicarboxylethyl)-N-alkylaryl-sulfosuccinamide derivatives are present at a concentration of ca. 0.001 to 2.0% (w/v).
The determination of lipase and in particular of human pancreatic lipase (E.C. 3.1.1.3.) has for a long time been of undisputed importance for the diagnosis and assessment of the course of pancreatic diseases (W. Steinberg et al., Annals of Internal Medicine 102 (1985), 576; M. Panteghini et al., Clin. Biochem. 24 (1991), 497; N. W. Tietz and D. F. Shuev. Clin. Chem. 39/5 (1993), 746). For example in acute pancreatitis an increase of the lipase in serum occurs within a few hours which is sometimes very massive.
The function of lipase in the body is essentially to cleave preferably &agr; ester bonds of triglycerides containing long chained fatty acid esters into a diglyceride component and the free fatty acid. Subsequently conversion to the monoglyceride takes place but considerably more slowly.
Usually enzyme-catalysed reactions proceed in an aqueous phase: however, due to its physiological importance, lipase only reacts at the oil/water interlace. In this respect it differs significantly from the esterases. Furthermore lipases can only attach to an interface emulsified by bile acids without denaturation and to cleave triglycerides with the aid of colipase. For this reason the reaction kinetics are substantially influenced by the quality of the interface presented to the enzyme in addition to chemical parameters.
Nowadays various methods are known for the determination of lipase. These are essentially titrimetric, turbidimetric and immunological test principles. In the titrimetric determination an excess of lipase substrate, such as for example olive oil, is added first and the amount of fatty acid released from this by the lipase is measured by titration with alkali using an indicator or by extracting the corresponding copper salts. Titrimetric methods are nowadays only seldom used in routine clinical chemistry laboratories because of the difficult handling in some cases, the long reaction period and the large quantity of sample required (W. Junge in Methods of Enzymatic Analysis, Weinheim VCH, U. Bergmeyer ed., vol. 4 (1984), 15).
The turbidimetric lipase determination in which the clearing of the turbidity of a triglyceride/water emulsion is monitored photometrically is nowadays a widespread method in routine clinical chemical laboratories (W. Rick and M. Hockeborn, J. Clin. Chem. Biochem. 20 (1982), 735; J. Ziegenhorn et al., “Medica Sonderheft” 11 (1980)). A problem with the turbidity measurement is that individual serum samples do not exhibit a linear decrease of the measured signal within the measurement window of the photometer or even exhibit an increase in turbidity. A further difficulty with this method of determination is the reproducible production of an emulsion which always reliably has the same droplet size.
A particular disadvantage of immunological methods is that in this procedure the mass and not the enzyme activity is measured (W. Uhl et al., Internat. J. Pancreatology 12/3 (1992), 253; G. E. Hoffmann et al., Ärztl. Lab. 30 (1984), 193; H. Herden and K. Walter, Klin. Lab. 38 (1992), 89).
Consequently test methods based on a colour development have been developed and are mainly used nowadays. For example 1,2-diglycerides are used as a substrate which are degraded by lipase to 2-mono-glycerides and subsequently cleaved to glycerol by a 2-monoglyceride lipase that is added to the test. The free glycerol that is formed in this manner is degraded with the aid of a glycerol phosphate oxidase to form dihydroxyacetone and hydrogen peroxide (H
2
O
2
). The H
2
O
2
that is formed is detected by an appropriate colour indicator system (P. Fossati et al., Clin. Chem. 38/2 (1992), 211).
Today it is also possible to directly convert a colour substrate for the determination of lipase (EP 0 207 252). In this method the activity of human pancreatic lipase directly releases a photometrically determinable dye from the substrate which hence avoids a complicated enzyme cascade to produce the dye.
Furthermore it has been known for a long time that the ester bond of the colour substrates used can generate an excessively high signal due to unspecific hydrolysis. For example it is problematic that the colour substrate para-nitrophenyl acetate is hydrolysed by albumin and gamma globulins (W. Downey and P. Andrews, Biochem. J. 96 (1965) 21). Such reactions result in a falsification of the measured value which can lead to a clinically false diagnosis especially in the case of serum samples.
SUMMARY AND OBJECTS OF THE INVENTIONS
The object of the invention is therefore to eliminate unspecific reactions in the determination of lipase and hence to increase the validity of the analytical result.
The object is achieved by a method for the determination of lipase by adding a lipase colour substrate and a lower molecular N-substituted carboxylic acid amide derivative and subsequently photometrically determining the dye released from the substrate. Compounds of the general formula (I) or (I
a
) have in particular proven to be suitable according to the invention as the carboxylic acid amide derivative
in which R
1
and R
2
independently of one another represent hydrogen or a saturated or unsaturated, substituted or unsubstituted, optionally carboxylated hydrocarbon residue with 2 to 24 carbon atoms.
Z denotes a saturated or unsaturated, substituted or unsubstituted, cyclic or straight-chained hydrocarbon residue with 1 to 10 carbon atoms,
X represents an atom or a group of atoms with positive charge and n is a number from 1 to 3.
Those carboxylic acid amide compounds according to formula (I) or (I
a
) are preferred according to the invention, wherein
Z is a methylene and/or ethylene group composed of one to ten C atoms and which is optionally substituted by an electron-attracting group such as a carboxyl, sulfonyl, phosphate, phosphonate, nitro, nitrite or nitrate, halogen or alkoxy group,
R
1
and R
2
independently of one another represent hydrogen, an optionally substituted straight-chained or branched, saturated or unsaturated alkyl, aryl, alkylaryl or alkylene group composed of three to 24 C atoms, in which a carboxyl alkyl or dicarboxyl-alkyl residue composed of two to 24 C atoms is particularly preferred and
X represents an atom or a group of atoms with a positive charge and n denotes the number 1 or 2, have proven to be suitable.
According to the invention compounds which come into particular consideration are those in which the residue Z carries a carboxyl and/or sulfonyl group but also those in which one of the residues R
1
or R
2
denotes a lower alkyl group such as for example a methyl, ethyl, propyl, butyl, pentyl, an ethylenyl or propylenyl group or a C 10 to C 18 alkyl group. The substitution of appropriate alkyl or alkylene groups is optionally carried out according to known methods. Furthermore it has proven to be advantageous if R
1
for example represents a dicarboxylic acid residue based on malonic acid, glutaric acid, adipic acid, pimelic acid, azelaic acid and sebacic acid and in particular succinic acid. The compounds that can be used according to the invention preferably have a molecular weight of ca. 200 to 1000 Daltons; however, compounds have also proven to be suitable of up to 3000 Daltons. Compounds or salts that have at least two acidic groups have proven to be quite particularly advantageous such as for example the tetrasodium salt based on N-(1,2-dicarboxy-ethyl)-N-alkyl-sulfosuccinamide with a lower alkyl group (1 to 6 C atoms) and/or with a higher alkyl group (12 to 18 C atoms) or N-(1,2-dicarboxyethyl)-N-alkylaryl-sulfosuccin
Prinzing Urban
Schelong Lieselotte
Zielenski Ralf
Amick Marilyn L.
Leary Louise N.
Roche Diagnostics Corporation
Roche Diagnostics GmbH
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