Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-05-01
2004-08-31
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S018000, C435S069200
Reexamination Certificate
active
06783949
ABSTRACT:
This applications claims benefit to German Patent Application No. 199 19 634.6 filed Apr. 30, 1999.
The invention relates to a simple continuous test for the identification of structures which favor the arrangement of aromatics to give charge-transfer complexes, such as, for example, complex phospholipid/lipid structures (bilayer, monolayer, aggregates, micelles), with the aid of synthetic fluorescence-labeled acylglycerides, and its use for determination of the activity of lipases/lipase inhibitors.
Lipases, phospholipases, and other lipolytic enzymes have great importance in the biotechnological and medical field. In certain metabolic disorders, increased lipase activity in the fatty tissue can be detected, which is held partly responsible for the pathogenesis of this disease. The greatest part of the energy reserves of the body is stored in cells of the fatty tissue as fatty acids of the triglycerides. The essential anabolic processes caused by insulin include the stimulation of the uptake of substrates for triglyceride synthesis and the increase in lipogenesis. A further important process caused by insulin is the inhibition of lipolysis, the process by means of which catabolic hormones, primarily catecholamines, stimulates the hydrolysis of triglycerides and thereby induce the release of fatty acids. An important problem which is linked with noninsulin-dependent diabetes mellitus (NIDDM) has its cause in the uninhibited lipolysis of the fat cells, which leads to increased levels of unesterified fatty acids in the plasma. According to a present idea, the fatty acids stimulate gluconeogenesis in the liver and decrease the glucose utilization in the skeletal muscle by means of still poorly characterized molecular mechanisms. In fact, it was possible to show that the suppression of lipolysis in fat cells by inhibitors of lipolysis, such as agonists of the nicotinic acid receptor of the fat cell, lowers both the fatty acid concentrations in the plasma and raised blood sugar in diabetic animals and patients. Unfortunately, these beneficial effects are not particularly strongly pronounced and only of relatively short duration. This may be based on a physiological counterregulation caused by intervention in the regulatory mechanism of the rate-determining enzyme of lipolysis, the hormone-sensitive lipase (HSL). There are good reasons to assume that the inhibition of the lipolytic reaction will lead to an improved therapy of NIDDM, at least with respect to the suppression of the fatty acid release from the fat cells. The direct inhibition of HSL by suitable inhibitors should in this case get around the obvious difficulties of an intervention into the complex regulation of HSL.
The activity of lipolytic enzymes is traditionally investigated using radiometric, titrimetric, enzymatic, or fluorimetric/photometric methods. Radiometric assays are the most sensitive, but they require expensive radiolabeled substrates, are discontinuous, and require the separation of the radiolabeled substrate from the radiolabeled product. Such separations are often troublesome and the avoidance/reduction of radioactive waste is of increasing importance (especially relevant if there are a large number of tests).
Titrimetric tests are continuous and can be carried out both with natural and synthetic substrates, but they frequently suffer from a fairly low sensitivity and are susceptible to conditions that influence the amount of protons released.
Enzymatic or chromatographic methods for the detection of one of the products of the lipolytic reaction (e.g., glycerol) are very sensitive and relatively robust, but are also complicated in terms of handling, as they demand the working-up of the incubation batch of the lipase reaction before the actual enzymatic/chromatographic detection. The coupling of an enzyme test allows only endpoint measurements (“time-stop” measurement). Furthermore, in the investigation of unknown substances (e.g., searching for potential inhibitors), an effect on the enzymes of the detection reaction cannot be excluded in principle and therefore necessitates appropriate controls.
These considerations gave the impetus to the development of fluorimetric/photometric processes. In principle, these achieve the sensitivity of radiometric methods but necessitate the use of synthetic substrates or samples modified with fluorophores or chromophores. Traditional fluorimetric/photometric methods, like the radiometric procedures, are discontinuous in course and necessitate the separation of the substrate from the product. Recently, continuous fluorimetric/photometric assays have been developed (S. Hendrickson,
Analyt. Biochem
219 (1994) 1-8), which are based on a shift in the fluorescence or extinction maximum of the product in comparison with the substrate. However, all these processes are restricted to the detection of phospholipases, lipoprotein lipase (LPL), cholesterol esterase, sphingomyelinase, and glucosylceramide glucosidase. Substrates having fluorophoric/chromophoric groups, suitable for the continuous activity measurement of tryglyceride-cleaving enzymes (e.g., HSL, monoglyceride lipase, diglyceride lipase, triglyceride lipase, LPL, pancreatic lipase, hepatic lipase, bacterial lipase, phospholipase A
2
(PLA
2
), phospholipase C (PLC), cholesterol esterase), are as yet unknown.
It is therefore the aim of the invention to develop a simple continuous test for the identification of structures which favor the arrangement of aromatics to give charge-transfer complexes, such as complex phospholipid/lipid structures (bilayer, monolayer, aggregates, micelles), with the aid of synthetic fluorescence-labeled acylglycerides, and a process for determination of the activity of lipid-binding proteins, such as lipases.
Lipid transporters are proteins that recognize lipids and do not cleave like lipases, but instead transport through biological membranes.
Lipases are understood here as meaning biologically relevant endogenous lipases, such as are defined, for example, in R. D. Schmid, R. Verger,
Angew. Chem
. 110 (1998) 1694-1720.
A hormone-sensitive enzyme is understood as meaning an enzyme that is influenced in its activity by secondary messengers (e.g., cyclic adenosine monophosphate (cAMP)) of dependent phosphorylation or by means of other allosteric mechanisms (e.g., protein-protein interaction) which are under hormone control. Hormones that regulate the CAMP level are, for example, adrenalin, noradrenalin, glucagon, and insulin.
The invention relates to a process for the preparation of a substrate, comprising
a) reacting a fatty acid provided with a fluorescent label with 2,3-epoxypropanol to give a monoacylglyceride in alcoholic solution, such as, for example, C
1
-C
4
-alkanol, preferably methanol, at room temperature with addition of a base, such as, for example, a non nucleophilic inorganic base, preferably alkali metal carbonates and alkali metal C
1
-C
4
-alkanolates, particularly preferably methanolates, such as sodium methanolate or potassium methanolate,
b) subjecting this monoacylglyceride to ultrasonic treatment with phospholipids in the ratio (mg/ml) 1:10 to 10:1, preferably 1:2 to 3:1, and particularly preferably 1:1 to 1.5:1, from which the substrate results, which is recognizable by a color change from yellow to red.
A fluorescent label is defined as a chemical group within a molecule, which, after excitation by light, is itself capable of emitting light. Such groups are employed here in order to prepare substances which themselves are still detectable in lowest concentrations of about 1 nM. Mention may be made, for example, of N,N-dimethylaminosulfonic acid (dansyl) or 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD), preferably NBD.
A fatty acid is understood, for example, as meaning a long-chain carboxylic acid, which is saturated or unsaturated and has a chain length of C-8 to C-20, preferably C-12, C-14, C-16, and C-18 which is saturated or unsaturated, particularly preferably C-12 and saturated.
A fatty acid provided with a fluorescent label was coupled to a monoacylglycerid
Jordan Holger
Kleine Horst
Müller Günther
Petry Stefan
Wenzel Horst
Aventis Pharma Deutschland GmbH
Gitomer Ralph
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