Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides

Definitions

• the present invention is concerned with a process and reagent for the determination of triglycerides by the saponification of glycerides and determination of the glycerol thereby liberated.
• triglycerides plays an increasingly important part in foodstuff analysis and also in medical diagnosis, especially in the diagnosis of hyperlipemias in clinical chemistry.
• the triglycerides are first saponified with an alcoholic solution of an alkali metal hydroxide and the glycerol formed is then determined, the determination preferably being carried out by enzymatic methods.
• the glycerol is phosphorylated with adenosine triphospate (ATP) in the presence of glycerokinase (GK) to give glycerol-l-phosphate and adenosine diphosphate (ADP).
• ATP adenosine triphospate
• GK glycerokinase
• ADP adenosine diphosphate
• the ADP formed is, in turn, converted by phosphoenol pyruvate (PEP), in the presence of pyruvate kinase, into pyruvate and ATP.
• PEP phosphoenol pyruvate
• the pyruvate hereby formed is hydrogenated by nicotinamide-adenine-dinucleotide in the reduced form (NADH), in the presence of lactate dehydrogenase (LDH), to give lactate, the NADH thereby being oxidized to nicotinamide-adeninedinucleotide (NAD).
• NADH nicotinamide-adenine-dinucleotide
• LDH lactate dehydrogenase
• NAD nicotinamide-adeninedinucleotide
• the amount of NADH utilized by the reaction is equivalent to the amount of glycerol.
• NADH can easily be determined quantitatively on the basis of its absorption at 366, 340 or 344 nm.
• a disadvantage of the enzymatic splitting is, however, that the saponification still takes quite a long time and, in addition, necessitates the use of considerable amounts of the very expensive enzyme. In order to achieve useful reaction times, it is necessary to use about 1 mg. of the enzyme per test. Furthermore, the reaction time is more than 30 minutes and thus is scarcely suitable for routine laboratory investigations, especially in the case of tests which have to be carried out frequently. In addition, the liberated fatty acids form insoluble soaps with calcium and magnesium ions which, in turn, give rise to turbidity and thus a falsification of the measurement results if centrifuging is not carried out.
• the present invention provides a method for the determination of triglycerides by enzymatic saponification by means of a lipase and measurement of the liberated glycerol, which process comprises carrying out the saponification in the presence of carboxylesterase and of an alkali metal or alkaline earth metal alkyl sulfate, the alkyl radical of which contains 10 to 15 carbon atoms.
• the new process according to the present invention is preferably carried out in the presence of serum albu-
• the reagent combination used according to the present invention enables the amount of lipase necessary for the saponification to be very considerably reduced from about 1 mg. per test 20 pg. per test, with a simultaneous reduction of the reaction temperature to ambient temperature and of the period of the reaction.
• a lipase from Rhizopus arrhizus is preferred.
• carboxylesterase EC 3.1.1.1 carboxyl ester hydrolase
• a mammalian liver preparation especially a pig liver esterase.
• carboxylesterases can also be used. An amount of about pg. esterase per test has proved to be completely sufficient.
• the alkali metal salts are preferred because they do not form insoluble soaps with the liberated fatty acids.
• the alkaline earth metal alkyl sulfates can also be used.
• the dodecyl sulfates are preferred because they accelerate the saponification of the triglycerides under these conditions the most (factor 5).
• the alkyl sulfate is effective in an amount of as small as 0.01 mg./ml in the test batch. Amounts above 1.0 mg./ml. can lead to a disturbing foam formation and slight inhibition and are, therefore, preferably not used.
• the process according to the present invention is preferably carried out at a pH between 6 and 9 and more preferably at at pH of 7 to 8.5.
• This has the advantage that, in the case of detection of the glycerol formed by the known reaction using ATP, GK, PEP, PK, NADH and LDH, no rebuffering is essential so that the saponification and measurement of the glycerol formed can be carried out in a single reaction batch.
• serum albumen preferably of bovine serum albumen
• a turbidity due to precipitated soaps is avoided so that such soaps do not have to be separated off, for example, by centrifuging.
• the serum albumen is preferably used in a concentration of between 0.1 and 2.0 mg./ml. test batch.
• Cobalt and/or magnesium ions possess a certain activating effect on the lipase and esterase and can, there fore, also be added for further acceleration of the reaction.
• any buffer can be used which is effective in the abovegiven pH range, with the exception of phosphate buffers.
• suitable buffers include triethanol-amine buffer, tris buffer, imidazole buffer, veronal buffer and glycine buffer, as well as, but less preferably, amediol buffer, borate buffer and collidine buffer.
• the new reagent according to the present invention for carrying out the process of the present invention comprises a system for the detection of glycerol and, in addition, lipase, carboxylesterase, an alkali metal or alkaline earth metal alkyl sulfate, the alkyl radical of which contains to carbon atoms, and optionally also serum albumen.
• any known system can be used for the detection of glycerol in the reagent according to the present invention.
• a preferred detection system comprises NADH, ATP, PEP, LDH, PK and GK, as well as magnesium ions and buffer.
• This detection system can be readily used in the presence of the saponification agent combination according to the present invention and has, in addition, the advantage that the components do not disturb each other and all the enzymes are active in the same pH range.
• an especially preferred reagent consists of:
• lipase from Rhizopus arrhizus 0.5 to 20.0 mg. carboxylesterase 0.01 to 0.2 mg. alkyl sulfate (preferably sodium dodecyl sulfate) 1 to mM NADH 10 to 100 mM ATP 2 to 20 mM PEP 0.5 to 5 mg.
• LDH lipase from Rhizopus arrhizus 0.5 to 20.0 mg. carboxylesterase 0.01 to 0.2 mg. alkyl sulfate (preferably sodium dodecyl sulfate) 1 to mM NADH 10 to 100 mM ATP 2 to 20 mM PEP 0.5 to 5 mg.
• LDH lipase from Rhizopus arrhizus
• serum albumen 3 to 30 mM magnesium ions optionally 0.5 to 1.0 mM cobalt ions and 0.03 to 0.3M buffer of pH 6 to 9.
• the reagent combination preferably contains a 0.1M triethanolamine buffer of pH 7 to 8.5
• the process and reagent according to the present invention permit an extraordinary acceleration and simplification of the triglyceride determination.
• the time of the determination is reduced from more than an hour to less than 30 minutes.
• EXAMPLE 1 There is prepared a storage-stable reagent, consisting of 5 components which are mixed prior to use, and is then storage-stable in a mixed state for l to 2 days:
• Component 1 0.1M triethanolamine buffer, pH 7.6, containing 3 mM magnesium sulfate, 1.5 mg. bovine serum albumen/ml. and 0.1 mg. sodium dodecyl sulfate/ml.
• Component 2 solution of6 mM NADl-l, 33 mM ATP and 11 mM PEP in distilled water
• Component 3 crystalline suspension of 2 mg. LDH/ml. and 1 mg. PK/ml. (commercially available)
• Component 4 solution of 0.2 mg. lipase from Rhizopus arrhizus/ml. and 4.0 mg. carboxylesterase/ml.
• Component 5 crystalline suspension of 2 mg. GK/ml. 2.9 ml. Component 1,0.1 ml. Component 2 and 0.02 ml.
• Component 3 were mixed and warmed to 25C.
• 0.1 ml. serum which contains the triglycerides to be determined, was then admixed therewith and incubated for 5 minutes at the given temperature.
• 0.1 ml. Component 4 was then admixed, the mixture was maintained for 15 to 20 minutes at the given temperature and subsequently the extinction was read off in a photometer at 366 nm or at 340 nmv The valve read off is E Subsequently, 0.02 ml.
• Component 5 was added to the test sample and, after 10 minutes, the extinction was again read off. The measurement value obtained was After a further 10 minutes, a further reading was made which was E The results were evaluated as follows:
• Reagent for the determination of triglycerides by enzymatic saponification which reagent comprises a saponification agent, a buffer, and a system for the de tection of glycerol, wherein the saponification agent comprises a lipase obtained from Rhizopus arrhizus, carboxylesterase, and an alkali metal or alkaline earth metal alkyl sulfate, wherein the alkyl group contains from 10 to 15 carbon atoms.
• Reagent as claimed in claim 1 wherein the said system for the detection of glycerol comprises nicotinamideadenine-dinucleotide in reduced form, adenosine triphosphate, phosphoenol pyruvate, lactate dehydrogenase, pyruvate kinase, glycerokinase, cobalt and/or magnesium ions and a buffer.
• Reagent as claimed in claim 1 wherein the buffer is a 0.1M triethanolamine buffer of pH 7 to 8.5.
• Reagent as claimed in claim 1 additionally comprising 0.5 to 1.0 mM of cobalt ions.
• Reagent as claimed in claim 1 comprising:
• Method for the determination of triglycerides by enzymatic saponification comprises saponifying a sample containing triglycerides with a saponification agent comprising a lipase obtained from Rhizopus arrhizus, carboxylesterase and an alkali metal or alkaline earth metal alkyl sulfate, wherein the alkyl radicals are of from 10 to 15 carbon atoms, and measuring the liberated glycerol.

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• Chemical & Material Sciences (AREA)
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• Life Sciences & Earth Sciences (AREA)
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• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Health & Medical Sciences (AREA)
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• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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Cited By (19)

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Citations (2)

• 1973
  • 1973-03-12 AT AT216473A patent/AT324282B/de active
  • 1973-04-10 IT IT22837/73A patent/IT990535B/it active
  • 1973-04-11 HU HUBO1423A patent/HU167097B/hu unknown
  • 1973-04-16 AR AR247572A patent/AR195328A1/es active
  • 1973-04-17 NL NL7305350.A patent/NL165845C/xx not_active IP Right Cessation
  • 1973-04-25 DD DD170421A patent/DD104367A5/xx unknown
  • 1973-05-04 DK DK245273A patent/DK144643C/da not_active IP Right Cessation
  • 1973-05-08 CS CS3274*BA patent/CS166842B2/cs unknown
  • 1973-05-30 US US365355A patent/US3862009A/en not_active Expired - Lifetime
  • 1973-06-11 GB GB2769573A patent/GB1395126A/en not_active Expired
  • 1973-06-13 CA CA174,004A patent/CA994658A/en not_active Expired
  • 1973-06-14 SE SE7308385A patent/SE413326B/xx unknown
  • 1973-06-14 CH CH864573A patent/CH573117A5/xx not_active IP Right Cessation
  • 1973-06-14 FR FR7321759A patent/FR2190277A5/fr not_active Expired
  • 1973-06-19 BE BE132423A patent/BE801106A/xx not_active IP Right Cessation
  • 1973-06-19 JP JP48069109A patent/JPS4964495A/ja active Pending
  • 1973-12-13 SU SU731975398A patent/SU639487A3/ru active

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