US3862009A - Determination of triglycerides - Google Patents
Determination of triglycerides Download PDFInfo
- Publication number
- US3862009A US3862009A US365355A US36535573A US3862009A US 3862009 A US3862009 A US 3862009A US 365355 A US365355 A US 365355A US 36535573 A US36535573 A US 36535573A US 3862009 A US3862009 A US 3862009A
- Authority
- US
- United States
- Prior art keywords
- reagent
- saponification
- buffer
- carboxylesterase
- lipase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 150000003626 triacylglycerols Chemical class 0.000 title claims abstract description 19
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 60
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 34
- 238000007127 saponification reaction Methods 0.000 claims abstract description 34
- 239000000872 buffer Substances 0.000 claims abstract description 25
- 108090001060 Lipase Proteins 0.000 claims abstract description 22
- 102000004882 Lipase Human genes 0.000 claims abstract description 22
- 239000004367 Lipase Substances 0.000 claims abstract description 22
- 235000019421 lipase Nutrition 0.000 claims abstract description 22
- 108010051152 Carboxylesterase Proteins 0.000 claims abstract description 20
- 102000013392 Carboxylesterase Human genes 0.000 claims abstract description 20
- -1 alkaline earth metal alkyl sulfate Chemical class 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 229910052784 alkaline earth metal Inorganic materials 0.000 claims abstract description 10
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 10
- 229910052783 alkali metal Inorganic materials 0.000 claims abstract description 8
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 8
- 150000001340 alkali metals Chemical class 0.000 claims abstract description 7
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 14
- 102000057621 Glycerol kinases Human genes 0.000 claims description 12
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims description 12
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims description 12
- 101710163410 Probable glycerol kinase Proteins 0.000 claims description 12
- 240000005384 Rhizopus oryzae Species 0.000 claims description 11
- 235000013752 Rhizopus oryzae Nutrition 0.000 claims description 11
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 10
- 102000007562 Serum Albumin Human genes 0.000 claims description 10
- 108010071390 Serum Albumin Proteins 0.000 claims description 10
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 10
- 229930029653 phosphoenolpyruvate Natural products 0.000 claims description 10
- 150000008051 alkyl sulfates Chemical class 0.000 claims description 9
- 108090000371 Esterases Proteins 0.000 claims description 8
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims description 8
- 229950006238 nadide Drugs 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- 229910001429 cobalt ion Inorganic materials 0.000 claims description 6
- 210000004185 liver Anatomy 0.000 claims description 6
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 125000004432 carbon atom Chemical group C* 0.000 claims description 5
- 108091000080 Phosphotransferase Proteins 0.000 claims description 4
- 102000020233 phosphotransferase Human genes 0.000 claims description 4
- 102000013009 Pyruvate Kinase Human genes 0.000 claims description 3
- 108020005115 Pyruvate Kinase Proteins 0.000 claims description 3
- 239000010941 cobalt Substances 0.000 claims description 3
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims description 3
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims description 3
- 229910052936 alkali metal sulfate Inorganic materials 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims 3
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 claims 3
- 238000005259 measurement Methods 0.000 abstract description 5
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 10
- 229960001456 adenosine triphosphate Drugs 0.000 description 10
- DTBNBXWJWCWCIK-UHFFFAOYSA-K phosphonatoenolpyruvate Chemical compound [O-]C(=O)C(=C)OP([O-])([O-])=O DTBNBXWJWCWCIK-UHFFFAOYSA-K 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 5
- 239000000344 soap Substances 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 229960004418 trolamine Drugs 0.000 description 4
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 3
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 3
- 230000001133 acceleration Effects 0.000 description 3
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical class CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 3
- 229940043264 dodecyl sulfate Drugs 0.000 description 3
- 150000004665 fatty acids Chemical class 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- 150000008044 alkali metal hydroxides Chemical class 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- UXFQFBNBSPQBJW-UHFFFAOYSA-N 2-amino-2-methylpropane-1,3-diol Chemical compound OCC(N)(C)CO UXFQFBNBSPQBJW-UHFFFAOYSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000004308 Carboxylic Ester Hydrolases Human genes 0.000 description 1
- 108090000863 Carboxylic Ester Hydrolases Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- HOPRXXXSABQWAV-UHFFFAOYSA-N anhydrous collidine Natural products CC1=CC=NC(C)=C1C HOPRXXXSABQWAV-UHFFFAOYSA-N 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 229960002319 barbital Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- UTBIMNXEDGNJFE-UHFFFAOYSA-N collidine Natural products CC1=CC=C(C)C(C)=N1 UTBIMNXEDGNJFE-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 201000005577 familial hyperlipidemia Diseases 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N glycerol 1-phosphate Chemical compound OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000012332 laboratory investigation Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- GFYHSKONPJXCDE-UHFFFAOYSA-N sym-collidine Natural products CC1=CN=C(C)C(C)=C1 GFYHSKONPJXCDE-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- 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)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2229849A DE2229849C2 (de) | 1972-06-19 | 1972-06-19 | Verfahren und Reagens zur Bestimmung von Triglyceriden |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| US3862009A true US3862009A (en) | 1975-01-21 |
Family
ID=5848134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US365355A Expired - Lifetime US3862009A (en) | 1972-06-19 | 1973-05-30 | Determination of triglycerides |
Country Status (17)
| Country | Link |
|---|---|
| US (1) | US3862009A (OSRAM) |
| JP (1) | JPS4964495A (OSRAM) |
| AR (1) | AR195328A1 (OSRAM) |
| AT (1) | AT324282B (OSRAM) |
| BE (1) | BE801106A (OSRAM) |
| CA (1) | CA994658A (OSRAM) |
| CH (1) | CH573117A5 (OSRAM) |
| CS (1) | CS166842B2 (OSRAM) |
| DD (1) | DD104367A5 (OSRAM) |
| DK (1) | DK144643C (OSRAM) |
| FR (1) | FR2190277A5 (OSRAM) |
| GB (1) | GB1395126A (OSRAM) |
| HU (1) | HU167097B (OSRAM) |
| IT (1) | IT990535B (OSRAM) |
| NL (1) | NL165845C (OSRAM) |
| SE (1) | SE413326B (OSRAM) |
| SU (1) | SU639487A3 (OSRAM) |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4012287A (en) * | 1975-11-18 | 1977-03-15 | Dr. Bruno Lange Gmbh | Method and reagent for the quantitative analysis of triglycerides |
| US4014744A (en) * | 1975-01-30 | 1977-03-29 | Miles Laboratories Inc. | Processes for measuring tri-, di- and monoglycerides |
| US4019961A (en) * | 1974-03-14 | 1977-04-26 | Boehringer Mannheim G.M.B.H. | Analytical enzymatic determination |
| US4045297A (en) * | 1975-12-15 | 1977-08-30 | Monsanto Company | Triglycerides determination method |
| US4056442A (en) * | 1976-06-01 | 1977-11-01 | The Dow Chemical Company | Lipase composition for glycerol ester determination |
| US4066508A (en) * | 1975-08-12 | 1978-01-03 | Boehringer Mannheim Gmbh | Process and reagent for determining triglycerides |
| US4168203A (en) * | 1974-04-30 | 1979-09-18 | Fujisawa Pharmaceutical Co., Ltd. | Quantitative analysis of neutral lipids and lecithin |
| US4178285A (en) * | 1978-12-20 | 1979-12-11 | Felts James M | Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system |
| US4179334A (en) * | 1976-08-19 | 1979-12-18 | Eastman Kodak Company | Hydrolysis of protein-bound triglycerides |
| US4229527A (en) * | 1975-12-24 | 1980-10-21 | Boehringer Mannheim Gmbh | Process and reagent for the kinetic determination of enzyme substrates |
| US4241178A (en) * | 1978-01-06 | 1980-12-23 | Eastman Kodak Company | Process and composition for the quantification of glycerol ATP and triglycerides |
| US4259440A (en) * | 1979-05-21 | 1981-03-31 | Miles Laboratories, Inc. | Hydrolysis and assay of triglycerides |
| US4264589A (en) * | 1978-12-20 | 1981-04-28 | Felts James M | Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system |
| US4273870A (en) * | 1978-07-18 | 1981-06-16 | Boehringer Mannheim Gmbh | Method and composition for the determination of glycerol |
| US4309502A (en) * | 1980-06-30 | 1982-01-05 | Beckman Instruments, Inc. | Enzymatic assay for glycerol and triglycerides and a reagent for use therein |
| EP0045031A1 (en) * | 1980-07-22 | 1982-02-03 | Baker Instruments Corporation | Glycerol detection reagent and its use |
| US4322496A (en) * | 1980-04-17 | 1982-03-30 | Eastman Kodak Company | Inhibition of lactate oxidase |
| US4368261A (en) * | 1979-12-14 | 1983-01-11 | Boehringer Mannheim Gmbh | Method and reagent for the determination of triglycerides |
| US4394445A (en) * | 1979-02-22 | 1983-07-19 | Nix Paul T | Enzymatic glyceride hydrolysis |
| US4425427A (en) | 1980-03-13 | 1984-01-10 | Vitafin N.V. | Simultaneous, kinetic, spectrophotometric analysis of blood serum for multiple components |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4275152A (en) | 1977-02-03 | 1981-06-23 | Eastman Kodak Company | Hydrolysis of protein-bound cholesterol esters |
| US4275151A (en) | 1977-02-03 | 1981-06-23 | Eastman Kodak Company | Hydrolysis of protein-bound cholesterol esters |
| FR2449726A1 (fr) * | 1979-02-22 | 1980-09-19 | Millipore Corp | Composition a base de lipases bacteriennes et son application a l'hydrolyse et au dosage d'un ester de glycerol |
| JPS55156598A (en) * | 1979-05-25 | 1980-12-05 | Mitsubishi Chem Ind Ltd | Enzymatic determination of monobasic fatty acid |
| JPS561895A (en) * | 1979-06-20 | 1981-01-10 | Mitsubishi Chem Ind Ltd | Enzymic determination of monofunctional fatty acid |
| DE3413118A1 (de) * | 1984-04-06 | 1985-10-24 | Miles Laboratories, Inc., Elkhart, Ind. | Analysenverfahren und mittel zum nachweis esterolytischer und/oder proteolytischer enzyme |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3703591A (en) * | 1970-12-16 | 1972-11-21 | Calbiochem | Triglyceride hydrolysis and assay |
| US3759793A (en) * | 1970-01-02 | 1973-09-18 | Boehringer Mannheim Gmbh | Process for the quantitative determination of tri di and monoglycerides |
-
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 CH CH864573A patent/CH573117A5/xx not_active IP Right Cessation
- 1973-06-14 SE SE7308385A patent/SE413326B/xx unknown
- 1973-06-14 FR FR7321759A patent/FR2190277A5/fr not_active Expired
- 1973-06-19 JP JP48069109A patent/JPS4964495A/ja active Pending
- 1973-06-19 BE BE132423A patent/BE801106A/xx not_active IP Right Cessation
- 1973-12-13 SU SU731975398A patent/SU639487A3/ru active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3759793A (en) * | 1970-01-02 | 1973-09-18 | Boehringer Mannheim Gmbh | Process for the quantitative determination of tri di and monoglycerides |
| US3703591A (en) * | 1970-12-16 | 1972-11-21 | Calbiochem | Triglyceride hydrolysis and assay |
Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4019961A (en) * | 1974-03-14 | 1977-04-26 | Boehringer Mannheim G.M.B.H. | Analytical enzymatic determination |
| US4168203A (en) * | 1974-04-30 | 1979-09-18 | Fujisawa Pharmaceutical Co., Ltd. | Quantitative analysis of neutral lipids and lecithin |
| US4014744A (en) * | 1975-01-30 | 1977-03-29 | Miles Laboratories Inc. | Processes for measuring tri-, di- and monoglycerides |
| US4066508A (en) * | 1975-08-12 | 1978-01-03 | Boehringer Mannheim Gmbh | Process and reagent for determining triglycerides |
| US4012287A (en) * | 1975-11-18 | 1977-03-15 | Dr. Bruno Lange Gmbh | Method and reagent for the quantitative analysis of triglycerides |
| US4045297A (en) * | 1975-12-15 | 1977-08-30 | Monsanto Company | Triglycerides determination method |
| US4229527A (en) * | 1975-12-24 | 1980-10-21 | Boehringer Mannheim Gmbh | Process and reagent for the kinetic determination of enzyme substrates |
| US4056442A (en) * | 1976-06-01 | 1977-11-01 | The Dow Chemical Company | Lipase composition for glycerol ester determination |
| JPS5747484A (en) * | 1976-08-19 | 1982-03-18 | Eastman Kodak Co | Hydrolysis of plasma phospholipid |
| US4179334A (en) * | 1976-08-19 | 1979-12-18 | Eastman Kodak Company | Hydrolysis of protein-bound triglycerides |
| US4241178A (en) * | 1978-01-06 | 1980-12-23 | Eastman Kodak Company | Process and composition for the quantification of glycerol ATP and triglycerides |
| US4273870A (en) * | 1978-07-18 | 1981-06-16 | Boehringer Mannheim Gmbh | Method and composition for the determination of glycerol |
| US4264589A (en) * | 1978-12-20 | 1981-04-28 | Felts James M | Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system |
| US4178285A (en) * | 1978-12-20 | 1979-12-11 | Felts James M | Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system |
| US4394445A (en) * | 1979-02-22 | 1983-07-19 | Nix Paul T | Enzymatic glyceride hydrolysis |
| US4259440A (en) * | 1979-05-21 | 1981-03-31 | Miles Laboratories, Inc. | Hydrolysis and assay of triglycerides |
| US4368261A (en) * | 1979-12-14 | 1983-01-11 | Boehringer Mannheim Gmbh | Method and reagent for the determination of triglycerides |
| US4425427A (en) | 1980-03-13 | 1984-01-10 | Vitafin N.V. | Simultaneous, kinetic, spectrophotometric analysis of blood serum for multiple components |
| US4322496A (en) * | 1980-04-17 | 1982-03-30 | Eastman Kodak Company | Inhibition of lactate oxidase |
| US4309502A (en) * | 1980-06-30 | 1982-01-05 | Beckman Instruments, Inc. | Enzymatic assay for glycerol and triglycerides and a reagent for use therein |
| EP0045031A1 (en) * | 1980-07-22 | 1982-02-03 | Baker Instruments Corporation | Glycerol detection reagent and its use |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2190277A5 (OSRAM) | 1974-01-25 |
| CS166842B2 (OSRAM) | 1976-03-29 |
| CA994658A (en) | 1976-08-10 |
| NL7305350A (OSRAM) | 1973-12-21 |
| BE801106A (fr) | 1973-12-19 |
| NL165845B (nl) | 1980-12-15 |
| JPS4964495A (OSRAM) | 1974-06-21 |
| HU167097B (OSRAM) | 1975-08-28 |
| SE413326B (sv) | 1980-05-19 |
| SU639487A3 (ru) | 1978-12-25 |
| CH573117A5 (OSRAM) | 1976-02-27 |
| DD104367A5 (OSRAM) | 1974-03-05 |
| AT324282B (de) | 1975-08-25 |
| DK144643C (da) | 1982-09-27 |
| AR195328A1 (es) | 1973-09-28 |
| DK144643B (da) | 1982-04-26 |
| NL165845C (nl) | 1981-05-15 |
| GB1395126A (en) | 1975-05-21 |
| IT990535B (it) | 1975-07-10 |
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