CA1202869A - Method for the quantitative measurement of the phosphatidyl glycerol - Google Patents
Method for the quantitative measurement of the phosphatidyl glycerolInfo
- Publication number
- CA1202869A CA1202869A CA000415626A CA415626A CA1202869A CA 1202869 A CA1202869 A CA 1202869A CA 000415626 A CA000415626 A CA 000415626A CA 415626 A CA415626 A CA 415626A CA 1202869 A CA1202869 A CA 1202869A
- Authority
- CA
- Canada
- Prior art keywords
- quantitative measurement
- measurement
- glycero
- hydrogen peroxide
- phosphatidyl glycerol
- 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
Links
- 238000005259 measurement Methods 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 47
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 title claims abstract description 40
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 title claims abstract description 39
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 27
- 239000010452 phosphate Substances 0.000 claims abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 210000001124 body fluid Anatomy 0.000 claims abstract description 3
- 239000010839 body fluid Substances 0.000 claims abstract description 3
- 102000000587 Glycerolphosphate Dehydrogenase Human genes 0.000 claims description 30
- 108010041921 Glycerolphosphate Dehydrogenase Proteins 0.000 claims description 30
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 30
- 102000014384 Type C Phospholipases Human genes 0.000 claims description 28
- 108010079194 Type C Phospholipases Proteins 0.000 claims description 28
- 239000000203 mixture Substances 0.000 claims description 27
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical group CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 12
- 239000001301 oxygen Substances 0.000 claims description 12
- 229910052760 oxygen Inorganic materials 0.000 claims description 12
- 102000003992 Peroxidases Human genes 0.000 claims description 9
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 9
- 150000002632 lipids Chemical class 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000002243 precursor Substances 0.000 claims 3
- 238000011481 absorbance measurement Methods 0.000 claims 2
- 238000002848 electrochemical method Methods 0.000 claims 2
- KRNUKKZDGDAWBF-UHFFFAOYSA-N 2-(n-ethyl-n-m-toluidino)ethanol Chemical compound OCCN(CC)C1=CC=CC(C)=C1 KRNUKKZDGDAWBF-UHFFFAOYSA-N 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 31
- 108090000790 Enzymes Proteins 0.000 abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 12
- 239000003054 catalyst Substances 0.000 abstract description 6
- 239000012530 fluid Substances 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 29
- 239000000243 solution Substances 0.000 description 19
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- 239000000872 buffer Substances 0.000 description 11
- 229960002163 hydrogen peroxide Drugs 0.000 description 10
- 229940072417 peroxidase Drugs 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 241000187747 Streptomyces Species 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 108010093096 Immobilized Enzymes Proteins 0.000 description 5
- 239000003580 lung surfactant Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000004809 thin layer chromatography Methods 0.000 description 5
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 4
- JLTDJTHDQAWBAV-UHFFFAOYSA-N N,N-dimethylaniline Chemical compound CN(C)C1=CC=CC=C1 JLTDJTHDQAWBAV-UHFFFAOYSA-N 0.000 description 4
- 210000004381 amniotic fluid Anatomy 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 229940067606 lecithin Drugs 0.000 description 4
- 239000000787 lecithin Substances 0.000 description 4
- 235000010445 lecithin Nutrition 0.000 description 4
- 150000003904 phospholipids Chemical class 0.000 description 4
- CIEYFQRZEULHJV-UHFFFAOYSA-N 1,1-dihydroxypropan-2-one;phosphoric acid Chemical compound OP(O)(O)=O.CC(=O)C(O)O CIEYFQRZEULHJV-UHFFFAOYSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 229960003964 deoxycholic acid Drugs 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 150000002989 phenols Chemical class 0.000 description 3
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 3
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 3
- TUAMRELNJMMDMT-UHFFFAOYSA-N 3,5-xylenol Chemical compound CC1=CC(C)=CC(O)=C1 TUAMRELNJMMDMT-UHFFFAOYSA-N 0.000 description 2
- XQXPVVBIMDBYFF-UHFFFAOYSA-N 4-hydroxyphenylacetic acid Chemical compound OC(=O)CC1=CC=C(O)C=C1 XQXPVVBIMDBYFF-UHFFFAOYSA-N 0.000 description 2
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- GGSUCNLOZRCGPQ-UHFFFAOYSA-N diethylaniline Chemical compound CCN(CC)C1=CC=CC=C1 GGSUCNLOZRCGPQ-UHFFFAOYSA-N 0.000 description 2
- QRMZSPFSDQBLIX-UHFFFAOYSA-N homovanillic acid Chemical compound COC1=CC(CC(O)=O)=CC=C1O QRMZSPFSDQBLIX-UHFFFAOYSA-N 0.000 description 2
- KUBCEEMXQZUPDQ-UHFFFAOYSA-N hordenine Chemical compound CN(C)CCC1=CC=C(O)C=C1 KUBCEEMXQZUPDQ-UHFFFAOYSA-N 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
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- 230000035484 reaction time Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 1
- JYSUYJCLUODSLN-UHFFFAOYSA-N 1,3-benzothiazol-2-ylhydrazine Chemical compound C1=CC=C2SC(NN)=NC2=C1 JYSUYJCLUODSLN-UHFFFAOYSA-N 0.000 description 1
- PQMRRAQXKWFYQN-UHFFFAOYSA-N 1-phenyl-2-sulfanylideneimidazolidin-4-one Chemical compound S=C1NC(=O)CN1C1=CC=CC=C1 PQMRRAQXKWFYQN-UHFFFAOYSA-N 0.000 description 1
- VPMMJSPGZSFEAH-UHFFFAOYSA-N 2,4-diaminophenol;hydrochloride Chemical compound [Cl-].NC1=CC=C(O)C([NH3+])=C1 VPMMJSPGZSFEAH-UHFFFAOYSA-N 0.000 description 1
- CCBICDLNWJRFPO-UHFFFAOYSA-N 2,6-dichloroindophenol Chemical compound C1=CC(O)=CC=C1N=C1C=C(Cl)C(=O)C(Cl)=C1 CCBICDLNWJRFPO-UHFFFAOYSA-N 0.000 description 1
- IDRHRIHJNRTYHD-UHFFFAOYSA-N 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-1h-tetrazole Chemical compound C1=CC([N+](=O)[O-])=CC=C1N1N(C=2C=CC(I)=CC=2)NC(C=2C=CC=CC=2)=N1 IDRHRIHJNRTYHD-UHFFFAOYSA-N 0.000 description 1
- UHGULLIUJBCTEF-UHFFFAOYSA-N 2-aminobenzothiazole Chemical compound C1=CC=C2SC(N)=NC2=C1 UHGULLIUJBCTEF-UHFFFAOYSA-N 0.000 description 1
- MNIQECRMTVGZBM-UHFFFAOYSA-N 3-(1-methylpyrrolidin-2-yl)pyridine;7h-purin-6-amine Chemical compound NC1=NC=NC2=C1NC=N2.CN1CCCC1C1=CC=CN=C1 MNIQECRMTVGZBM-UHFFFAOYSA-N 0.000 description 1
- DIVQKHQLANKJQO-UHFFFAOYSA-N 3-methoxytyramine Chemical compound COC1=CC(CCN)=CC=C1O DIVQKHQLANKJQO-UHFFFAOYSA-N 0.000 description 1
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 241000193798 Aerococcus Species 0.000 description 1
- 241000193792 Aerococcus viridans Species 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 206010003598 Atelectasis Diseases 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241001663154 Electron Species 0.000 description 1
- 241001547070 Eriodes Species 0.000 description 1
- 108010022355 Fibroins Proteins 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 101710112684 Non-hemolytic phospholipase C Proteins 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 241000192001 Pediococcus Species 0.000 description 1
- 101710124951 Phospholipase C Proteins 0.000 description 1
- 208000007123 Pulmonary Atelectasis Diseases 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000187220 Streptomyces albireticuli Species 0.000 description 1
- 241000499056 Streptomyces griseocarneus Species 0.000 description 1
- 241000936729 Streptomyces hachijoensis Species 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 241000082085 Verticillium <Phyllachorales> Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
- 239000011609 ammonium molybdate Substances 0.000 description 1
- 229940010552 ammonium molybdate Drugs 0.000 description 1
- 235000018660 ammonium molybdate Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000009841 combustion method Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- DZAUWHJDUNRCTF-UHFFFAOYSA-N dihydrocaffeic acid Natural products OC(=O)CCC1=CC=C(O)C(O)=C1 DZAUWHJDUNRCTF-UHFFFAOYSA-N 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 230000009429 distress Effects 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- ACGUYXCXAPNIKK-UHFFFAOYSA-N hexachlorophene Chemical compound OC1=C(Cl)C=C(Cl)C(Cl)=C1CC1=C(O)C(Cl)=CC(Cl)=C1Cl ACGUYXCXAPNIKK-UHFFFAOYSA-N 0.000 description 1
- 229940071490 hordenine Drugs 0.000 description 1
- HBSYJULDYOVADU-UHFFFAOYSA-N hydrazine;3-methyl-1,3-benzothiazol-2-one Chemical compound NN.C1=CC=C2SC(=O)N(C)C2=C1 HBSYJULDYOVADU-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- FSVCQIDHPKZJSO-UHFFFAOYSA-L nitro blue tetrazolium dichloride Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 FSVCQIDHPKZJSO-UHFFFAOYSA-L 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- UYDLBVPAAFVANX-UHFFFAOYSA-N octylphenoxy polyethoxyethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCOCCOCCO)C=C1 UYDLBVPAAFVANX-UHFFFAOYSA-N 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 229960003903 oxygen Drugs 0.000 description 1
- 229940086255 perform Drugs 0.000 description 1
- 230000009984 peri-natal effect Effects 0.000 description 1
- 125000001095 phosphatidyl group Chemical group 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000002952 polymeric resin Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 1
- HFQQZARZPUDIFP-UHFFFAOYSA-M sodium;2-dodecylbenzenesulfonate Chemical compound [Na+].CCCCCCCCCCCCC1=CC=CC=C1S([O-])(=O)=O HFQQZARZPUDIFP-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- WRPWWVNUCXQDQV-UHFFFAOYSA-N vanillylamine Chemical compound COC1=CC(CN)=CC=C1O WRPWWVNUCXQDQV-UHFFFAOYSA-N 0.000 description 1
- 229940053939 vanillylamine Drugs 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
- C12Q1/34—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
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/90—Developer
- C12Q2326/96—4-Amino-antipyrine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
- G01N2405/04—Phospholipids, i.e. phosphoglycerides
Landscapes
- 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)
Abstract
ABSTRACT
METHOD FOR THE QUANTITATIVE MEASUREMENT OF
PHOSPHATIDYL GLYCEROL
A method for quantitative measurement of phosphatidyl glycerol in the liquid such as body fluid in which the fluid is treated with an enzyme to obtain glycero-3-phos-phate, said enzyme being able to play as a catalyst in a reaction to produce said glycero-3-phosphate and diglyceride from phosphatidyl glycerol and water, and the liberated glycero-3-phosphate is observed.
METHOD FOR THE QUANTITATIVE MEASUREMENT OF
PHOSPHATIDYL GLYCEROL
A method for quantitative measurement of phosphatidyl glycerol in the liquid such as body fluid in which the fluid is treated with an enzyme to obtain glycero-3-phos-phate, said enzyme being able to play as a catalyst in a reaction to produce said glycero-3-phosphate and diglyceride from phosphatidyl glycerol and water, and the liberated glycero-3-phosphate is observed.
Description
~2();Z~69 METHOD FOR THE QUANTITATIV~ MEASUREMENT OF
PHOSPHATIDYL GLYCEROL
The present invention relates to a novel method for the quantitative measurement of phosphatidyl glycerol.
Recently, in the management of newborn babies, the respi~atory distress syndrome (RDS) in the perinatal period constitutes a big problem and the deaths due to it occupy a high percentage of total deaths. It is regarded that RDS occurs due to the lack of pulmonary surfactant. This material is important to prevent contraction of alveoli pulmonum which expands immediately after the birth. The lack of this pulmonary surfactant brings about atelectasis in respiration and occurrence of RDS. Consequently, if the occurrence of RDS can be estimated in an earlv ~eriod and the treatment o~ the newborn baby can be starte~ early, th~
occurrence of RDS may be prevented or it may be restrained to a mild case. Accordingly, the measurement of this pul-monary surfactant is the means of necessity. The means are roughly classified into the method for the measurement wherein the physical property of pulmonary surfactant is utili~ed and the one wherein compositive components of pulmonary surfactant are biochemically resolved. The lat-ter method has been practised by measuring the ratio of lipids such as the ratio of lecithin to sphingomyelin, that of phosphatidyl glycerol to phosphatidyl inositol or the quantitative measurement of dipalmitoyl phosphatidyl cho-line by means of the thin-layer chromatography ~Am. J.
Obstet. Gynecol., 440: 109 (1971); Am. J. Obstet. Gynecol., 613: 125 (1976); Am. J. Obstet. ~ynecol., ~94: 133 (1979);
Obstet. & Gynecol., 295: 57 (1981); Am. J. Obstet. Gynecol., 697: 138 (1980); etc.]. From the results of these various inspections, it has been recently recognized that the ex-istence of phosphatidyl glycerol is an important factor ~2V2869 for the occurrence of RDS. However, the quantitative measurement of phosphatidyl glycerol was very difficult because the quantity thereof in existence is as little as about one tenth of the amount of lecithin. Heretofore, as the method for the quantitative measurement of phosphatidyl glycerol, there has been known the method of thin-layer chromatography wherein, after the removal of cell components by the centrifugal separation from the liquor amnii, lipid components are extracted from the supernatant, the compo-n~nts of this extract are separated by the thin-layer chro-matography, each phospholipid is quantitatively measured, the ratios of various phospholipids are obtained and the quantity of phosphatidyl glycerol is obtained indirectly from the total phospholipid value preliminarily obtained by the quantitative measurement of phosphoric acid accord-ing to the wet combustion method [Am. J. O~stet. Gynecol., 613: 125 (1976); Am. J. Obstet. Gynecol., 1079: 135 (1979);
Am. J. Obstet. Gynecol., 899: 133 (1979); Am. J. Obstet.
Gynesol., 440: 109 (1971)], or the method for the quantita-tive measurement wherein the high speed liquid chromato-graphy is used [Journal of Chromatography, 277: 223 (1981)].
However, this method of thin-layer chromatography had dis-advantages that it takes two to three days for the extrac-tion of lipid components from liquor amnii, it requires complicated operations and, further, a heating at a high temperature is necessary because of the spot detection.
Also it had such other defects that it is an indirect method for the measurement based on the ratios of relative quanti-ties from spots and total phospholipid and it can not per-form simultaneous treatment of many samples because itemploys thin-layer chromatography.
The present inventors carried out various studies about the method whereby, in a liquid containing various lipid components to be inspected such as liquor amnii, only 12(~;~869 phosphatidyl glycerol can be quantitatively measured simp-ly, conveniently and accurately in a short time. As the result, they found ~uite unexpectedly that phospholipase C (phospholipase C: 3.1.4.3 phosphatidyl choline choline phosphohydrolase), which has the enzymatic action to act on lecithin and catalyze the reaction to produce 1.2-digly-ceride and phosphatidyl choline acts on phosphatidyl glycerol producing glycero-3-phosphate and diglyceride and they achieved a satisfactory method for the quantitative measurement of phosphatidyl glycerol only by quantitatively measuring this glycero-3-phosphate produced by the reaction and extricated. More preferably, they have achieved a method whereby only phosphatidyl glycerol may be quantita-tively measured highly satisfactorily by allowing phos-pholipase C to act on the liquid to be inspected to produce glycero-3-phosphate allowing glycerophosphate oxidase to act on this glycero-3-phosphate and measuring the quantity of oxygen consumed or hydrogen peroxide produced in the reaction. Thus the present invention relates to a method for the quantitative measurement of phosphatidyl glycerol in the liquid to be inspected, which comprises liberating glycero-3-phosphate by the action of an enzyme which plays a role of the catalyst in the reaction to produce glycero-3-phosphate and diglyceride from phosphatidyl ~lycerol and water and then quantitatively measuring glycero-3-yhosphate produced; preferably, it relates to a method for the quan-titative measurement of phosphatidyl glycerol in the liquid to be inspected, which comprises carrying out the quantita-tive measurement by the combination of the following steps ~0 (a), (b) and (c):
(a) liberating glycero-3-phosphate by allowing phos-pholipase C to act on phosphatidyl glycerol, (b3 allowing glycerophosphate oxidase to act on glycero-3-phosphate,and then (c) measuring the quantity of oxygen consumed or .., hydrogen peroxide produced in the reaction.
In the present invention, it is advantageous to pre-pare each reagent in an adjusted kit for the quantitative measurement and besides the operation for the quantitative measurement can be conducted at the room temperature of about 37C and it is a very simple operation and in addi-tion, the time required for the reaction is very short.
Moreover, based on the present invention, phosphatidyl glycerol can be accurately measured down to a markedly low concentration and the value of phosphatidyl glycerol can be directly measured revealing a great usefulness. Also, since the measurement can be conducted simply and con-veniently in a short time, many samples can be simultane-ously measured. Thus, the present invention provides a useful method for the quantitative measurement of phos-phatidyl glycerol.
First, as an example of the enzyme which plays a role of the catalyst in the reaction to produce glycero-3-phos-phate and diglyceride from phosphatidyl glycerol and water in the present invention, phospholipase C may be included.
This phospholipase C (E.C.3.1.4.3) has been known as an enzyme which is a catalyst in the reaction to produce each 1 mole of diglyceride and phosphatidyl choline from each 1 mole of lecithin and water. As far as the substance can be a catalyst in the enzyme reaction mentioned above, there may be used any one such as the one obtained by the ext~ac-tion of phospholipase C-containing cells or an enzyme re-agent sold on the market, for example, a microorganism-originated enzyme obtained from the culture of a phos-pholipase C-producing microorganisms which belongs to a Streptomyces genus such as Streptomyces hachijoensis A-1143 strain ¦FERM-P No. 1329), Streptomyces albireticuli IFO 12737, Streptomyces cinn~mnneum IFO 12852 (Strepto-~20286g verticillium cinnamoneum subsp. cinnamoneum IFO 12852).
Streptomyces griseocarinensis IFO 12776 (Streptoverticil-lium griseocarneum IFO 12776~ [among the bacteria belong-ing to Streptomyces genus, those which form whirl in its aerobacteria system are separated from Streptomyces genus to be named Streptoverticillium genus (Baldacci; 1958)]
or Clostridium welchii and Bacillus cereus [Japanese Ex-amined Published Patent Application No. 1356/1978, I,ipid Metabolism P214 (1960)] or enzyme reagent of phospholipase C sold on the market. The quantity of phospholipase C to be used should be appropriately modified and designed ac-cording to the time required for the measurement and the concentration of phosphatidyl glycerol and no particular limitation is to be imposed on it. For instance, per one test, there may be used phospholipase C of usually not less than 0.1 unit, preferably about 1 - 100 units. In addition, this phospholipase C is preferably used after it is dis-solved in a buffer such as weakly acidic to weakly alkaline Tris-HCl buffer, citric acid buffer, boric acid buffer, PIPES-NaOH buffer or imidazole buffer andl if necessary, it may be adjusted by adding thereto a surfactant such as sodium deoxycholate or serum albumin. Then, the enzyme solution containing phospholipase C thus adjusted and the liquid to be inspected are mixed and glycero-3-phosphate is produced from phosphatidyl glycerol in the liquid to be inspected with the consumption of water. The mixing ratio of the both is not particularly limited, and they may be mixed at such ratio that preferably about 1 - 100 units of phospholipase C is contained per one test of the liquid to be inspected. The reaction temperature may be about 37C and the reaction time may be what is sufficient for the liberation of glycero-3-phosphate,usually not less than 5 minutes, preferably not less than 10 minutes. Then, glycero-3-phosphate liberated by the reaction is quantita-tively measured. From this value of quan~itative ~easure-., , ~ i, 2136~
~ 6 -ment, the value of phosphatidyl glycerol in the liquid to be inspected is obtained.
For the quantitative measurement of glycero-3-phos-phate, various known methods for the quantitative chemicalmeasurement or those using enzymes may be employed. Pre-ferably, an enzymatic method ~or the quantitative measure-ment wherein one species or more of enzymes whose substrate is glycero-3-phosphate is employed for action and the de-tectable change of enzyme action in the reaction is quanti-tatively measured is simple and convenient. For instance, the GPO type method for quantitative measurement wherein glycerophosphate oxidase (GPO) is allowed to act on the liberated glycero-3-phosphate so that the oxygen in the re action mixture is consumed to produce h~drogen peroxide is particularly simple and convenient.
Further, in this GPO type method for the quantitative measurement, the quantity of oxygen consumed in the reac-tion liquid is measured with an oxygen electrode; thequantity of hydrogen peroxide produced is measured with a hydrogen peroxide electrode as the electrical change. The quantity of oxygen or h~drogen peroxide can be measured by an enzyme electrode wherein an oxygen electrode or a hydrogen peroxide electrode and immobilized enzyme are assembled.
Immobilized enzymes may be formed by various means such as inclusion, adsorption and bonding; for instance, GPO or GPO
and phospholipase C are immobilized by a known immobiliza-tion means such as a method wherein GPO or GPO and phos-pholipase C are included and immobilized with acrylamide;or a method wherein GPO or GPO and phospholipase C are blended with a protein such as albumin and the proteins are cross linked with each other to cause immobilization; or a method wherein GPO or GPO and phospholipase C are included with collagen or fibroin or are covalent-bonded with them; or a l~U2~;9 method wherein GPO or GPO and phospholipase C are immobiliz-ed by the adsorption onto a porous organic polymer resin or by the covalent bonding with the same, a method wherein GPO
or GPO and phospholipase C are included and immobilized us-ing a photosetting resin. The form of the immobilizedemzyme may be the one wherein the immobilized enzyme is processed into a membranelike, fibrous, granular or tubular shape which is preferable in use for an enzyme electrode to be assembled into an oxygen electrode or a hydrogen peroxide electrode and the immobilized enzyme may be used as an enzyme electrode wherein this immobilized enzyme is incorporated into the detecting part of the electrode to be used so that the amount of the effective enzyme used for the quantitative measurement by an electrical means is markedly small.
Further, the quantitative measurement of hydrogen per-oxide may be carried out by using an indicator composition which produces a detecta~le material by the reaction with hydrogen peroxide. As the indicator composition,there usually are used the compositions whose change may be quantitatively - measured by a spectroscopic means, for instance, a colora-tion reagent composition whose color change occurs in the visible range, a fluorescence reagent composition which fluoreSces by ultraviolet rays irradiation or a photogenic reagent composition which gives forth light. For example, as a coloration reagent composition, a material containing a substance having peroxidase action and chromogen may be used. As the substance having the peroxidase action, usual-ly the peroxidase originated from horseradish is often usedand, as the chromogen, usually the combination of an elec-tron acceptor and a phenolic compound is often used.
Further, as an electron acceptor, such compound is used as, for example, 4-aminoantipyrine, 2~hydrazinobenzothiazole, 3-methyl-2-benzothiazolone hydrazine, 2-aminobenzothiazole ~,' ~ ~Z~Z869 . _ ~
or the like. As a phenolic compound, such compound is used as, for example~ phenol, 3-methyl-N-ethyl~N-(B-hydroxyethylJaniline , 3,5-xylenol, N,N-dimethylaniline, N,N-diethylaniline or the like.
As luminous substrates in a fluorescence reagent com-position or a photogenic reagent composition, there may be illustrated various known ones, for example, bis(2,4,6-trichlorophenol)oxalate, phenylthiohydantoin, homovanillic acid, 4-hydroxyphenylacetic acid, vanillylamine, 3-methoxy-tyramine, phloretic acid, hordenine, luminol monoanion, lucigenine, wa~in and the like. Each of them may be, if necessary, used together with an electron acceptor and/or a substance having peroxidase action for the quantitative measurement of hydrogen peroxide.
There is no particular limitation on the quantity of the enzyme reagent or the chromogen used. For instance, there may be used per one test usually not less than 0.05 unit, preferably 0.1 - 200 units of GPO and usually not less than 0.05 unit, preferably 0.1 - 500 units of per-oxidase. ~lso there may be used a solution so adjusted by distilled water or weakly acidic or weakly alkaline buffer solution that the concentration of an electron ac-ceptor or a phenolic compound i5 usually not less than 0.1 mM. These reagents may be used separately from the enzyme solution of phospholipase C mentioned above or may be blended therewith, or further, these reagents may be formed into such composition for quantitative measurement as a freeze-dried composition or an integrate~ laminate by ap-plying them onto filter papers, films or the li~e.
The GPO type method for the quantitative measurement thus composed shows a high sensitivity to the glycero-3-phosphate in the liquid to be inspected and, since v ~ZV'~136~
g it is not affected by the impurity in the liquid to be inspected, it is an excellent method whereby an accurate measurement can be carried out.
The GP0 used for this GPO type method may be any enzymes as far as it plays the role of a catalyst in the reaction to produce dihydroxyacetone phosphoric acid and hydrogen peroxide from glycero-3-phosphate and oxy-gen, for example, Streptococcus genus, Lactobacillus genus, Lenconostoc genus, glycerophosphate oxidase producing bacteria which belong to Pediococcus genus (Japanese unexamined patent publication No. 7289211978), a enzyme obtained from the culture of glycerophosphate oxidase-producing bacteria which belongs to Aerococcus genus (Aerococcus viridans IFO 12219 strain and IFO 12317 strain, Japanese unexamined patent publication No. 15746/1980) and enzymes sold on the market.
As another enzymatic method for the quantitative measurement of glycero-3-phosphate, glycerophosphate dihydrogenase which catalyses the reac~ion to produce dihydroxyacetone phosphoric acid and reduced NAD
from glycero-3-phosphate and nicotine adenine dinucleotide (NAD) is allowed to act on glycero-3-phosphate liberated in the solution to be tested in the presence of NA~ to produce dihydroxyacetone phosphoric acid and re-duced NAD and then this reduced NAD is quan-titatively measured so that the quantity of glycero-3-phosphate may be determined. When the quantitative measurement of this reduced NAD is conducted, such method may be usually used as the measurement of absorbance by a wave length of about 340 nm or, after it is aIlowed to develop color by a water-soluble tetrazolium salt such as 3-(4,5-dimethyl)-2-thiazolyl-2H-tetrazolium bromide,
PHOSPHATIDYL GLYCEROL
The present invention relates to a novel method for the quantitative measurement of phosphatidyl glycerol.
Recently, in the management of newborn babies, the respi~atory distress syndrome (RDS) in the perinatal period constitutes a big problem and the deaths due to it occupy a high percentage of total deaths. It is regarded that RDS occurs due to the lack of pulmonary surfactant. This material is important to prevent contraction of alveoli pulmonum which expands immediately after the birth. The lack of this pulmonary surfactant brings about atelectasis in respiration and occurrence of RDS. Consequently, if the occurrence of RDS can be estimated in an earlv ~eriod and the treatment o~ the newborn baby can be starte~ early, th~
occurrence of RDS may be prevented or it may be restrained to a mild case. Accordingly, the measurement of this pul-monary surfactant is the means of necessity. The means are roughly classified into the method for the measurement wherein the physical property of pulmonary surfactant is utili~ed and the one wherein compositive components of pulmonary surfactant are biochemically resolved. The lat-ter method has been practised by measuring the ratio of lipids such as the ratio of lecithin to sphingomyelin, that of phosphatidyl glycerol to phosphatidyl inositol or the quantitative measurement of dipalmitoyl phosphatidyl cho-line by means of the thin-layer chromatography ~Am. J.
Obstet. Gynecol., 440: 109 (1971); Am. J. Obstet. Gynecol., 613: 125 (1976); Am. J. Obstet. ~ynecol., ~94: 133 (1979);
Obstet. & Gynecol., 295: 57 (1981); Am. J. Obstet. Gynecol., 697: 138 (1980); etc.]. From the results of these various inspections, it has been recently recognized that the ex-istence of phosphatidyl glycerol is an important factor ~2V2869 for the occurrence of RDS. However, the quantitative measurement of phosphatidyl glycerol was very difficult because the quantity thereof in existence is as little as about one tenth of the amount of lecithin. Heretofore, as the method for the quantitative measurement of phosphatidyl glycerol, there has been known the method of thin-layer chromatography wherein, after the removal of cell components by the centrifugal separation from the liquor amnii, lipid components are extracted from the supernatant, the compo-n~nts of this extract are separated by the thin-layer chro-matography, each phospholipid is quantitatively measured, the ratios of various phospholipids are obtained and the quantity of phosphatidyl glycerol is obtained indirectly from the total phospholipid value preliminarily obtained by the quantitative measurement of phosphoric acid accord-ing to the wet combustion method [Am. J. O~stet. Gynecol., 613: 125 (1976); Am. J. Obstet. Gynecol., 1079: 135 (1979);
Am. J. Obstet. Gynecol., 899: 133 (1979); Am. J. Obstet.
Gynesol., 440: 109 (1971)], or the method for the quantita-tive measurement wherein the high speed liquid chromato-graphy is used [Journal of Chromatography, 277: 223 (1981)].
However, this method of thin-layer chromatography had dis-advantages that it takes two to three days for the extrac-tion of lipid components from liquor amnii, it requires complicated operations and, further, a heating at a high temperature is necessary because of the spot detection.
Also it had such other defects that it is an indirect method for the measurement based on the ratios of relative quanti-ties from spots and total phospholipid and it can not per-form simultaneous treatment of many samples because itemploys thin-layer chromatography.
The present inventors carried out various studies about the method whereby, in a liquid containing various lipid components to be inspected such as liquor amnii, only 12(~;~869 phosphatidyl glycerol can be quantitatively measured simp-ly, conveniently and accurately in a short time. As the result, they found ~uite unexpectedly that phospholipase C (phospholipase C: 3.1.4.3 phosphatidyl choline choline phosphohydrolase), which has the enzymatic action to act on lecithin and catalyze the reaction to produce 1.2-digly-ceride and phosphatidyl choline acts on phosphatidyl glycerol producing glycero-3-phosphate and diglyceride and they achieved a satisfactory method for the quantitative measurement of phosphatidyl glycerol only by quantitatively measuring this glycero-3-phosphate produced by the reaction and extricated. More preferably, they have achieved a method whereby only phosphatidyl glycerol may be quantita-tively measured highly satisfactorily by allowing phos-pholipase C to act on the liquid to be inspected to produce glycero-3-phosphate allowing glycerophosphate oxidase to act on this glycero-3-phosphate and measuring the quantity of oxygen consumed or hydrogen peroxide produced in the reaction. Thus the present invention relates to a method for the quantitative measurement of phosphatidyl glycerol in the liquid to be inspected, which comprises liberating glycero-3-phosphate by the action of an enzyme which plays a role of the catalyst in the reaction to produce glycero-3-phosphate and diglyceride from phosphatidyl ~lycerol and water and then quantitatively measuring glycero-3-yhosphate produced; preferably, it relates to a method for the quan-titative measurement of phosphatidyl glycerol in the liquid to be inspected, which comprises carrying out the quantita-tive measurement by the combination of the following steps ~0 (a), (b) and (c):
(a) liberating glycero-3-phosphate by allowing phos-pholipase C to act on phosphatidyl glycerol, (b3 allowing glycerophosphate oxidase to act on glycero-3-phosphate,and then (c) measuring the quantity of oxygen consumed or .., hydrogen peroxide produced in the reaction.
In the present invention, it is advantageous to pre-pare each reagent in an adjusted kit for the quantitative measurement and besides the operation for the quantitative measurement can be conducted at the room temperature of about 37C and it is a very simple operation and in addi-tion, the time required for the reaction is very short.
Moreover, based on the present invention, phosphatidyl glycerol can be accurately measured down to a markedly low concentration and the value of phosphatidyl glycerol can be directly measured revealing a great usefulness. Also, since the measurement can be conducted simply and con-veniently in a short time, many samples can be simultane-ously measured. Thus, the present invention provides a useful method for the quantitative measurement of phos-phatidyl glycerol.
First, as an example of the enzyme which plays a role of the catalyst in the reaction to produce glycero-3-phos-phate and diglyceride from phosphatidyl glycerol and water in the present invention, phospholipase C may be included.
This phospholipase C (E.C.3.1.4.3) has been known as an enzyme which is a catalyst in the reaction to produce each 1 mole of diglyceride and phosphatidyl choline from each 1 mole of lecithin and water. As far as the substance can be a catalyst in the enzyme reaction mentioned above, there may be used any one such as the one obtained by the ext~ac-tion of phospholipase C-containing cells or an enzyme re-agent sold on the market, for example, a microorganism-originated enzyme obtained from the culture of a phos-pholipase C-producing microorganisms which belongs to a Streptomyces genus such as Streptomyces hachijoensis A-1143 strain ¦FERM-P No. 1329), Streptomyces albireticuli IFO 12737, Streptomyces cinn~mnneum IFO 12852 (Strepto-~20286g verticillium cinnamoneum subsp. cinnamoneum IFO 12852).
Streptomyces griseocarinensis IFO 12776 (Streptoverticil-lium griseocarneum IFO 12776~ [among the bacteria belong-ing to Streptomyces genus, those which form whirl in its aerobacteria system are separated from Streptomyces genus to be named Streptoverticillium genus (Baldacci; 1958)]
or Clostridium welchii and Bacillus cereus [Japanese Ex-amined Published Patent Application No. 1356/1978, I,ipid Metabolism P214 (1960)] or enzyme reagent of phospholipase C sold on the market. The quantity of phospholipase C to be used should be appropriately modified and designed ac-cording to the time required for the measurement and the concentration of phosphatidyl glycerol and no particular limitation is to be imposed on it. For instance, per one test, there may be used phospholipase C of usually not less than 0.1 unit, preferably about 1 - 100 units. In addition, this phospholipase C is preferably used after it is dis-solved in a buffer such as weakly acidic to weakly alkaline Tris-HCl buffer, citric acid buffer, boric acid buffer, PIPES-NaOH buffer or imidazole buffer andl if necessary, it may be adjusted by adding thereto a surfactant such as sodium deoxycholate or serum albumin. Then, the enzyme solution containing phospholipase C thus adjusted and the liquid to be inspected are mixed and glycero-3-phosphate is produced from phosphatidyl glycerol in the liquid to be inspected with the consumption of water. The mixing ratio of the both is not particularly limited, and they may be mixed at such ratio that preferably about 1 - 100 units of phospholipase C is contained per one test of the liquid to be inspected. The reaction temperature may be about 37C and the reaction time may be what is sufficient for the liberation of glycero-3-phosphate,usually not less than 5 minutes, preferably not less than 10 minutes. Then, glycero-3-phosphate liberated by the reaction is quantita-tively measured. From this value of quan~itative ~easure-., , ~ i, 2136~
~ 6 -ment, the value of phosphatidyl glycerol in the liquid to be inspected is obtained.
For the quantitative measurement of glycero-3-phos-phate, various known methods for the quantitative chemicalmeasurement or those using enzymes may be employed. Pre-ferably, an enzymatic method ~or the quantitative measure-ment wherein one species or more of enzymes whose substrate is glycero-3-phosphate is employed for action and the de-tectable change of enzyme action in the reaction is quanti-tatively measured is simple and convenient. For instance, the GPO type method for quantitative measurement wherein glycerophosphate oxidase (GPO) is allowed to act on the liberated glycero-3-phosphate so that the oxygen in the re action mixture is consumed to produce h~drogen peroxide is particularly simple and convenient.
Further, in this GPO type method for the quantitative measurement, the quantity of oxygen consumed in the reac-tion liquid is measured with an oxygen electrode; thequantity of hydrogen peroxide produced is measured with a hydrogen peroxide electrode as the electrical change. The quantity of oxygen or h~drogen peroxide can be measured by an enzyme electrode wherein an oxygen electrode or a hydrogen peroxide electrode and immobilized enzyme are assembled.
Immobilized enzymes may be formed by various means such as inclusion, adsorption and bonding; for instance, GPO or GPO
and phospholipase C are immobilized by a known immobiliza-tion means such as a method wherein GPO or GPO and phos-pholipase C are included and immobilized with acrylamide;or a method wherein GPO or GPO and phospholipase C are blended with a protein such as albumin and the proteins are cross linked with each other to cause immobilization; or a method wherein GPO or GPO and phospholipase C are included with collagen or fibroin or are covalent-bonded with them; or a l~U2~;9 method wherein GPO or GPO and phospholipase C are immobiliz-ed by the adsorption onto a porous organic polymer resin or by the covalent bonding with the same, a method wherein GPO
or GPO and phospholipase C are included and immobilized us-ing a photosetting resin. The form of the immobilizedemzyme may be the one wherein the immobilized enzyme is processed into a membranelike, fibrous, granular or tubular shape which is preferable in use for an enzyme electrode to be assembled into an oxygen electrode or a hydrogen peroxide electrode and the immobilized enzyme may be used as an enzyme electrode wherein this immobilized enzyme is incorporated into the detecting part of the electrode to be used so that the amount of the effective enzyme used for the quantitative measurement by an electrical means is markedly small.
Further, the quantitative measurement of hydrogen per-oxide may be carried out by using an indicator composition which produces a detecta~le material by the reaction with hydrogen peroxide. As the indicator composition,there usually are used the compositions whose change may be quantitatively - measured by a spectroscopic means, for instance, a colora-tion reagent composition whose color change occurs in the visible range, a fluorescence reagent composition which fluoreSces by ultraviolet rays irradiation or a photogenic reagent composition which gives forth light. For example, as a coloration reagent composition, a material containing a substance having peroxidase action and chromogen may be used. As the substance having the peroxidase action, usual-ly the peroxidase originated from horseradish is often usedand, as the chromogen, usually the combination of an elec-tron acceptor and a phenolic compound is often used.
Further, as an electron acceptor, such compound is used as, for example, 4-aminoantipyrine, 2~hydrazinobenzothiazole, 3-methyl-2-benzothiazolone hydrazine, 2-aminobenzothiazole ~,' ~ ~Z~Z869 . _ ~
or the like. As a phenolic compound, such compound is used as, for example~ phenol, 3-methyl-N-ethyl~N-(B-hydroxyethylJaniline , 3,5-xylenol, N,N-dimethylaniline, N,N-diethylaniline or the like.
As luminous substrates in a fluorescence reagent com-position or a photogenic reagent composition, there may be illustrated various known ones, for example, bis(2,4,6-trichlorophenol)oxalate, phenylthiohydantoin, homovanillic acid, 4-hydroxyphenylacetic acid, vanillylamine, 3-methoxy-tyramine, phloretic acid, hordenine, luminol monoanion, lucigenine, wa~in and the like. Each of them may be, if necessary, used together with an electron acceptor and/or a substance having peroxidase action for the quantitative measurement of hydrogen peroxide.
There is no particular limitation on the quantity of the enzyme reagent or the chromogen used. For instance, there may be used per one test usually not less than 0.05 unit, preferably 0.1 - 200 units of GPO and usually not less than 0.05 unit, preferably 0.1 - 500 units of per-oxidase. ~lso there may be used a solution so adjusted by distilled water or weakly acidic or weakly alkaline buffer solution that the concentration of an electron ac-ceptor or a phenolic compound i5 usually not less than 0.1 mM. These reagents may be used separately from the enzyme solution of phospholipase C mentioned above or may be blended therewith, or further, these reagents may be formed into such composition for quantitative measurement as a freeze-dried composition or an integrate~ laminate by ap-plying them onto filter papers, films or the li~e.
The GPO type method for the quantitative measurement thus composed shows a high sensitivity to the glycero-3-phosphate in the liquid to be inspected and, since v ~ZV'~136~
g it is not affected by the impurity in the liquid to be inspected, it is an excellent method whereby an accurate measurement can be carried out.
The GP0 used for this GPO type method may be any enzymes as far as it plays the role of a catalyst in the reaction to produce dihydroxyacetone phosphoric acid and hydrogen peroxide from glycero-3-phosphate and oxy-gen, for example, Streptococcus genus, Lactobacillus genus, Lenconostoc genus, glycerophosphate oxidase producing bacteria which belong to Pediococcus genus (Japanese unexamined patent publication No. 7289211978), a enzyme obtained from the culture of glycerophosphate oxidase-producing bacteria which belongs to Aerococcus genus (Aerococcus viridans IFO 12219 strain and IFO 12317 strain, Japanese unexamined patent publication No. 15746/1980) and enzymes sold on the market.
As another enzymatic method for the quantitative measurement of glycero-3-phosphate, glycerophosphate dihydrogenase which catalyses the reac~ion to produce dihydroxyacetone phosphoric acid and reduced NAD
from glycero-3-phosphate and nicotine adenine dinucleotide (NAD) is allowed to act on glycero-3-phosphate liberated in the solution to be tested in the presence of NA~ to produce dihydroxyacetone phosphoric acid and re-duced NAD and then this reduced NAD is quan-titatively measured so that the quantity of glycero-3-phosphate may be determined. When the quantitative measurement of this reduced NAD is conducted, such method may be usually used as the measurement of absorbance by a wave length of about 340 nm or, after it is aIlowed to develop color by a water-soluble tetrazolium salt such as 3-(4,5-dimethyl)-2-thiazolyl-2H-tetrazolium bromide,
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium 12~)Z8~9 chloride, 3,3'-(3,3'-dimethoxy-4,4'-biphenylene)-bis[2-(p-nitrophenyl-5-phenyl-2H-tetra~olium chloride)](Nitrotetra-zolium Blue) or 2,6-dichlorophenolindophenol in the pre-sence of diaphorase or phenazine methosulfate, the color may be measured in accordance with absorbances using their special absorption wave lengths.
In addition, as to these enzymes, reagents and the like, it is simple and convenient to use those sold on the market and the quantity to be used may be appropriately designed. Also, if necessary, a surfactant or a stabilizer may be used and they may ~e processed into various composi-tions.
In addition, as the liquid to be inspected which is the object in the present invention, any sample may be used as far as it contains phosphatidyl glycerol, for in-stance, body fluids such as amniotic fluid sampled.
When amniotic fluid is the liquid to be inspected, the sample obtained may be used as it is or when the content of phosphatidyl glycerol is remarkably small 7 the sample may be concentrated for instance, 5 - 6 ml of liquor amniotic fluid sampled is extracted with 15 - 18 ml of chloroform-methanol (2:1), the chloroform layer is collected by cen-trifuging at 2000 rpm and it is evaporated to dryness in nitrogen gas to obtain the total lipid. Then, after dis-solving this total lipid in a definite amount of 1%"Triton X-lOO"solution, the obtained solution may be used as the sample of amniotic fluid. In this way, each enzyme solu-tion of phospholipase C and other enzymes such as used in GPO type methods of quantitative measurement mentioned above and other reagents may be allowed to act on the solu-tion to be inspected successively or simultaneously. In this case, there is no particular limitation on the use * Trademark for octylphenoxy polyethoxy ethanol; it is a nonionic surfactant.
12{)Z869 ratio of the liquld to be inspected to the enzyme reagent and the like and usually, about 0.1 - 3 ml of the solution of enzyme reagent or the like is used for 0.01 ml - 1 ml of the liquid to be inspected. As the reaction condition, it is preferable to conduct the reaction at about 37C and, as the reaction time, any length may be selected as far as the reaction is completely terminated; usually the reaction is continued for not less than 5 minutes, preferably not less than 10 minutes. Also as the reaction medium, water or a weakly acidic to weakly alkaline buffer as a solvent of each rea~ent and the like is used.
Thus, by the quantitative measurement of the liquid to be inspected, phosphatidyl glycerol may be directly and quantitatively measured in a very short time down to an extremely low concentration. In addition, no complicated operation is needed and the operation may be carried out at the normal temperature. Consequently, this is a good method for the quantitative measurement.
Also there is no particular limitation to the method for the measurement of the activity of phospholipase C and GPO used in the practical examples mentioned later in the present invention and they may be illustrated by examples as follows:
(a] Method for the measurement of the activity of phospholipase C
0.1 ml of 4% phosphatidyl choline(separated from egg yolk and purified) solution, 0.3 ml of 0.1 M Tris-hydro-chloric acid buffer (pH 8.5) and 0.1 ml of 20 mM aqueous solution of MgC12 are mixed together and to this is added 0.1 ml of enzyme solution containing phospholipase C.
The mixture is allowed to react at 37C for 15 minutes and the reaction is suspended by adding 0.8 ml of 3.6%
~L2~;~8~9 trichloroacetic acid and 0.1 ml of 5.5~ serum albumin and the reaction mixture is allowed to stand in water bath for 20 min.,then filtered by Toyo filter paper (No. 5B).
0.5 ml of the filtrate is taken into a Kjeldahl flask, 0.55 ml of 60% HC104 is added to decompose for 2 hours at 170GC, and liberated inorganic phosphor is allowed to develop color by addition of 0.5 ml of amidol sulfite testing solution, 0.25 ml of 3.3% ammonium molybdate and 3.2 ml of water. After being left to stand at room temperature for 20 min.,the absorbance is measured at OD 650 m~. The activity of enzyme which extricates 1 ~mole of inor~anic phosphor per 15 minutes is defined as 1 unit (U).
(b3 Method for the measurement of the activity of GPO
0.2 M Tris-hydrochloric acid buffer (pH 8.0) 0.2 ml peroxidase (0.5 mg/ml, 45 U/ml) 0.1 ml 0.3% tW/V) 4-aminoantipyrine 0.1 ml 0.1 M DL-glycero-3-phosphate 0.1 ml 0.2% (V/V) N,N-dimethylaniline 0.2 ml Distilled water 0.3 ml One mililiter of the reaction liquid having the com-position mentioned above is charged in a small test tube and preheated at 37C for 3 minutes. To this is added 20 ~1 of enzyme solution containing GPO and the mixture is allowed to react for 10 minutes. Then, the reaction is suspended by the addition of 2.0 ml of 0.25% (W/V) sodium laurylbenzene sulfonate and the absorbance of the product is measured at a wave length of 565 nm.
The activity of the enzyme is calculated in accordance with the following equation:
Activity of enzyme (U/ml) = ( 6 o ) x ( 10 ) .~
, .
l~Z86g wherein ~A shows the absorbance for 10 minutes at a wave length of 565 nm.
The ~mbodiment of the present invention will be men-S tioned in the following examples. However, the present in-vention is not intended to be limited by them.
Fig. 1 and Fig. 2 indicate the quantitative measure-ment curve of phosphatidyl glycerol.
Example 1 0.2 M dimethylglutarate-NaOH buffer (pH 7.5) 0.2 ml 10 mM CaC12 0.2 ml 10% sodium deoxycholate 0.05 ml 1% Bovine serum albumin 0.1 ml Solution containing 40 U/ml phospholipase C
and 50 U/ml GPO 0.1 ml 45 U/ml peroxidase 0.1 ml 0.3% 4-aminoantipyrine 0.2 ml 0.3% 3-methyl-N-ethyl-N-~-hydroxyethyl~aniline 0.2 ml Distilled water 0.~5 ml Total 2.0 ml 2.0 ml of reaction composition for quantitative measure-ment of phosphatidyl glycerol having the above compositionis prepared and thereto is added 50 ~1 of solution to be in-spected which contains various phosphatidyl glycerol ~con-tent of phosphatidyl glycerol being 20 n moles ~ 100 n moles~
and the mixture is reacted at 37C for 15 minutes and then the color development of the pigment produced after reaction is measured by absorbance at the wave length of 550 nm (OD
550).
The results are as shown by Fig. 1. A satisfactory tendency of linear relation was obtained as against the , ~;~0~8~g content of phosphatidyl glycerol of the inspected solution.
Example 2 0.2 M dimethyl glutarate-NaOH buffer (pH 7-5) 0 2 ml 10% sodium deoxycholate 0.05 ml Solution containing 20 U/ml phospholipase C
and 50 U/ml GPO 0.1 ml ~5 U/ml peroxidase 0.1 ml 0.3% 4-aminoantipyrine 0.2 ml 0.3~ phenol 0.2 ml 1% Bovine serum albumin 0.1 ml Distilled water 1.05 ml Total 2.0 ml 2.0 ml of reaction composition for quantitative measurement of phosphatidyl glycerol having the aforesaid composition is prepared, to such solution is added 50 ~l of the solution to be inspected containing various phos-phatidyl glycerol (content of phosphatidyl glycerol being10 n moles ~100 n moles), the mixture is allowed to react for 15 minutes at 37C and the color shade of the pigment produced after reaction was measured by absorbance at wave length of 500 nm (OD 500).
The results are as shown in Fig. 2, where a quantita-tive measurement curve having satisfactory linearity against the content of phosphatidyl glycerol was obtained.
In addition, as to these enzymes, reagents and the like, it is simple and convenient to use those sold on the market and the quantity to be used may be appropriately designed. Also, if necessary, a surfactant or a stabilizer may be used and they may ~e processed into various composi-tions.
In addition, as the liquid to be inspected which is the object in the present invention, any sample may be used as far as it contains phosphatidyl glycerol, for in-stance, body fluids such as amniotic fluid sampled.
When amniotic fluid is the liquid to be inspected, the sample obtained may be used as it is or when the content of phosphatidyl glycerol is remarkably small 7 the sample may be concentrated for instance, 5 - 6 ml of liquor amniotic fluid sampled is extracted with 15 - 18 ml of chloroform-methanol (2:1), the chloroform layer is collected by cen-trifuging at 2000 rpm and it is evaporated to dryness in nitrogen gas to obtain the total lipid. Then, after dis-solving this total lipid in a definite amount of 1%"Triton X-lOO"solution, the obtained solution may be used as the sample of amniotic fluid. In this way, each enzyme solu-tion of phospholipase C and other enzymes such as used in GPO type methods of quantitative measurement mentioned above and other reagents may be allowed to act on the solu-tion to be inspected successively or simultaneously. In this case, there is no particular limitation on the use * Trademark for octylphenoxy polyethoxy ethanol; it is a nonionic surfactant.
12{)Z869 ratio of the liquld to be inspected to the enzyme reagent and the like and usually, about 0.1 - 3 ml of the solution of enzyme reagent or the like is used for 0.01 ml - 1 ml of the liquid to be inspected. As the reaction condition, it is preferable to conduct the reaction at about 37C and, as the reaction time, any length may be selected as far as the reaction is completely terminated; usually the reaction is continued for not less than 5 minutes, preferably not less than 10 minutes. Also as the reaction medium, water or a weakly acidic to weakly alkaline buffer as a solvent of each rea~ent and the like is used.
Thus, by the quantitative measurement of the liquid to be inspected, phosphatidyl glycerol may be directly and quantitatively measured in a very short time down to an extremely low concentration. In addition, no complicated operation is needed and the operation may be carried out at the normal temperature. Consequently, this is a good method for the quantitative measurement.
Also there is no particular limitation to the method for the measurement of the activity of phospholipase C and GPO used in the practical examples mentioned later in the present invention and they may be illustrated by examples as follows:
(a] Method for the measurement of the activity of phospholipase C
0.1 ml of 4% phosphatidyl choline(separated from egg yolk and purified) solution, 0.3 ml of 0.1 M Tris-hydro-chloric acid buffer (pH 8.5) and 0.1 ml of 20 mM aqueous solution of MgC12 are mixed together and to this is added 0.1 ml of enzyme solution containing phospholipase C.
The mixture is allowed to react at 37C for 15 minutes and the reaction is suspended by adding 0.8 ml of 3.6%
~L2~;~8~9 trichloroacetic acid and 0.1 ml of 5.5~ serum albumin and the reaction mixture is allowed to stand in water bath for 20 min.,then filtered by Toyo filter paper (No. 5B).
0.5 ml of the filtrate is taken into a Kjeldahl flask, 0.55 ml of 60% HC104 is added to decompose for 2 hours at 170GC, and liberated inorganic phosphor is allowed to develop color by addition of 0.5 ml of amidol sulfite testing solution, 0.25 ml of 3.3% ammonium molybdate and 3.2 ml of water. After being left to stand at room temperature for 20 min.,the absorbance is measured at OD 650 m~. The activity of enzyme which extricates 1 ~mole of inor~anic phosphor per 15 minutes is defined as 1 unit (U).
(b3 Method for the measurement of the activity of GPO
0.2 M Tris-hydrochloric acid buffer (pH 8.0) 0.2 ml peroxidase (0.5 mg/ml, 45 U/ml) 0.1 ml 0.3% tW/V) 4-aminoantipyrine 0.1 ml 0.1 M DL-glycero-3-phosphate 0.1 ml 0.2% (V/V) N,N-dimethylaniline 0.2 ml Distilled water 0.3 ml One mililiter of the reaction liquid having the com-position mentioned above is charged in a small test tube and preheated at 37C for 3 minutes. To this is added 20 ~1 of enzyme solution containing GPO and the mixture is allowed to react for 10 minutes. Then, the reaction is suspended by the addition of 2.0 ml of 0.25% (W/V) sodium laurylbenzene sulfonate and the absorbance of the product is measured at a wave length of 565 nm.
The activity of the enzyme is calculated in accordance with the following equation:
Activity of enzyme (U/ml) = ( 6 o ) x ( 10 ) .~
, .
l~Z86g wherein ~A shows the absorbance for 10 minutes at a wave length of 565 nm.
The ~mbodiment of the present invention will be men-S tioned in the following examples. However, the present in-vention is not intended to be limited by them.
Fig. 1 and Fig. 2 indicate the quantitative measure-ment curve of phosphatidyl glycerol.
Example 1 0.2 M dimethylglutarate-NaOH buffer (pH 7.5) 0.2 ml 10 mM CaC12 0.2 ml 10% sodium deoxycholate 0.05 ml 1% Bovine serum albumin 0.1 ml Solution containing 40 U/ml phospholipase C
and 50 U/ml GPO 0.1 ml 45 U/ml peroxidase 0.1 ml 0.3% 4-aminoantipyrine 0.2 ml 0.3% 3-methyl-N-ethyl-N-~-hydroxyethyl~aniline 0.2 ml Distilled water 0.~5 ml Total 2.0 ml 2.0 ml of reaction composition for quantitative measure-ment of phosphatidyl glycerol having the above compositionis prepared and thereto is added 50 ~1 of solution to be in-spected which contains various phosphatidyl glycerol ~con-tent of phosphatidyl glycerol being 20 n moles ~ 100 n moles~
and the mixture is reacted at 37C for 15 minutes and then the color development of the pigment produced after reaction is measured by absorbance at the wave length of 550 nm (OD
550).
The results are as shown by Fig. 1. A satisfactory tendency of linear relation was obtained as against the , ~;~0~8~g content of phosphatidyl glycerol of the inspected solution.
Example 2 0.2 M dimethyl glutarate-NaOH buffer (pH 7-5) 0 2 ml 10% sodium deoxycholate 0.05 ml Solution containing 20 U/ml phospholipase C
and 50 U/ml GPO 0.1 ml ~5 U/ml peroxidase 0.1 ml 0.3% 4-aminoantipyrine 0.2 ml 0.3~ phenol 0.2 ml 1% Bovine serum albumin 0.1 ml Distilled water 1.05 ml Total 2.0 ml 2.0 ml of reaction composition for quantitative measurement of phosphatidyl glycerol having the aforesaid composition is prepared, to such solution is added 50 ~l of the solution to be inspected containing various phos-phatidyl glycerol (content of phosphatidyl glycerol being10 n moles ~100 n moles), the mixture is allowed to react for 15 minutes at 37C and the color shade of the pigment produced after reaction was measured by absorbance at wave length of 500 nm (OD 500).
The results are as shown in Fig. 2, where a quantita-tive measurement curve having satisfactory linearity against the content of phosphatidyl glycerol was obtained.
Claims (11)
1. A method for the quantitative measurement of phos-phatidyl glycerol, in a liquid containing lipid components which is to be inspected, which method comprises the sequential steps of (a) liberating glycero-3-phosphate by allowing phos-pholipase C to act on phosphatidyl glycerol, (b) allowing glycerophosphate oxidase to act on glycero-3-phosphate, and then (c) measuring the quantity of oxygen consumed or hydrogen peroxide produced in the reaction.
2. The method for the quantitative measurement according to Claim 1, wherein the liquid to be inspected is a body fluid.
3. The method for the quantitative measurement accord-ing to Claim 1, wherein the quantitative measurement of oxygen is made by an electrochemical method using an oxygen electrode.
4. The method for the quantitative measurement accord-ing to Claim 1, wherein the quantitative measurement of hydrogen peroxide is carried out by an electrochemical method using an hydrogen peroxide electrode.
5. The method for the quantitative measurement accord-ing to Claim 1, wherein the quantitative measurement of hydrogen peroxide is carried out by using an indicator com-position which changes into a detectable product produced by the reaction with hydrogen peroxide.
6. The method for the quantitative measurement accord-ing to Claim 5, wherein the indicator composition is a color developing composition, fluorescent composition or luminating composition.
7. The method for quantitative measurement according to Claim 6, wherein the color developing composition con-tains a substance having peroxidase action and a dye pre-cursor.
8. The method for quantitative measurement according to Claim 7, wherein the dye precursor is 4-aminoantipyrine and phenol.
9. The method for quantitative measurement according to Claim 7, wherein the dye precursor is 4-aminoantipyrine and 3-methyl-N-ethyl-N-(.beta.-hydroxyethyl) aniline.
10. The method for quantitative measurement according to Claim 8, wherein the measurement consists of an absorbance measurement at a wave length of about 500 nm.
11. The method for quantitative measurement according to Claim 9, wherein the measurement consists of an absorbance measurement at a wave length of about 550 nm.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP56187486A JPS5889199A (en) | 1981-11-20 | 1981-11-20 | Quantitative analysis of phosphatidylglycerol |
JP187486/81 | 1981-11-20 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1202869A true CA1202869A (en) | 1986-04-08 |
Family
ID=16206903
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000415626A Expired CA1202869A (en) | 1981-11-20 | 1982-11-16 | Method for the quantitative measurement of the phosphatidyl glycerol |
Country Status (6)
Country | Link |
---|---|
JP (1) | JPS5889199A (en) |
CA (1) | CA1202869A (en) |
DE (1) | DE3242652A1 (en) |
FR (1) | FR2516936B1 (en) |
GB (1) | GB2110822B (en) |
IT (1) | IT1153093B (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS58107199A (en) * | 1981-12-16 | 1983-06-25 | Toyo Jozo Co Ltd | Qualitative analysis of phosphatidyl glycerol |
JPS59140900A (en) * | 1983-01-28 | 1984-08-13 | Toyo Jozo Co Ltd | Novel method for highly sensitive, enzymatic determination |
JPH0751482B2 (en) * | 1987-03-25 | 1995-06-05 | ア−ス製薬株式会社 | Pest control material |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS604716B2 (en) * | 1976-04-26 | 1985-02-06 | 東洋醸造株式会社 | Novel choline oxidase and its production method |
US4241178A (en) * | 1978-01-06 | 1980-12-23 | Eastman Kodak Company | Process and composition for the quantification of glycerol ATP and triglycerides |
DE2938737A1 (en) * | 1978-09-26 | 1980-04-03 | Toyo Jozo Kk | DEVICE AND METHOD FOR SIMULTANEOUSLY DETERMINING MULTIPLE COMPONENTS IN A SAMPLE BY MEANS OF ENZYMATIC ANALYSIS |
-
1981
- 1981-11-20 JP JP56187486A patent/JPS5889199A/en active Pending
-
1982
- 1982-11-10 GB GB08232079A patent/GB2110822B/en not_active Expired
- 1982-11-16 FR FR8219135A patent/FR2516936B1/en not_active Expired
- 1982-11-16 CA CA000415626A patent/CA1202869A/en not_active Expired
- 1982-11-18 DE DE19823242652 patent/DE3242652A1/en not_active Withdrawn
- 1982-11-19 IT IT24350/82A patent/IT1153093B/en active
Also Published As
Publication number | Publication date |
---|---|
FR2516936B1 (en) | 1987-04-30 |
GB2110822B (en) | 1985-01-30 |
IT1153093B (en) | 1987-01-14 |
GB2110822A (en) | 1983-06-22 |
DE3242652A1 (en) | 1984-01-19 |
IT8224350A0 (en) | 1982-11-19 |
FR2516936A1 (en) | 1983-05-27 |
JPS5889199A (en) | 1983-05-27 |
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