JPH03160367A - Treating agent for urine - Google Patents
Treating agent for urineInfo
- Publication number
- JPH03160367A JPH03160367A JP1299572A JP29957289A JPH03160367A JP H03160367 A JPH03160367 A JP H03160367A JP 1299572 A JP1299572 A JP 1299572A JP 29957289 A JP29957289 A JP 29957289A JP H03160367 A JPH03160367 A JP H03160367A
- Authority
- JP
- Japan
- Prior art keywords
- urine
- physiologically active
- metal salt
- added
- active substance
- 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.)
- Granted
Links
- 210000002700 urine Anatomy 0.000 title claims abstract description 41
- 239000013543 active substance Substances 0.000 claims abstract description 19
- 229910052751 metal Inorganic materials 0.000 claims abstract description 18
- 239000002184 metal Substances 0.000 claims abstract description 18
- 150000003839 salts Chemical class 0.000 claims abstract description 18
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 8
- 229910052725 zinc Inorganic materials 0.000 claims abstract description 8
- 239000011701 zinc Substances 0.000 claims abstract description 8
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical class [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910052782 aluminium Chemical class 0.000 claims abstract description 6
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical class [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims abstract description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical class [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims abstract description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical class [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052788 barium Inorganic materials 0.000 claims abstract description 5
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical class [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052791 calcium Inorganic materials 0.000 claims abstract description 5
- 239000011575 calcium Chemical class 0.000 claims abstract description 5
- 229910052802 copper Inorganic materials 0.000 claims abstract description 5
- 239000010949 copper Chemical class 0.000 claims abstract description 5
- 239000006228 supernatant Substances 0.000 abstract description 8
- 238000004587 chromatography analysis Methods 0.000 abstract description 3
- 238000004255 ion exchange chromatography Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- XTEGARKTQYYJKE-UHFFFAOYSA-M Chlorate Chemical compound [O-]Cl(=O)=O XTEGARKTQYYJKE-UHFFFAOYSA-M 0.000 abstract 2
- 229910002651 NO3 Inorganic materials 0.000 abstract 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 abstract 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- 239000013049 sediment Substances 0.000 abstract 1
- 239000003463 adsorbent Substances 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 239000002244 precipitate Substances 0.000 description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 239000011550 stock solution Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 4
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- IWOUKMZUPDVPGQ-UHFFFAOYSA-N barium nitrate Chemical compound [Ba+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O IWOUKMZUPDVPGQ-UHFFFAOYSA-N 0.000 description 4
- 238000000354 decomposition reaction Methods 0.000 description 4
- 239000011345 viscous material Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000011592 zinc chloride Substances 0.000 description 4
- 235000005074 zinc chloride Nutrition 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 102000001399 Kallikrein Human genes 0.000 description 2
- 108060005987 Kallikrein Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 101710162629 Trypsin inhibitor Proteins 0.000 description 2
- 229940122618 Trypsin inhibitor Drugs 0.000 description 2
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 2
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 229940024546 aluminum hydroxide gel Drugs 0.000 description 2
- SMYKVLBUSSNXMV-UHFFFAOYSA-K aluminum;trihydroxide;hydrate Chemical compound O.[OH-].[OH-].[OH-].[Al+3] SMYKVLBUSSNXMV-UHFFFAOYSA-K 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 238000010908 decantation Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 239000002753 trypsin inhibitor Substances 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- 229960005356 urokinase Drugs 0.000 description 2
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 235000003591 Tagetes lucida Nutrition 0.000 description 1
- 240000002670 Tagetes lucida Species 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- YKTSYUJCYHOUJP-UHFFFAOYSA-N [O--].[Al+3].[Al+3].[O-][Si]([O-])([O-])[O-] Chemical compound [O--].[Al+3].[Al+3].[O-][Si]([O-])([O-])[O-] YKTSYUJCYHOUJP-UHFFFAOYSA-N 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000003841 chloride salts Chemical class 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 229940047120 colony stimulating factors Drugs 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 230000000938 luteal effect Effects 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 235000013706 tagetes lucida Nutrition 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は尿の処理剤に関する。さらに詳しくはトリブシ
ンインヒビター、コロニー刺激因子等の生理活性物質を
取得する際にその生理活性物質を尿から分離しやすくす
るために用いられる尿の処理剤に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a urine treatment agent. More specifically, the present invention relates to a urine processing agent that is used to facilitate the separation of physiologically active substances such as tribucin inhibitors and colony stimulating factors from urine when the physiologically active substances are obtained.
尿中に様々な生理活性物質、例えばウロキナーゼ、カリ
クレイン、トリブシンインヒビター、エリスロボエチン
、コロニー刺激因子、黄体形或ホルモン、卵胞刺激ホル
モン等が存在することは、既に知られている。実際に、
大量の尿からこれらの物質が単離・精製されており、医
薬品としても応用されている。It is already known that various physiologically active substances, such as urokinase, kallikrein, tribusin inhibitor, erythroboetin, colony stimulating factor, luteinizing hormone, follicle stimulating hormone, etc., exist in urine. actually,
These substances have been isolated and purified from large amounts of urine, and are also used as pharmaceuticals.
大量の尿からこれら生理活性物質を抽出する場合は、限
外濾過濃縮、吸着剤による回収、タンパク質沈澱等の操
作を組み合わせるのが一般的である。When extracting these physiologically active substances from a large amount of urine, it is common to combine operations such as ultrafiltration concentration, recovery using an adsorbent, and protein precipitation.
また、尿中には、コンドロイチン硫酸、ヘパラン硫酸を
はじめとするムコ多［１が存在することも知られている
。これらは粘性が高く、濃縮、精製工程において悪影響
があり、望ましくない。It is also known that mukopolymer [1] including chondroitin sulfate and heparan sulfate are present in urine. These are highly viscous and have an adverse effect on concentration and purification processes, making them undesirable.
そのため、大量の尿から生理活性物質を回収する際に、
（ｉ）操作が簡便である、（ ｉｉ ）効率がよい、（
ｉｉｉ）再現性がよい、（ ｉｖ ）生理活性物質の分
解・失活がない、等が望まれている。Therefore, when recovering physiologically active substances from large amounts of urine,
(i) Easy to operate, (ii) Efficient, (
iii) good reproducibility, and (iv) no decomposition or deactivation of physiologically active substances, etc. are desired.
そこで、このような要望に応えたものとして特開昭６３
−１９８７００号公報では尿中コロニ刺激因子の精製工
程にｐＨ８〜９処理の工程が採用されている。このよう
に、尿をｐＨ８〜９に調整し、生じる不溶物を除去する
ことで粘性物質を取り除くことは可能である。Therefore, in response to such requests, the Japanese Patent Laid-open Publication No. 63
In the publication No. 198700, a process of pH 8 to 9 treatment is adopted in the purification process of the urinary colony stimulating factor. In this way, it is possible to remove viscous substances by adjusting the pH of urine to 8 to 9 and removing the resulting insoluble matter.
しかしながら、このｐＨ８〜９処理のみではムコ多糖類
をはじめとする粘性物質の除去は完全ではなく、尿を大
量に濃縮するにつれ限外濾過膜やファイバーの目詰まり
が発生する。また、クロマト精製工程においては、カラ
ム樹脂上部パッキングが起こり、十分な分離が行われな
いばかりでなく、トラブル発生の原因となる。However, this pH 8-9 treatment alone does not completely remove viscous substances such as mucopolysaccharides, and as urine is concentrated in large quantities, ultrafiltration membranes and fibers become clogged. Furthermore, in the chromatographic purification process, packing occurs in the upper part of the column resin, which not only prevents sufficient separation but also causes trouble.
さらに、弱アルカリ性条件下で安定性の悪い生理活性物
質に適用することはできない。Furthermore, it cannot be applied to physiologically active substances that are poorly stable under weakly alkaline conditions.
本発明者は、尿中から抽出する生理活性物質が損失する
ことなく、尿中に存在する粘性物質が簡便かつ効率よく
除去される方法について、鋭意研究を重ねた結果、尿に
特定戒分の金属塩を添加してから処理すれば良いことを
見い出し本発明を完成した。The inventor of the present invention has conducted intensive research into a method for easily and efficiently removing viscous substances present in urine without losing physiologically active substances extracted from urine. The present invention was completed by discovering that the treatment can be carried out after adding a metal salt.
すなわち、本発明は、亜鉛、カルシウム、銅、バリウム
、及びアル≧ニウムの金属塩から選ばれた少なくとも１
種以上からなることを特徴とする尿中生理活性物質を取
得する際に用いられる尿の処理剤である。That is, the present invention provides at least one metal salt selected from zinc, calcium, copper, barium, and aluminum≧nium metal salts.
This is a urine treatment agent used to obtain a physiologically active substance in urine, which is characterized by comprising more than one type of urinary physiologically active substance.
以下、さらに本発明について詳しく説明する。The present invention will be further explained in detail below.
本発明において、尿はそのまま用いてもよいが、大量の
尿から例えばウロキナーゼ、カリクレイン、トリブシン
インヒビター、エリスロポエチン、コロニー刺激因子、
黄体形或ホルモン、卵胞刺激ホルモン等の生理活性物質
を抽出する場合は、濃縮または、尿を吸着剤で処理しそ
の吸着画分を溶出して得られる尿原液を用いることが好
ましい。In the present invention, urine may be used as it is, but from a large amount of urine, for example, urokinase, kallikrein, tribusin inhibitor, erythropoietin, colony stimulating factor, etc.
When extracting physiologically active substances such as luteal hormones and follicle-stimulating hormones, it is preferable to use a urine stock solution obtained by concentration or by treating urine with an adsorbent and eluting the adsorbed fraction.
その手段としては限外濾過濃縮、水酸化アルミニウムゲ
ル、合或ケイ酸アルミニウム、カオリン、シリカゲル、
イオン交換樹脂、キトサン等の吸着剤による回収・濃縮
が実施可能である。また、尿の処理剤として用いる金属
塩は、亜鉛、カルシウム、銅、バリウム、アルξニウム
の硝酸塩、硫酸塩、酢酸塩、塩化物等が利用可能であり
、添加濃度は１０ｍＭ〜５Ｍ程度まで、目的とする物質
の種類により選択することができるが、好ましくは、０
．１Ｍ〜１．０Ｍである。添加方法は特に限定されない
が、添加した金属塩が均一に溶解する条件が望ましく、
そのためには４〜８０゜Ｃの温度範囲の中で目的とする
生理活性物質の安定性を考慮しつつ設定された温度条件
下で、撹拌及び／又は振とうしつつ金属塩を添加し、０
．５〜２４時間放置するのが好ましい。The methods include ultrafiltration concentration, aluminum hydroxide gel, aluminum silicate, kaolin, silica gel,
Recovery and concentration can be carried out using adsorbents such as ion exchange resins and chitosan. In addition, metal salts used as urine treatment agents include nitrates, sulfates, acetates, and chlorides of zinc, calcium, copper, barium, and aluminum, with addition concentrations ranging from 10mM to 5M. It can be selected depending on the type of target substance, but preferably 0
．． It is 1M to 1.0M. The addition method is not particularly limited, but it is desirable that the added metal salt be dissolved uniformly.
To do this, metal salts are added with stirring and/or shaking under temperature conditions set in the temperature range of 4 to 80°C, taking into account the stability of the target physiologically active substance.
．． It is preferable to leave it for 5 to 24 hours.
次いで遠心分離機により沈澱を除去し、得られた上清を
さらに、イオン交換クロマトグラフィー疎水クロマトグ
ラクィー、ゲル濾過クロマトグラフィー、アフィニティ
ークロマトグラフイー等の繁用のクロマトグラフィーを
組み合せ実施することにより目的とする生理活性物質を
単離することが可能である。Next, the precipitate is removed using a centrifugal separator, and the resulting supernatant is subjected to a combination of commonly used chromatography such as ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, and affinity chromatography. It is possible to isolate physiologically active substances.
以下、実施例をあげてさらに具体的に説明するが、本発
明は以下の実施例に限定されるものではない。Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
１ 〜 ５ 六 １ 〜 ３正常人尿
２２Ｎに酢酸を添加し、ｐＨを４．０に調整後生した沈
澱を遠心分離（８ ０ ０ ０回転、１５分）で除去し
て酸処理尿を得た。この酸処理尿に’１１０ｇの水酸化
アルξニウムゲル（キョーワード２００Ｂ：協和化学工
業■製）を添加し、室温にて２時間撹拌した。１時間の
放置後、上清と吸着剤をデカンテーションで分離し、さ
らに吸引濾過により水で吸着剤を洗浄した。この洗浄吸
着剤を１％アンモニア１ｌで１時間撹拌溶出を行ない、
遠心分離（８ ０ ０ ０回転、１５分）により溶出液
を得た。溶出液に硫酸アンモニウムを８０％飽和となる
ように加え、５時間放置後、遠心分離（８ ０ ０ ０
回転、３０分）により得られた沈澱を、水５０ｄに溶解
し尿原液とした。この尿原液１ｍ１に各種金属塩をそれ
ぞれＩＭとなるように添加し、３０分間室温放置後、微
量高速遠心分離機（エッペンドルフ製）にて沈澱を除去
した。上清中のトリプシンインヒビター活性を、Ｍｕｒ
ａｍａ ｔｓｕ等の方法（Ｊ．Ｂｉｏｃｈｅｍ．， ５
７，４０２．（１９６５））に準拠し、カゼイン分解法
で測定した。1-5 6 1-3 Acetic acid was added to 22N normal human urine, the pH was adjusted to 4.0, and the resulting precipitate was removed by centrifugation (8000 rpm, 15 minutes) to obtain acid-treated urine. . To this acid-treated urine, 110 g of aluminum ξ hydroxide gel (Kyoward 200B, manufactured by Kyowa Kagaku Kogyo ■) was added and stirred at room temperature for 2 hours. After standing for 1 hour, the supernatant and adsorbent were separated by decantation, and the adsorbent was further washed with water by suction filtration. This washed adsorbent was stirred and eluted with 1 liter of 1% ammonia for 1 hour.
An eluate was obtained by centrifugation (8000 rpm, 15 minutes). Ammonium sulfate was added to the eluate to make it 80% saturated, and after standing for 5 hours, centrifugation (8000
The precipitate obtained by spinning for 30 minutes was dissolved in 50 d of water to obtain a urine stock solution. Various metal salts were added to 1 ml of this urine stock solution to form IM, and after being left at room temperature for 30 minutes, the precipitate was removed using a high-speed microcentrifuge (manufactured by Eppendorf). Trypsin inhibitor activity in the supernatant was determined by Mur
The method of Amatsu et al. (J.Biochem., 5
7,402. (1965)), it was measured by the casein decomposition method.
すなわち、各試料とトリブシンを２０″Ｃで１５分間反
応させた後、残存するトリブシン活性をカゼイン分解を
指標として測定した。カゼイン分解は、遊離したチロシ
ン残基をフェノール試薬を用いて定量した。尚、トリプ
シンインヒビターの１単位（Ｕ）はトリプシン１ｕｇの
カゼイン分解能を完全に阻害する活性として定義した。That is, after each sample was reacted with tribucin at 20''C for 15 minutes, the remaining tribucin activity was measured using casein decomposition as an indicator. Casein decomposition was determined by quantifying the released tyrosine residue using a phenol reagent. , one unit (U) of trypsin inhibitor was defined as the activity of completely inhibiting the casein-degrading ability of 1 ug of trypsin.
また、タンパク質定量は、Ｂ’ＣＡ法タンパク質定量キ
ット（ピアス社製）を用いた。For protein quantification, a B'CA method protein quantification kit (manufactured by Pierce) was used.
添加する金属塩を変化させた場合の沈澱量、トリブシン
インヒビター活性、蛋白質量を測定した。The amount of precipitation, tribusin inhibitor activity, and protein amount were measured when the metal salt added was changed.
また比活性は活性／蛋白質量より算出した。それらの結
果を第１表に示す。Further, specific activity was calculated from activity/protein amount. The results are shown in Table 1.
なお、金属塩としては、実施例１は、硝酸バリウム（Ｂ
ａＮＯｓ ）を、実施例２は、硫酸！Ｍ （ＣｕＳＯ４
）を、実施例３は、塩化カルシウム（ＣａＣ１ｚ）を、
実施例４は、塩化亜鉛（ＺｎＣ１ｚ）を、実施例５は、
硫酸アンモニウム（ＡＩ２（５０４）２）を用いた。ま
た、比較例ｌは、無添加とした。比較例１は無添加であ
り、比較例２については、金属塩無添加で３．５Ｎ水酸
化ナトリウム添加でｐｌｌ８．５とし、生じる沈澱を除
去後、同様に測定を行なった．
塩化マグネシウム（ＭｇＣｌｚ）を用いた。In addition, as the metal salt, in Example 1, barium nitrate (B
aNOs), and Example 2 sulfuric acid! M (CuSO4
), in Example 3, calcium chloride (CaC1z),
Example 4 uses zinc chloride (ZnC1z), and Example 5 uses
Ammonium sulfate (AI2(504)2) was used. Moreover, in Comparative Example 1, no additives were added. Comparative Example 1 had no additives, and Comparative Example 2 had no metal salts added and 3.5N sodium hydroxide was added to give a pll of 8.5, and after removing the resulting precipitate, measurements were conducted in the same manner. Magnesium chloride (MgClz) was used.
比較例３は
＋＋＋：８ｉめて多い 士：少ない＋＋：非常に多
い 一二ない
＋ ：多い
裏嵐孤立二エー此教員土
実施例ｌ〜５と同様にして得た尿原液５０ｍに、各種濃
度の塩化亜鉛を添加し、室温１時間放置後遠心分離（
１００００回転、１５分）により沈澱を除去した。上清
中のトリブシンインヒビター活性およびタンパク質量を
測定した。Comparative example 3 is +++: 8i very high, shi: low ++: very high, 12 no +: high Ura-arashi isolated, 2A. of zinc chloride, left at room temperature for 1 hour, and then centrifuged (
The precipitate was removed by rotating at 10,000 rpm for 15 minutes. Tribusin inhibitor activity and protein amount in the supernatant were measured.
結果を第２表に示す。The results are shown in Table 2.
正常人尿２２Ｊ２に、５０ｇの粒状含水珪酸（ホワイト
カーボン：徳山曹達社製）を添加し、室温にて５時間撹
拌した。２時間の放置後、上清と吸着剤をデカンテーシ
ゴンで分離し、さらに吸引濾過により水で吸着剤を洗浄
した。この洗浄吸着剤を１％アンモニア１２で１時間撹
拌溶出を行ない、遠心分離（８ ０ ０ ０回転、１５
分）により溶出液を得た。溶出液に硫酸アンモニウムを
８０％飽和となるように加え、
５時間放置後、遠心分離（８ ０ ０ ０回転、３０分
）により得られた沈澱を、水５０ｍｌに溶解し尿原液と
した。この尿原液ＩＩｒＩ１に各種金属塩をそれぞれＩ
Ｍとなるように添加し、室温３０分間放置後、微量高速
遠心機（エッペンドルフ製）にて沈澱を除去し、上清を
リン酸緩衝生理食塩水（ＰＢＳ）に対して透析して、そ
のコロニー刺激因子（ＣＳＦ）活性およびタンパク質量
を測定した。50 g of granular hydrated silicic acid (white carbon, manufactured by Tokuyama Soda Co., Ltd.) was added to normal human urine 22J2, and the mixture was stirred at room temperature for 5 hours. After standing for 2 hours, the supernatant and adsorbent were separated using a decant, and the adsorbent was further washed with water by suction filtration. This washed adsorbent was stirred and eluted with 1% ammonia 12 for 1 hour, and then centrifuged (8000 rpm, 15
The eluate was obtained by 1 minute). Ammonium sulfate was added to the eluate to achieve 80% saturation, and after standing for 5 hours, the precipitate obtained by centrifugation (8000 rpm, 30 minutes) was dissolved in 50 ml of water to obtain a urine stock solution. Various metal salts were added to this urine stock solution IIrI1.
After standing at room temperature for 30 minutes, the precipitate was removed using a high-speed microcentrifuge (manufactured by Eppendorf), and the supernatant was dialyzed against phosphate buffered saline (PBS) to isolate the colonies. Stimulating factor (CSF) activity and protein levels were measured.
ＣＳＦ活性の測定方法は、Ｐｌｕｚｎｉｋ，Ｓａｃｈｓ
（Ｊ．Ｃｅｌ ｌ． Ｐｈｙｓｉｏｌ． ＋ ６６＋
３１９ （１９６５） ）， Ｂｒａｄｌｅｙ， Ｍｅ
ｔｃａｌｆ（Ａｕｓｔ，Ｊ．Ｅｘｐ．Ｂｉｏ１，Ｍｅｄ
．，４４，２８７（１９６６））、Ｔｓｕｎｅｏｋａ，
Ｓｈｉｋｉｔａ（ＦＥＢＳ Ｌｅｔｔｅｒｓ， ７７，
２４３（１９７７））に準拠して行なった。具体的には
、直径３５ｍｍのプラスチック培養皿に２０％馬血清、
各濃度のＣＳＦ試料、０．３％の寒天およびＩＸＩＯ’
個のマウス骨髄細胞を含むＭｃＣｏｙ’ｓ ５Ａ培地１
ｍｌを加え、７日間３？゜Ｃで５％ＣＯ■を含む飽和
水蒸気下で培養した。The method for measuring CSF activity is described by Pluznik, Sachs.
(J. Cell. Physiol. + 66+
319 (1965)), Bradley, Me.
tcalf (Aust, J. Exp. Bio1, Med
．． , 44, 287 (1966)), Tsuneoka,
Shikita (FEBS Letters, 77,
243 (1977)). Specifically, 20% horse serum was placed in a plastic culture dish with a diameter of 35 mm.
CSF samples of each concentration, 0.3% agar and IXIO'
McCoy's 5A medium containing mouse bone marrow cells 1
Add ml and 3? for 7 days. Cultures were incubated at .degree. C. under saturated steam containing 5% CO.sub.2.
培養後、倒立顕微鏡下で検鏡し、５０個以上の細胞集塊
をコロニーの数とし、コロニーを１個形威させる活性を
１単位（Ｕ）と定義した。After culturing, the cells were examined under an inverted microscope, and 50 or more cell aggregates were counted as a colony, and the activity that caused one colony to form was defined as 1 unit (U).
なお、金属塩としては、実施例１０は、硝酸バリウム（
８ａＮＯｉ）を、実施例ｌ１は、硫酸銅（ＣｕＳ０４冫
を、実施例１２は、塩化カルシウム（ＣａＣｌｚ）を、
実施例１３は、塩化亜鉛（ＺｎＣ１■）を、実施例１４
は、硫酸アルミニウム（ＡＩ！（ＳＯ４）２）を用いた
。In addition, as the metal salt, in Example 10, barium nitrate (
8aNOi), Example 11 used copper sulfate (CuS04), Example 12 used calcium chloride (CaClz),
In Example 13, zinc chloride (ZnC1■) was added to Example 14.
used aluminum sulfate (AI!(SO4)2).
また、比較例５は、無添加であり、比較例６は、金属塩
無添加で３．５Ｎ水酸化ナトリウム添加でｐＨ８．５と
し、生じる沈澱を除去後、同様に測定を行なった。比較
例７は、塩化マグネシウム（ＭｇＣ１ｚ）を用いた。Further, in Comparative Example 5, no addition was made, and in Comparative Example 6, no metal salt was added, the pH was adjusted to 8.5 by adding 3.5N sodium hydroxide, and after removing the resulting precipitate, measurements were performed in the same manner. Comparative Example 7 used magnesium chloride (MgC1z).
結果を第３表に示す。The results are shown in Table 3.
芽≦Ｌ聚
＋＋＋：極めて多い 士：少ない
十＋：非常に多い −：ない
＋ ：多い
８
正常人尿２００Ｉｌに酢酸を添加し、ｐＨを４−０に調
整後、生じた沈澱を遠心分！（８０００回転、３０分）
で除去して酸処理尿を得た。この酸処理尿にｌｋｇの水
酸化アルミニウムゲル（キヨーワード２００Ｂ：ｖＡ和
化学工業■製）を添加し、室温にて３時間撹拌した。１
時間の放置後、上清と吸着剤をデカンテーションで分離
し、さらに吸引濾過により水１０１！．で吸着剤を洗浄
した。この洗浄吸着剤を１％アンモニアｌＯｌで２時間
撹拌溶出を行ない、遠心分離（８ ０ ０ ０回転、３
０分）により溶出液を得た。溶出液を酢酸で中和後、０
．５Ｍとなるように塩化亜鉛を添加し、室温１時間放置
後、遠心分離（８ ５ ０ ０回転、６０分）により沈
澱を除去して亜鉛処理尿原液とした。Buds ≦ L + + +: Extremely many Shi: Few 10+: Very many -: None +: Many 8 After adding acetic acid to 200 Il of normal human urine and adjusting the pH to 4-0, the resulting precipitate was centrifuged! (8000 rpm, 30 minutes)
to obtain acid-treated urine. 1 kg of aluminum hydroxide gel (Kyoward 200B: manufactured by vA Wa Kagaku Kogyo ■) was added to the acid-treated urine, and the mixture was stirred at room temperature for 3 hours. 1
After standing for a period of time, the supernatant and adsorbent are separated by decantation, and then filtered using suction. ．． The adsorbent was washed with This washed adsorbent was stirred and eluted with 1% ammonia lOl for 2 hours, and then centrifuged (8000 rpm, 3
0 minutes) to obtain an eluate. After neutralizing the eluate with acetic acid, 0
．． Zinc chloride was added to give a concentration of 5M, and after being left at room temperature for 1 hour, the precipitate was removed by centrifugation (8500 rpm, 60 minutes) to obtain a zinc-treated urine stock solution.
この亜鉛処理尿原液１０Ｎをペリコンカセット（ミリポ
ア社製）による限外濾過で濃縮を行なった。１０ｆから
Ｉｆまで濃縮される時間を、亜鉛処理尿原液（実施例１
５）と未処理尿原液（比較例８）とで富周べた。This zinc-treated urine stock solution (10N) was concentrated by ultrafiltration using a Pericon cassette (manufactured by Millipore). The concentration time from 10f to If was calculated using the zinc-treated urine stock solution (Example 1).
5) and untreated urine stock solution (Comparative Example 8).
結果を第４表に示す。The results are shown in Table 4.
鯨シＬ表
〔発明の効果］
本発明による、尿の処．理剤を用いることにより、大量
の尿から生理活性物質を取得する際に、これらを損失す
ることなく夾雑する粘性物質を除去することが可能とな
り、限外濾過等による濃縮時間が短縮され膜トラブルが
防止されると同時に、目的とする生理活性物質を工業的
規模で効率よ《、再現性よく取得することが可能となっ
た。Whale urine L table [Effects of the invention] Treatment of urine according to the present invention. By using a physical agent, when obtaining physiologically active substances from a large amount of urine, it is possible to remove contaminating viscous substances without losing them, shorten the concentration time by ultrafiltration, etc., and prevent membrane trouble. At the same time, it has become possible to efficiently and reproducibly obtain the desired physiologically active substance on an industrial scale.
Claims (1)
ウムの金属塩から選ばれた少なくとも１種以上からなる
ことを特徴とする尿中生理活性物質を取得する際に用い
られる尿の処理剤。1. A urine treatment agent used to obtain a physiologically active substance in urine, characterized by comprising at least one selected from metal salts of zinc, calcium, copper, barium, and aluminum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1299572A JPH0823556B2 (en) | 1989-11-20 | 1989-11-20 | Urine treatment agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1299572A JPH0823556B2 (en) | 1989-11-20 | 1989-11-20 | Urine treatment agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03160367A true JPH03160367A (en) | 1991-07-10 |
| JPH0823556B2 JPH0823556B2 (en) | 1996-03-06 |
Family
ID=17874368
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1299572A Expired - Lifetime JPH0823556B2 (en) | 1989-11-20 | 1989-11-20 | Urine treatment agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0823556B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021047056A (en) * | 2019-09-17 | 2021-03-25 | 株式会社ファンケル | METHOD OF ESTIMATING NON-INVASIVE HbA1c VALUE |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5750649A (en) * | 1980-09-12 | 1982-03-25 | Hitachi Ltd | Analyzing method for chloride ion in urine |
| JPS5972058A (en) * | 1982-10-18 | 1984-04-23 | Daicel Chem Ind Ltd | Quick detection of medicine in urine |
-
1989
- 1989-11-20 JP JP1299572A patent/JPH0823556B2/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5750649A (en) * | 1980-09-12 | 1982-03-25 | Hitachi Ltd | Analyzing method for chloride ion in urine |
| JPS5972058A (en) * | 1982-10-18 | 1984-04-23 | Daicel Chem Ind Ltd | Quick detection of medicine in urine |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021047056A (en) * | 2019-09-17 | 2021-03-25 | 株式会社ファンケル | METHOD OF ESTIMATING NON-INVASIVE HbA1c VALUE |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0823556B2 (en) | 1996-03-06 |
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