JPH0154669B2 - - Google Patents
Info
- Publication number
- JPH0154669B2 JPH0154669B2 JP14944183A JP14944183A JPH0154669B2 JP H0154669 B2 JPH0154669 B2 JP H0154669B2 JP 14944183 A JP14944183 A JP 14944183A JP 14944183 A JP14944183 A JP 14944183A JP H0154669 B2 JPH0154669 B2 JP H0154669B2
- Authority
- JP
- Japan
- Prior art keywords
- bilirubin
- conjugated
- present
- solution
- unconjugated
- 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
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 claims description 75
- 238000000034 method Methods 0.000 claims description 12
- 239000002736 nonionic surfactant Substances 0.000 claims description 11
- 230000000087 stabilizing effect Effects 0.000 claims description 3
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 238000008789 Direct Bilirubin Methods 0.000 description 8
- 239000012086 standard solution Substances 0.000 description 8
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- BPYKTIZUTYGOLE-UHFFFAOYSA-N billirubin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(C=C3C(=C(C=C)C(=O)N3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-UHFFFAOYSA-N 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 239000003638 chemical reducing agent Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 4
- 108010024636 Glutathione Proteins 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- DQYSALLXMHVJAV-UHFFFAOYSA-M 3-heptyl-2-[(3-heptyl-4-methyl-1,3-thiazol-3-ium-2-yl)methylidene]-4-methyl-1,3-thiazole;iodide Chemical compound [I-].CCCCCCCN1C(C)=CS\C1=C\C1=[N+](CCCCCCC)C(C)=CS1 DQYSALLXMHVJAV-UHFFFAOYSA-M 0.000 description 2
- RCNSAJSGRJSBKK-NSQVQWHSSA-N Biliverdin IX Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(\C=C/2C(=C(C)C(=C/C=3C(=C(C=C)C(=O)N=3)C)/N\2)CCC(O)=O)N1 RCNSAJSGRJSBKK-NSQVQWHSSA-N 0.000 description 2
- 239000006171 Britton–Robinson buffer Substances 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- 206010023126 Jaundice Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical group OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- -1 aromatic diazonium salt Chemical class 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229940097043 glucuronic acid Drugs 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 229920001451 polypropylene glycol Polymers 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 229960003080 taurine Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- SCJLWMXOOYZBTH-BTVQFETGSA-N (2s,3s,4s,5r,6s)-6-[3-[2-[[3-[3-[(2s,3r,4s,5s,6s)-6-carboxy-3,4,5-trihydroxyoxan-2-yl]oxy-3-oxopropyl]-5-[(z)-(3-ethenyl-4-methyl-5-oxopyrrol-2-ylidene)methyl]-4-methyl-1h-pyrrol-2-yl]methyl]-5-[(z)-(4-ethenyl-3-methyl-5-oxopyrrol-2-ylidene)methyl]-4-meth Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(=O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@H](O2)C(O)=O)O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(=O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@H](O2)C(O)=O)O)N1 SCJLWMXOOYZBTH-BTVQFETGSA-N 0.000 description 1
- FDCJDKXCCYFOCV-UHFFFAOYSA-N 1-hexadecoxyhexadecane Chemical compound CCCCCCCCCCCCCCCCOCCCCCCCCCCCCCCCC FDCJDKXCCYFOCV-UHFFFAOYSA-N 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- GWZYPXHJIZCRAJ-UHFFFAOYSA-N Biliverdin Natural products CC1=C(C=C)C(=C/C2=NC(=Cc3[nH]c(C=C/4NC(=O)C(=C4C)C=C)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O GWZYPXHJIZCRAJ-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- CIWBSHSKHKDKBQ-DUZGATOHSA-N D-isoascorbic acid Chemical compound OC[C@@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-DUZGATOHSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010023129 Jaundice cholestatic Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 201000005267 Obstructive Jaundice Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- QBUVFDKTZJNUPP-UHFFFAOYSA-N biliverdin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(C=C2C(=C(C)C(C=C3C(=C(C=C)C(=O)N3)C)=N2)CCC(O)=O)N1 QBUVFDKTZJNUPP-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001875 compounds Chemical group 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 229960002433 cysteine Drugs 0.000 description 1
- 239000012954 diazonium Substances 0.000 description 1
- 238000006193 diazotization reaction Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000010350 erythorbic acid Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 229930182480 glucuronide Natural products 0.000 description 1
- 150000008134 glucuronides Chemical class 0.000 description 1
- 235000003969 glutathione Nutrition 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229940026239 isoascorbic acid Drugs 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 229940032330 sulfuric acid Drugs 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/728—Bilirubin; including biliverdin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Saccharide Compounds (AREA)
Description
（産業上の利用分野）
本発明は抱合型ビリルビンの安定化法に関する
ものである。
（従来の技術）
体液中のビリルビンには、非抱合型と抱合型が
あり、抱合型ビリルビンは、非抱合型ビリルビ
ン、すなわちビリルビンそのものに対し、その側
鎖のプロピオン酸残基に水可溶性の化合物（ヒト
体液中では主として、グルクロン酸）が結合した
ものである。これらビリルビンの、特に血中での
動態は、黄疽の鑑別診断のために、その定量的把
握が重要とされてきた。すなわち例えば溶血性黄
疽では非抱合型のビリルビンの血中濃度が上昇
し、また閉塞性黄疽では抱合型ビリルビンの血中
濃度が上昇する。従来の定量法において、直接型
ビリルビンと称されるものは、抱合型ビリルビン
の濃度を反映し、間接型ビリルビンと称されるも
のは、非抱合型ビリルビンに対応している。
体液中のビリルビン及びその分画（直接型およ
び間接型）の測定法として最も広く用いられてい
る方法は、芳香族ジアゾニウム塩とビリルビンと
の反応により生成する色素（アゾビリルビンピグ
メント）の光吸収を利用したものであり、これは
1910年代にHIJMANS VAN DEN BERGHら
により考案され、1930年代にMALLOYと
EVELYNおよびJENDRASSIKとGR′OFらによ
り定量法として確立されたものである。そして近
年、種々の改良法が実施されているが、いずれも
分画法は、溶解性の差を利用している。すなわち
メタノール、尿素、ジメチルスルホキシド、カフ
エインと安息香酸、ダイフイリン、非イオン界面
活性剤等の促進剤と呼ばれるもの−これらは水系
には溶解しにくい非抱合体を可溶化する−を添加
し、抱合体と非抱合体の総計を発色定量すること
により測定される総ビリルビン値と上記促進剤の
非存在下で測定される抱合体に対応した直接ビリ
ルビンに分画される。
これら、測定法において用いられる標準液とし
ては、従来抱合型ビリルビンが入手困難であつた
ために、非抱合型ビリルビンを用いて総ビリルビ
ンの検量線を描き、これを直接ビリルビン測定に
転用するという方法が実施されてきた。係る方法
は、前記促進剤の存在が、アゾビリルビンピグメ
ントの吸光係数に影響しないという仮定のもと
に、直接ビリルビン濃度が算定されることにな
り、理論的明確さに欠ける点がある。また実用的
側面においても、自動分析機の多くのものは、１
つの測定項目に対し対応する標準液があるべきシ
ステムとなつており、直接ビリルビンの如き、従
来対応する標準液として適当なものがない場合に
は、代用標準を用いることが多く行われてきた。
（発明の解決しようとする課題）
本発明の目的は直接ビリルビン及び総ビリルビ
ンの各々の測定に対して使用し得る抱合型ビリル
ビンを用いた安定な標準液を提供することであ
る。
一般にビリルビンは、化学的に不安定である。
抱合型ビリルビンは、酸化に不安定（ビリベルジ
ンに変化）、光に比較的安定であり、非抱合型ビ
リルビンは、酸化に比較的安定、光に不安定であ
る〔臨床検査技術全書６、臨床化学検査332頁、
1980年医学書院発行〕。定量分析に用いる標準液
としては特に安定性が優れていることが望まれて
いる。
最近、ポルフイリン、プロダクツ社から、合成
ジタウリンビリルビン誘導体、商品名ビリルビン
コンジユゲートが発売されている。このものは、
ヒト胆汁から精製されたビリルビンジグルクロナ
イドと種々のジアゾ法において同様の反応性を示
すことが示された。ところがこのビリルビンコン
ジユゲート水溶液の安定性を検討したところ、極
めて悪いことが明らかとなつた。このビリルビン
コンジユゲートの不安定さは、タウリンに依る誘
導体に限られず、水溶解性を促進するための抱合
化を行つたもの一般に言い得ることがあつて、グ
ルクロン酸抱合体も同様に安定性が悪い。ビリル
ビン骨格が酸化されて、ビリベルジン骨格に変化
することは、良く知られている事実であるから、
還元剤の添加により安定化されることは容易に類
推される。事実ビリルビンコンジユゲートの水溶
液に、アスコルビン酸、イソアスコルビン酸、ジ
チオスレイトール、システイン、グルタチオン等
の還元剤を添加することにより安定化される。然
しながらこれらの還元剤は、ジアゾ反応を著しく
阻害するためビリルビン標準液としては、十分な
安定化を確保するための還元剤の十分な量を添加
することは適当でない。
（課題を解決するための手段）
本発明者らは上記欠点を改善するため、種々鋭
意検討したところHLB13〜19の非イオン性界面
活性剤を添加することにより抱合型ビリルビンが
著しく安定化されることを見出し、本発明に到達
した。すなわち本発明は抱合型ビリルビンに
HLB13〜19の非イオン型界面活性剤を共存させ
ることを特徴とする抱合型ビリルビンの安定化法
である。
本発明ではHLB13〜19の非イオン界面活性剤
を共存させることにより抱合型ビリルビンの安定
性を著しく向上させることができる。
本発明に用いる抱合型ビリルビンとはビリルビ
２個プロピオン酸残基の１個又は２個に、水溶性
化合物が結合したものであり、生体中であり、グ
ルクロン酸、タウリン、硫酸等が結合したものが
知られている。本発明では直接ジアゾ化反応をす
るために、水溶性化される基であれば何でもよ
い。
本発明に用いる抱合型ビリルビンは固体でもあ
るいは溶液であつてもよい。抱合型ビリルビン溶
液はPH6.0〜10.5であることが好ましい。
本発明に用いるHLB13〜19の非イオン界面活
性剤の具体例としてはリエチレングリコール
（PEGと略する）セチルエーテル（HLB13.0）、
PEG−ノニルフエニルエーテル、PEG−アルキ
ルエーテル、PEG−ラウリルエーテル、PEG−
PPG（ポリプロピレングリコール）共重合体、
PEG−ステアリルエーテル、商標名パイオニン
Ｄ−1420（竹本油脂製）などがある。
本発明に用いる非イオン界面活性剤の添加量は
抱合型ビリルビン溶液に全して0.2〜10W／Ｖ％
好ましくは１〜3W／Ｖ％である。非イオン界面
活性剤に還元性物質、例えばグルタチオン、シス
テイン、アスコルビン酸、イスアスコルビン酸な
どを併用することも好ましい。その添加量は抱合
型ビリルビン溶液に対して0.0005〜0.02W／Ｖ％
である。
抱合型および非抱合型ビリルビンのジアゾ発色
の色調（最大吸収波長）は、EVERYN−
MALLOY法を端緒とする酸アゾ法においては、
蛋白質の存在量により影響（低波長シフト）す
る。したがつて血清を検体として行う分析の場合
には該標準液中に蛋白質を共存させることが好ま
しい。特に動物またはヒトの正常血清の添加が好
ましい。
（発明の効果）
本発明の抱合型ビリルビン溶液は、凍結乾燥し
た状態で保管及び流通することが長期間使用し得
る状態に保つために好ましい。
（実施例）
以下、本発明を実施例により具体的に説明す
る。実施例中、単に％とあるのはＷ／Ｖ％を示
す。
実施例 １
次の組成のジタウロビリルビン溶液を作製し
た。
ブリツトン・ロビンソン緩衝液（PH9.5）
EDTA・2Na 0.1mM
ジタウロビリルビン 14.2mg／ｄ
添加物（第１表の通り）
上記溶液作製初期及び250℃にて３日間保管し
た後に、総ビリルビン測定試薬（東洋紡績(株)製
“ダイヤカラー（登録商標名）TB”）にて総ビリ
ルビン値を測定し、総ビリルビン値の保管前後の
保持率を求めた。その結果は第１表に示す通りで
あり、本発明の方法が安定性が高いことが示され
た。
(Industrial Application Field) The present invention relates to a method for stabilizing conjugated bilirubin. (Prior Art) There are two types of bilirubin in body fluids: unconjugated and conjugated. (mainly glucuronic acid in human body fluids) is bound thereto. Quantitative understanding of the dynamics of bilirubin, particularly in blood, has been considered important for the differential diagnosis of jaundice. That is, for example, in hemolytic jaundice, the blood concentration of unconjugated bilirubin increases, and in obstructive jaundice, the blood concentration of conjugated bilirubin increases. In conventional quantitative methods, what is called direct bilirubin reflects the concentration of conjugated bilirubin, and what is called indirect bilirubin corresponds to unconjugated bilirubin. The most widely used method for measuring bilirubin and its fractions (direct and indirect) in body fluids is to measure the light absorption of a pigment (azobilirubin pigment) produced by the reaction of an aromatic diazonium salt with bilirubin. This is what I used, and this is
Invented by HIJMANS VAN DEN BERGH and others in the 1910s, and developed by MALLOY in the 1930s.
This was established as a quantitative method by EVELYN, JENDRASSIK, and GR'OF. In recent years, various improved methods have been implemented, but all fractionation methods utilize differences in solubility. That is, by adding accelerators such as methanol, urea, dimethyl sulfoxide, caffein and benzoic acid, daifylin, and nonionic surfactants, which solubilize unconjugated substances that are difficult to dissolve in aqueous systems, the conjugate is The total bilirubin value is measured by colorimetric quantification of the sum of unconjugated and unconjugated bilirubin, which corresponds to the conjugated and directly fractionated bilirubin measured in the absence of the promoter. Since conjugated bilirubin has traditionally been difficult to obtain as a standard solution used in these measurement methods, a method has been developed in which a calibration curve for total bilirubin is drawn using unconjugated bilirubin and this is used directly for bilirubin measurement. It has been implemented. Such a method lacks theoretical clarity because the bilirubin concentration is directly calculated on the assumption that the presence of the promoter does not affect the extinction coefficient of the azobilirubin pigment. Also, from a practical standpoint, many automatic analyzers are
The system requires a standard solution that corresponds to each measurement item, and when there is no suitable conventional standard solution, such as direct bilirubin, substitute standards have often been used. (Problems to be Solved by the Invention) An object of the present invention is to provide a stable standard solution using conjugated bilirubin that can be used for the measurement of direct bilirubin and total bilirubin. Bilirubin is generally chemically unstable.
Conjugated bilirubin is unstable to oxidation (converted to biliverdin) and relatively stable to light, while unconjugated bilirubin is relatively stable to oxidation and unstable to light [Clinical Laboratory Techniques Book 6, Clinical Chemistry 332 pages of inspection,
Published by Igaku Shoin in 1980]. As a standard solution used for quantitative analysis, it is desired that it has particularly excellent stability. Recently, a synthetic ditaurin-bilirubin derivative, trade name bilirubin conjugate, has been released by Porphyrin Products. This thing is
It was shown that bilirubin diglucuronide purified from human bile showed similar reactivity in various diazo methods. However, when the stability of this bilirubin conjugate aqueous solution was investigated, it became clear that it was extremely poor. This instability of bilirubin conjugates is not limited to taurine-based derivatives, but can be said to generally apply to bilirubin conjugates that have been conjugated to promote water solubility, and glucuronide conjugates are similarly unstable. It's bad. It is a well-known fact that the bilirubin skeleton is oxidized and changes to the biliverdin skeleton.
It is easily inferred that it is stabilized by adding a reducing agent. In fact, it can be stabilized by adding a reducing agent such as ascorbic acid, isoascorbic acid, dithiothreitol, cysteine, or glutathione to an aqueous solution of bilirubin conjugate. However, since these reducing agents significantly inhibit the diazo reaction, it is not appropriate to add a sufficient amount of the reducing agent to ensure sufficient stabilization as a bilirubin standard solution. (Means for Solving the Problems) In order to improve the above-mentioned drawbacks, the present inventors conducted various intensive studies and found that conjugated bilirubin was significantly stabilized by adding nonionic surfactants of HLB13 to HLB19. They discovered this and arrived at the present invention. In other words, the present invention uses conjugated bilirubin.
This is a method for stabilizing conjugated bilirubin, which is characterized by coexisting a nonionic surfactant with HLB13 to HLB19. In the present invention, the stability of conjugated bilirubin can be significantly improved by coexisting a nonionic surfactant with HLB13 to HLB19. Conjugated bilirubin used in the present invention is one in which a water-soluble compound is bound to one or two bilirubin bipropionic acid residues, and is present in living organisms, and is bound to glucuronic acid, taurine, sulfuric acid, etc. It has been known. In the present invention, since a direct diazotization reaction is carried out, any group that can be made water-soluble may be used. The conjugated bilirubin used in the present invention may be a solid or a solution. The conjugated bilirubin solution preferably has a pH of 6.0 to 10.5. Specific examples of the nonionic surfactants HLB13 to HLB19 used in the present invention include lyethylene glycol (abbreviated as PEG), cetyl ether (HLB13.0),
PEG-nonylphenyl ether, PEG-alkyl ether, PEG-lauryl ether, PEG-
PPG (polypropylene glycol) copolymer,
Examples include PEG-stearyl ether, trade name Pionin D-1420 (manufactured by Takemoto Yushi). The amount of nonionic surfactant used in the present invention is 0.2 to 10 W/V% in the conjugated bilirubin solution.
Preferably it is 1 to 3 W/V%. It is also preferable to use a reducing substance such as glutathione, cysteine, ascorbic acid, isascorbic acid, etc. in combination with the nonionic surfactant. The amount added is 0.0005 to 0.02 W/V% to the conjugated bilirubin solution.
It is. The diazo color tone (maximum absorption wavelength) of conjugated and unconjugated bilirubin is EVERYN-
In the acid azo method, which originated from the MALLOY method,
Affected by the amount of protein present (lower wavelength shift). Therefore, in the case of analysis using serum as a specimen, it is preferable that proteins coexist in the standard solution. Particularly preferred is the addition of normal animal or human serum. (Effects of the Invention) The conjugated bilirubin solution of the present invention is preferably stored and distributed in a freeze-dried state in order to maintain a usable state for a long period of time. (Example) Hereinafter, the present invention will be specifically explained with reference to Examples. In the examples, the term % simply indicates W/V%. Example 1 A ditaurobilirubin solution having the following composition was prepared. Britton-Robinson buffer (PH9.5) EDTA・2Na 0.1mM Ditaurobilirubin 14.2mg/d Additives (as shown in Table 1) Total bilirubin measurement reagent at the beginning of the above solution preparation and after storage at 250℃ for 3 days. ("Diacolor (registered trademark) TB" manufactured by Toyobo Co., Ltd.) was used to measure the total bilirubin value, and the retention rate of the total bilirubin value before and after storage was determined. The results are shown in Table 1, indicating that the method of the present invention has high stability.
【表】【table】
【表】
実施例 ２
実施例１と同様の組成において、添加物として
第２表に示される種々の界面活性剤を選んだ（添
加量２％）。調製液を40℃に保管して、ビリルビ
ン値の保持率をみた。[Table] Example 2 In the same composition as in Example 1, various surfactants shown in Table 2 were selected as additives (addition amount: 2%). The prepared solution was stored at 40°C and the retention rate of bilirubin value was examined.
【表】【table】
【表】
但し試料No.16，17，18は、それぞれシステイ
ン、グルタチオン、シチオスレイトール0.01％を
追加した。
第２表に示されたように、種々の非イオン界面
活性剤の添加によりまた、還元性物質を併用する
ことにより、抱合型ビリルビンの著しい安定化が
達成された。
実施例 ３
実施例１と同様の組成で添加物として、システ
イン0.02％、更に、パイオニンＤ−1420（竹本油
脂製非イオン界面活性剤）の添加量を第３表の如
く、変えて安定性をみた。[Table] However, for samples No. 16, 17, and 18, 0.01% of cysteine, glutathione, and cythiothreitol were added, respectively. As shown in Table 2, significant stabilization of conjugated bilirubin was achieved by the addition of various nonionic surfactants and by the combined use of reducing substances. Example 3 The composition was the same as in Example 1, but the additives were 0.02% cysteine and the amount of Pionin D-1420 (nonionic surfactant manufactured by Takemoto Yushi) was changed as shown in Table 3 to improve stability. saw.
【表】
実施例 ４
次の組成の溶液を作製し、凍結乾燥した。
ブリツトン・ロビンソン緩衝液 PH9.5
マンニトール 2.0％
リピツドセーラム（登録商標名）
（栄研化学製管理血清） 2.0％
アスコルビン酸 0.01％
非イオン界面活性剤Ｄ―1420 2.0％
ジタウロビリルビン 14.2mg／dl
凍結乾燥したバイアルを冷蔵保管した。蒸溜水
で再溶解した後、９℃及び25℃で保存テストを行
つた。総ビリルビン測定試薬及び直接ビリルビン
測定試薬〔東洋紡績(株)製ダイヤカラー（登録商標
名）TBおよびDB〕にて発色せしめた。発色度
の安定性は第１図に示す如く、良好で、総および
直接ビリルビン測定用として安定な測定を行うた
めに十分なものであつた。
なお、発色後の吸収極大は、総ビリルビン
560nm、直接ビリルビン540nmで、患者血清のも
のと一致した。
実施例 ５
実施例４で作製した標準液を市販試薬キツトを
用いて発色した。第４表に示すように総ビリルビ
ン、直接ビリルビン共、良好な発色を示した。[Table] Example 4 A solution with the following composition was prepared and freeze-dried. Britton-Robinson buffer PH9.5 Mannitol 2.0% Lipid Serum (registered trademark) (Eiken Chemical controlled serum) 2.0% Ascorbic acid 0.01% Nonionic surfactant D-1420 2.0% Ditaurobilirubin 14.2mg/dl Freeze-dried The vials were kept refrigerated. After redissolving with distilled water, storage tests were conducted at 9°C and 25°C. Color was developed using a total bilirubin measurement reagent and a direct bilirubin measurement reagent (Diacolor (registered trademark) TB and DB manufactured by Toyobo Co., Ltd.). As shown in FIG. 1, the stability of color development was good and sufficient for stable measurement of total and direct bilirubin. In addition, the maximum absorption after color development is based on total bilirubin.
560nm, direct bilirubin 540nm, which matched that of patient serum. Example 5 The standard solution prepared in Example 4 was colored using a commercially available reagent kit. As shown in Table 4, both total bilirubin and direct bilirubin showed good color development.
第１図は、本発明によつて作製された抱合型ビ
リルビン溶液の安定性を示す。図中、は総ビリ
ルビンの場合、は直接ビリルビンの場合を示
す。
FIG. 1 shows the stability of conjugated bilirubin solutions made according to the present invention. In the figure, indicates the case of total bilirubin, and indicates the case of direct bilirubin.
Claims (1)
界面活性剤を共存させることを特徴とする抱合型
ビリルビンの安定化法。1. A method for stabilizing conjugated bilirubin, which comprises coexisting a nonionic surfactant of HLB13 to 19 with conjugated bilirubin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14944183A JPS6040957A (en) | 1983-08-15 | 1983-08-15 | Stabilization of conjugation-type bilirubin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14944183A JPS6040957A (en) | 1983-08-15 | 1983-08-15 | Stabilization of conjugation-type bilirubin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6040957A JPS6040957A (en) | 1985-03-04 |
| JPH0154669B2 true JPH0154669B2 (en) | 1989-11-20 |
Family
ID=15475181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14944183A Granted JPS6040957A (en) | 1983-08-15 | 1983-08-15 | Stabilization of conjugation-type bilirubin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6040957A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69123848T2 (en) * | 1990-10-22 | 1997-07-10 | Abbott Lab | STABLE OXYGEN-FREE REAGENT SOLUTION FOR DIAGNOSTICS |
| DE69120373T2 (en) * | 1990-10-30 | 1997-02-06 | Wako Pure Chem Ind Ltd | Procedure for the detection of bilirubin |
| WO1996017251A1 (en) * | 1994-12-02 | 1996-06-06 | Nitto Boseki Co., Ltd. | Method for determining bilirubin |
-
1983
- 1983-08-15 JP JP14944183A patent/JPS6040957A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6040957A (en) | 1985-03-04 |
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