JPH01199166A - Method for simple and precise differential measurement of saccharified hemoglobin in blood - Google Patents
Method for simple and precise differential measurement of saccharified hemoglobin in bloodInfo
- Publication number
- JPH01199166A JPH01199166A JP2451288A JP2451288A JPH01199166A JP H01199166 A JPH01199166 A JP H01199166A JP 2451288 A JP2451288 A JP 2451288A JP 2451288 A JP2451288 A JP 2451288A JP H01199166 A JPH01199166 A JP H01199166A
- Authority
- JP
- Japan
- Prior art keywords
- concentration
- eluent
- phosphate
- salt
- cation
- 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.)
- Pending
Links
- 210000004369 blood Anatomy 0.000 title claims abstract description 21
- 239000008280 blood Substances 0.000 title claims abstract description 21
- 108010054147 Hemoglobins Proteins 0.000 title claims abstract description 13
- 102000001554 Hemoglobins Human genes 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 title abstract description 12
- 238000005259 measurement Methods 0.000 title abstract description 7
- 239000003480 eluent Substances 0.000 claims abstract description 53
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 42
- 150000001768 cations Chemical class 0.000 claims abstract description 21
- 239000000243 solution Substances 0.000 claims abstract description 19
- 239000007853 buffer solution Substances 0.000 claims abstract description 13
- 150000003839 salts Chemical class 0.000 claims abstract description 11
- 238000002835 absorbance Methods 0.000 claims abstract description 10
- 102000017011 Glycated Hemoglobin A Human genes 0.000 claims abstract description 9
- 150000001447 alkali salts Chemical class 0.000 claims abstract description 9
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910019142 PO4 Inorganic materials 0.000 claims description 39
- 235000021317 phosphate Nutrition 0.000 claims description 37
- 239000010452 phosphate Substances 0.000 claims description 35
- 150000008043 acidic salts Chemical class 0.000 claims description 8
- 230000002378 acidificating effect Effects 0.000 claims description 6
- 239000003729 cation exchange resin Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 238000000691 measurement method Methods 0.000 claims description 6
- 108091005995 glycated hemoglobin Proteins 0.000 claims description 5
- 150000002500 ions Chemical class 0.000 claims description 5
- 238000005194 fractionation Methods 0.000 claims description 3
- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims description 3
- 235000002639 sodium chloride Nutrition 0.000 abstract description 12
- 108010014663 Glycated Hemoglobin A Proteins 0.000 abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract description 4
- 239000011780 sodium chloride Substances 0.000 abstract description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract 2
- 239000002253 acid Substances 0.000 abstract 1
- 239000003456 ion exchange resin Substances 0.000 abstract 1
- 229920003303 ion-exchange polymer Polymers 0.000 abstract 1
- 235000011007 phosphoric acid Nutrition 0.000 abstract 1
- 238000000926 separation method Methods 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 238000011067 equilibration Methods 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- 206010012601 diabetes mellitus Diseases 0.000 description 3
- 239000006167 equilibration buffer Substances 0.000 description 3
- 230000003750 conditioning effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- FRZJZRVZZNTMAW-UHFFFAOYSA-N n,n-diethyl-3-(hydroxymethyl)benzamide Chemical compound CCN(CC)C(=O)C1=CC=CC(CO)=C1 FRZJZRVZZNTMAW-UHFFFAOYSA-N 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- -1 cationic ion Chemical class 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
本発明は、人の血液中に存在する糖化ヘモグロビンの筒
易精密分画測定法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a precise fractional measurement method for glycated hemoglobin present in human blood.
成人のヘモグロビンの糖化体、即ちＨｂＡ Ｓ画分は、
ヘモグロビン分子と糖及び糖誘導体分子が結合したもの
であり、この反応は赤血球の循環過程を通じて進行する
ので、その生成量は血中のＨ濃度を反映し、血糖値が高
いほど、またその期間が長いほど増加する。従って、Ｈ
ｂＡｌｃの量は、正常人の血液中より糖尿病患者の血液
中に多いことが知られている。The glycated form of adult hemoglobin, that is, the HbA S fraction, is
It is a combination of hemoglobin molecules and sugar and sugar derivative molecules, and since this reaction progresses through the circulation process of red blood cells, the amount of H produced reflects the H concentration in the blood, and the higher the blood sugar level is, the longer the reaction period is. Increases as the length increases. Therefore, H
It is known that the amount of bAlc is higher in the blood of diabetic patients than in the blood of normal people.
ＨｂＡ 、は３榎の成分、即ちＨｂＡｓ−、ＨｂＡｓｂ
及びＨｂＡ ＋ ｅからなり、この中で一番多く存在す
るのはＨｂＡｌ。である。１（ｂＡｌｃは、血液中のグ
ルコース量の経時的変化を反映する最も信頼できる指標
であることが知られており、糖尿病患者の血糖値管理の
経時的モニターとして極めて重要な意味をもつ。HbA is the three components, namely HbAs-, HbAsb
and HbA + e, of which HbAl is the most abundant. It is. 1 (bAlc) is known to be the most reliable index that reflects changes in the amount of glucose in the blood over time, and has extremely important meaning as a time-course monitor of blood sugar level management in diabetic patients.
従来の血中ヘモグロビンの簡易分画測定法としては、リ
ン酸塩でなる酸性塩及び塩基性塩を含み、その濃度比に
よりｐＨ７，０以下に調整され、かつ陽イオン濃度が０
．０３Ｍ〜０．０７Ｍの緩衝液からなる第１の溶離液と
、同じくリン酸塩でなる酸性塩及び塩基性塩を含み、そ
の濃度比によりＰＨ６，４〜７．７に調整され、かつ陽
イオン濃度が０．０８Ｍ〜０．１８Ｍの緩衝液からなる
第２の溶ａ液と、これらの溶離液と同じ塩類を含み、少
なくてもＰＨ，陽イオン濃度において前記第１のそれを
超えないカラム調整液とを用意し、該カラム調整液で平
衡化した弱酸性陽イオン交換樹脂（２００〜４００メツ
シユ）を充填してなるカラムに溶血血液を添加し、しか
る後、前記第１の溶離液を加えて）ＩｂＡｔ （ａ＋ｂ
）を溶出させ（第１画分とする）、次いで前記第２の溶
離液を加えて）ＩｂＡ 、、を溶出させて（第２画分と
する）、それぞれの溶出操作によって得られるそれぞれ
の画分について吸光度を測定しヘモグロビン濃度を求め
るか、又は前記第１の溶離液を単に予備洗浄に用い、第
２の溶離液による溶出液についてのみ吸光度を測定して
ヘモグロビン濃度を求めるという方法が知られている（
特開昭５６−９８６５８）。The conventional simple fractional measurement method for blood hemoglobin includes an acidic salt and a basic salt made of phosphate, and the pH is adjusted to 7.0 or less by the concentration ratio, and the cation concentration is 0.
．． The first eluent consists of a buffer solution of 0.03M to 0.07M, and contains an acidic salt and a basic salt also made of phosphate, and the pH is adjusted to 6.4 to 7.7 depending on the concentration ratio, and cationic ion A second solution a consisting of a buffer solution with a concentration of 0.08M to 0.18M, and a column containing the same salts as these eluents, and which does not exceed that of the first in terms of pH and cation concentration at least. Hemolyzed blood is added to a column packed with a weakly acidic cation exchange resin (200 to 400 mesh) equilibrated with the column adjustment solution, and then the first eluent is added to the column. In addition) IbAt (a+b
) is eluted (referred to as the first fraction), and then the second eluent is added to elute IbA, , (referred to as the second fraction), and each fraction obtained by each elution operation is There are known methods in which the hemoglobin concentration is determined by measuring the absorbance of the eluate with a second eluent, or by simply using the first eluent for preliminary washing and measuring the absorbance of only the eluate with the second eluent. ing(
Japanese Patent Publication No. 56-98658).
しかし、この方法は第１画分と第２画分の分離が良くな
く、また溶出率も悪く溶離液を多量必要とするため、測
定時間が長いという問題点があり、このため第１画分と
第２画分の分離が良好で、しかも測定時間が短いＨｂＡ
ｌ−の測定方法の開発が望まれていた。However, this method has the problem that the separation of the first and second fractions is poor, the elution rate is also poor, and a large amount of eluent is required, resulting in a long measurement time. HbA with good separation of the first and second fractions and short measurement time
It has been desired to develop a method for measuring l-.
本発明方法の目的は、第１画分と第２画分の分離が良好
で、かつ測定時間が短い血中糖化ヘモグロビンの筒易精
密分画測定方法を提供することにある。即ち、平衡化緩
衝液のリン酸塩濃度と第１溶ｔａ液のｐＨ及びリン酸塩
濃度を種々変化させて実験を行なった結果、平衡化緩衝
液のリン酸塩濃度を高くし、第１溶離液のＰＨを上げて
リン酸塩濃度を低くするとき、特に緩衝液の陽イオン濃
度を０．０８Ｍ〜０．１２Ｍ、第１溶離液のｐ）ｌを７
．２〜７．５、陽イオン濃度を０．０１６Ｍ〜０．０２
７Ｍになるように調整するとき、第１画分と第２画分の
分離が良好であることを見出した。また、分離能が非常
に良いので、樹脂容量が１ｍＬの場合でも第１溶離液は
約４１１３！、、第２溶離液は約５ｍＬと少量であって
も分離され、測定時間が大幅に短縮できる測定方法を提
供することにある。An object of the method of the present invention is to provide a method for easily and precisely fractionating blood glycated hemoglobin in which the first and second fractions are separated well and the measurement time is short. That is, as a result of conducting experiments by varying the phosphate concentration of the equilibration buffer and the pH and phosphate concentration of the first solution, we found that the phosphate concentration of the equilibration buffer was increased and the first When increasing the pH of the eluent to lower the phosphate concentration, the cation concentration of the buffer should be 0.08M to 0.12M, and the p)l of the first eluent should be 7.
．． 2-7.5, cation concentration 0.016M-0.02
It was found that when the concentration was adjusted to 7M, the first and second fractions were well separated. In addition, the separation power is very good, so even when the resin volume is 1 mL, the first eluent is approximately 4113! The object of the present invention is to provide a measurement method in which the second eluent can be separated even if it is as small as about 5 mL, and the measurement time can be significantly shortened.
本発明は、リン酸塩でなる酸性塩及び塩基性塩を含み、
その濃度比によりｐＨ７，２〜７．５に￥Ａ整され、か
つ陽イオン濃度が０．０１６Ｍ〜０．０２７Ｍでリン酸
塩濃度が０．ＯＩＭ〜０．０１５Ｍの陰イオン性緩衝液
からなる第１の溶離液と、同じくリン酸塩でなる酸性塩
及び塩基性塩を含み、その濃度比によりｐＨ６，７に調
整され、かつリン酸塩濃度が０．０３０Ｍ〜０．０５０
Ｍで、それに食塩を加えてイオン濃度を調整し陽イオン
濃度が０．１０Ｍ〜０．１１Ｍの緩衝液からなる第２の
溶離液と、これらの溶離液と同じリン酸塩でなり、その
濃度比によりｐＨ６，５〜６．６に調整きれ、かつ陽イ
オン濃度が０、０８Ｍ〜０．１２Ｍ、好ましくは０．０
９３Ｍで、リン酸塩濃度が０．０６Ｍ〜０．０９Ｍ、好
ましくは０．０７Ｍであるカラム調整波とを用意し、該
カラム調整液で平衡化した弱酸性陽イオン交換樹脂を充
填してなるカラムに溶血血液を添加し、しかる後、前記
第１の溶離液を加えてＨｂＡ＋ （ａ＋ｂ）を溶出させ
、次いで前記第２の溶離液を加えて）ＩｂＡｔ−を溶出
させ、それぞれの）富山操作によって得られる溶出液に
ついて吸光度を測定してヘモグロビン濃度を求めること
を特徴とし、更に１００−２００メツシユ及び２００〜
４００メツシユの弱酸性陽イオン交換樹脂を約１：９−
約２：３（Ｖ／Ｖ）の割合で、好ましくは１：２（Ｖ／
Ｖ）の割合で混合することにより、分離能を低下させず
に溶出速度を速めることを特徴とする。The present invention includes acidic salts and basic salts consisting of phosphates,
Depending on the concentration ratio, the pH is adjusted to 7.2-7.5, the cation concentration is 0.016M-0.027M, and the phosphate concentration is 0.0. A first eluent consisting of an anionic buffer of OIM ~ 0.015M, and an acidic salt and a basic salt also consisting of phosphate, adjusted to pH 6.7 by their concentration ratio, and phosphate Concentration is 0.030M~0.050
A second eluent consisting of a buffer solution with a cation concentration of 0.10M to 0.11M by adding salt to it and a second eluent consisting of a buffer solution with a cation concentration of 0.10M to 0.11M, and a second eluent consisting of a buffer solution containing the same phosphate as these eluents, whose concentration The pH can be adjusted to 6.5 to 6.6 depending on the ratio, and the cation concentration is 0.08M to 0.12M, preferably 0.0
93M and a column conditioning wave having a phosphate concentration of 0.06M to 0.09M, preferably 0.07M, and packed with a weakly acidic cation exchange resin equilibrated with the column conditioning solution. Add hemolysed blood to the column, then add the first eluent to elute HbA+ (a+b), then add the second eluent to elute IbAt-, and perform the respective Toyama operations. The hemoglobin concentration is determined by measuring the absorbance of the eluate obtained by
400 mesh of weakly acidic cation exchange resin at approximately 1:9-
The ratio is approximately 2:3 (V/V), preferably 1:2 (V/V).
By mixing at the ratio V), the elution rate is increased without reducing the separation ability.
また、本発明は糖化ヘモグロビンの中でもＨｂＡ ｉ
ｃ値は、例えば糖尿病における血糖管理の指標として有
効であることに鑑み、前記第１の溶離液を単に予備洗浄
に用い、第２の溶離液による溶出液についてのみ吸光度
を測定してヘモグロビン濃度を求めることを特徴とする
。In addition, the present invention provides HbA i among glycated hemoglobins.
Considering that the c value is effective as an index of blood sugar control in diabetes, for example, the first eluent is simply used for preliminary washing, and the absorbance is measured only for the eluate with the second eluent to determine the hemoglobin concentration. Characterized by seeking.
弱酸性陽イオン交換樹脂としては室町化学社製（ＤＪａ
ＯマックＭＷＣ−１ヲ用い、１００−２００ メッシ
：Ｌ （Ｌｏｔ。As a weakly acidic cation exchange resin, Muromachi Kagaku Co., Ltd. (DJa
Using Omac MWC-1, 100-200 Messi: L (Lot.
５４１５６）及び２００−４００メッシ：Ｌ （Ｌｏｔ
、５４１８６）を約Ｃ９（Ｖ／Ｖ）−約２：３（Ｖ／Ｖ
）の割合で混合し、好ましくは１：２（Ｖ／Ｖ）の割合
で混合し、カラム法により平衡化する。１００〜２００
メツシユ及び２００〜４００メツシユの割合が１：９以
下の場合は、流速が遅くなり測定時間が長くなる。また
、この割合が２：３以上の場合は、第１画分と第２画分
の分離が悪くなり、ＨｂＡｓｃのみの溶出が難しくなる
。54156) and 200-400 Messi: L (Lot
, 54186) to about C9 (V/V) - about 2:3 (V/V
), preferably at a ratio of 1:2 (V/V), and equilibrated by a column method. 100-200
If the ratio of mesh and 200 to 400 mesh is less than 1:9, the flow rate will be slow and the measurement time will be long. Furthermore, if this ratio is 2:3 or more, the separation of the first and second fractions becomes poor, making it difficult to elute only HbAsc.
平衡化種（釘液としては、リン酸−水素ナトリウム（Ｈ
ａＨｚＰＯａ）及びリン酸二水素ナトリウム（Ｎａ２Ｈ
ＰＯ＋）を含む水溶液を用い、ｐＨ６，５−６，８、か
つ陽イオン濃度カ０．０８Ｍ−０．１２Ｍテ、リン酸塩
濃度力０．０６Ｍ〜０．０９Ｍになるように調整する。Equilibrated species (as nail solution, sodium phosphate-hydrogen (H
aHzPOa) and sodium dihydrogen phosphate (Na2H
Using an aqueous solution containing PO+), adjust the pH to 6.5-6.8, the cation concentration to 0.08M to 0.12M, and the phosphate concentration to 0.06M to 0.09M.
ＰＨが６．７以上ではＨｂＡ 、。HbA when pH is 6.7 or higher.
のみの分離が不可能となる。It becomes impossible to separate the two.
第１溶離液は、ｐ）１７．２〜７．５、陽イオン濃度が
０．０１８Ｍ〜０．０２７Ｍで、リン酸塩濃度が０．Ｏ
ＩＭ〜０．０１５Ｍになるように調整する。The first eluent has a p) of 17.2 to 7.5, a cation concentration of 0.018M to 0.027M, and a phosphate concentration of 0. O
Adjust so that IM ~ 0.015M.
第２溶離液は、ｐＨ６，７、リン酸塩濃度が０．０３Ｍ
〜０．０５翼になるように調整し、更に食塩を加えてイ
オン濃度を調整する。食塩を用いるのは、リン酸塩だけ
でイオン濃度を調整すると、ＨｂＡｓが）容出しやす（
なってしまうためである〇
実施例１
朋」Ｌ狡１」」乱立−
平衡化緩衝液で予め処理したムロマックＭＭＣ−１。The second eluent has a pH of 6.7 and a phosphate concentration of 0.03M.
Adjust to ~0.05 wings, and further add salt to adjust the ion concentration. The reason for using table salt is that if you adjust the ion concentration with only phosphate, HbAs can be easily excreted (
Example 1 Muromak MMC-1 pre-treated with equilibration buffer.
１００〜２００メツシユ１容、２００−４００メツシユ
２容の混合物０．９＋＋＋Ｉ！、ｔ−図１のカラムに充
填し、溶血血ｆｆ１ｏ。A mixture of 1 volume of 100-200 mesh and 2 volumes of 200-400 mesh 0.9+++I! , t-packed into the column of Figure 1 and lysed hemolyzed blood ff1o.
μＬｆ−添加、第１溶離液４１１１Ｊ！、、次いで第２
溶離液５ＩＩＩＬを添加し流速を測定した。溶出時間は
第１溶離液１２分、第２溶離液１５分であり、分画も良
好であ）た。μLf-added, 1st eluent 4111J! ,, then the second
Eluent 5IIIL was added and the flow rate was measured. The elution time was 12 minutes for the first eluent and 15 minutes for the second eluent, and the fractionation was good.
実施例２
パ と １ の
・ムロマックＭＷＣ−１，１００−２００メツシユ１容
、２００〜４００メツシユ２容をリン酸塩（Ｎａ２）Ｉ
ｒＯ２−ＮａＨ２ＰＯ４）緩衝液０．０６Ｍ〜０．１０
Ｍ（陽イオン濃度０．０８）１〜０．１３５）１）、ｐ
Ｈ６，５で平衡化し、図１のカラムに０．９ｍＬ充填し
た。Example 2 Pa and 1
・Muromac MWC-1, 1 volume of 100-200 mesh, 2 volumes of 200-400 mesh with phosphate (Na2) I
rO2-NaH2PO4) buffer 0.06M~0.10
M (cation concentration 0.08) 1-0.135) 1), p
It was equilibrated with H6,5 and 0.9 mL was packed into the column shown in FIG.
第１溶離液としてリン酸塩（Ｎａ２ＨＰＯ４−ＮａＨ２
ＰＯ４）緩衝ｉｏ、ｏＩＭ〜０．０１５Ｍ（ｇＪイオン
濃度０．０１６Ｍ〜０．０２７Ｍ）、ｐ）Ｉ７．２〜７
．５を４＋ａＬ用いた。図２に第１画分の比率を示した
が、何れの第１ｉｆ鑓液を用いた場合にも、平衡化８１
回液として０．０７Ｍリン酸塩（Ｎａ２）ＩＰＯａ−Ｈ
ａＨｚＰＯａ）［［液（ｐＨ６，５）を用いた場合にお
いて、ＨＰＬＣによるＨｂＡ１．とＨｂＡ ｔ ｂの合
計値と一致する値が得られた。Phosphate (Na2HPO4-NaH2) was used as the first eluent.
PO4) Buffer io, oIM~0.015M (gJ ion concentration 0.016M~0.027M), p) I7.2~7
．． 5 was used for 4+aL. Although the ratio of the first fraction is shown in FIG. 2, the equilibration 81
0.07M phosphate (Na2) IPOa-H as liquid solution
aHzPOa)[[HbA1. A value consistent with the total value of and HbA t b was obtained.
実施例３
２゛ の組 とＨｂＡ イ會
０．０７Ｍ〜０．０９Ｍリン酸塩（Ｎａ２ＨＰＯ４−Ｈ
ａＨｚＰＯａ）緩衝液（ｐ）１６．５）テ平衡化し？１
． ムｌ：ｌ マックＭＷＣ−１、１００−２００メツ
シュｌ容、２００−４００メツシユ２容の混合物０．９
ｍＬを図１のカラムに充填し、第１溶＃２１！としてリ
ン酸塩（Ｎａ２）ＩｒＯ２−ＨａＨｚＰＯａ） Ｗｌ
ａ液０．ＯＩＭ〜０．０１５Ｍ（ｐＨ７．２〜７．５）
を４ｍＪ−第２溶離液として０．０４Ｍリン酸塩（Ｎａ
２ＨＰＯ４−ＨａＨｚＰＯａ） ｉｉ ？ｔｆ液に０．
０２Ｍ〜０．０７Ｍノ食塩を加えた溶液５＋ａｌを用い
て、溶血血液を分画した場合の第２画分の比率を図３に
示した。第２画分の比率は、何れの平衡化条件において
も第２溶離液の食塩濃度の増加に伴りて上昇したが、食
塩０．０３Ｍ〜０．０６Ｍの範囲で）ＩｂＡ＋ｃ値が妥
当な値を示した。Example 3 A combination of 2゛ and HbA 0.07M to 0.09M phosphate (Na2HPO4-H
aHzPOa) Buffer (p) 16.5) Equilibrate? 1
．． Mixture of MWC-1, 100-200 mesh 1 volume, 200-400 mesh 2 volumes 0.9
mL was packed into the column shown in Figure 1, and the first solution #21! as phosphate (Na2)IrO2-HaHzPOa) Wl
A liquid 0. OIM~0.015M (pH7.2~7.5)
4 mJ - 0.04 M phosphate (Na
2HPO4-HaHzPOa) ii? 0.0 to tf solution.
FIG. 3 shows the ratio of the second fraction when hemolyzed blood was fractionated using solution 5+al to which 0.02M to 0.07M salt was added. The ratio of the second fraction increased as the salt concentration of the second eluent increased under all equilibration conditions, but the IbA+c value (in the range of salt 0.03M to 0.06M) was reasonable. showed that.
実施例４
ヘモグロビンの 画像
０．０７Ｍリン酸塩（Ｎａ２）ＩＰＯ４−ＮａＨ２ＰＯ
４）緩衝液（ｐＨ６，５）で平衡化した樹脂０．９Ｊを
図１のカラムに充填、第１溶離液としてリン酸塩（Ｈａ
２ＨＰＯ４−ＮａＨ２ＰＯ４） ｆｌ ｉＵＵＯ３ＯＩ
Ｍ〜０．０１５Ｍ（ｐＨ７．２〜７．５）’ｔ ４ ｌ
ｌ１ｒｌ／、ｇ２ｉＷ離ｉとして０．０４Ｍリン酸塩（
Ｎａ２）ＩＰ０４−Ｈａ）１２ＰＯ４）緩衝液に０．０
５Ｍ〜０．０５５Ｍの食塩を加えた溶液５ｍＬを用いた
場合の溶血検体の分画像を図４に示した。何れの場合に
も第１画分（ＨｂＡｌ（−＋ｂ＞）と第２画分（ＨｂＡ
ｔｃ）の分離は良好であり、かつ第２画分には目的以外
のヘモグロビン分画の混入は殆ど認められなかった。Example 4 Image of hemoglobin 0.07M phosphate (Na2) IPO4-NaH2PO
4) Fill the column shown in Figure 1 with 0.9 J of resin equilibrated with buffer solution (pH 6,5), and use phosphate (Ha) as the first eluent.
2HPO4-NaH2PO4) fl iUUO3OI
M~0.015M (pH7.2~7.5)'t4l
l1rl/, g2iW separation i as 0.04M phosphate (
Na2)IP04-Ha)12PO4)0.0 in buffer
FIG. 4 shows separate images of hemolyzed specimens obtained when 5 mL of a solution containing 5 M to 0.055 M of sodium chloride was used. In either case, the first fraction (HbAl(-+b>) and the second fraction (HbA
The separation of tc) was good, and almost no hemoglobin fraction other than the target was found in the second fraction.
第１図は、本発明方法で用いるミニカラムを示す。
第２図は、平衡化条件の異なる樹脂の第１溶離液による
分画を示す。第２図の左図は第１溶離液１０ｍＭ、右図
は１５ｍＭの場合を示す。
第３図は、第２溶ｓ液の食塩濃度と）ｌｂＡｌ。値との
関係を示す図である。第３図の左図、中央図、右図は、
それぞれ平衡化リン酸塩濃度が７０１１ＩＭ、８０ＩＩ
Ｍ、９０ｍＭの場合を示す。
第４図は、７０ｍＭリン酸塩平衡化樹脂によるクロマト
グラムを示す。
図１ ミニカラムの寸法
（単位ｍ＠）
１．０
図２ 平衡化条件の異なる樹脂の
第１溶Ｉｌ！を液による分画
吸光度（４１５止）
平衡化緩、樹液のリン酸塩濃度（ｐＨ６，５）１ｆｆ１
４ ７０ｍＭリン酸塩平衡化（Ｓ）脂によるクロマトグ
ラム第１溶離液 第２溶離液
吸光度（４１５ｎＩＩｌ） ｒ） ン１ｉ３ｊｆｉ
ｐＨ食塩Ｍ１溶１ｉｉＩＩ液 第２溶
離液手 続 補 正 書 （方式）％式％
１、事件の表示
昭和６３年特許９２４５１２号
２、発明の名称
血中糖化ヘモグロビンの簡易精密分画測定法３、補正を
する者
事件との関係 特許出願人
住所 東京都千代田区岩本町２丁目２番３号昭和６３年
４月２６日（発送臼）
５、補正の対象
図面
６、補正の内容
第３図を鮮明に描いたものFIG. 1 shows a minicolumn used in the method of the invention. FIG. 2 shows the fractionation of resins with different equilibration conditions using the first eluent. The left diagram in FIG. 2 shows the first eluent at 10 mM, and the right diagram at 15 mM. Figure 3 shows the salt concentration of the second solution and ) lbAl. It is a figure showing the relationship with a value. The left, center, and right figures in Figure 3 are
Equilibrated phosphate concentrations are 7011IM and 80II, respectively.
The case of M, 90mM is shown. Figure 4 shows a chromatogram with 70mM phosphate equilibrated resin. Figure 1 Dimensions of mini-column (unit m@) 1.0 Figure 2 First solution Il of resin with different equilibration conditions! Fractional absorbance by liquid (415 stop) Slow equilibration, phosphate concentration of sap (pH 6,5) 1ff1
4 Chromatogram with 70mM phosphate equilibrated (S) fat 1st eluent 2nd eluent absorbance (415nIIl) r) N1i3jfi
pH salt M1 solution 1iiiII solution 2nd eluent procedure amendment (method) % formula % 1, Incident indication 1988 Patent No. 924512 2, Name of the invention Simple precise fractional measurement method for glycated hemoglobin in blood 3, Relationship with the case of the person making the amendment Patent Applicant Address: 2-2-3 Iwamoto-cho, Chiyoda-ku, Tokyo April 26, 1988 (Delivery mortar) 5. Drawing 6 subject to amendment, content of amendment Fig. 3 clearly drawn
Claims (2)
濃度比によりｐＨ７．２〜７．５に調整され、かつ陽イ
オン濃度が０．０１６Ｍ〜０．０２７Ｍの緩衝液からな
る第１の溶離液と、同じくリン酸塩でなる酸性塩及び塩
基性塩を含み、その濃度比によりｐＨ６．７に調整され
、かつリン酸塩濃度が０．０３０Ｍ〜０．０５０Ｍで、
それに食塩を加えイオン濃度を調整して陽イオン濃度が
０．１０Ｍ〜０．１１Ｍの緩衝液からなる第２の溶離液
と、これらの溶離液と同じリン酸塩でなり、その濃度比
によりｐＨ６．５〜６．６に調整され、かつ陽イオン濃
度が０．０８Ｍ〜０．１２Ｍのカラム調整液とを用意し
、該カラム調整液で平衡化した弱酸性陽イオン交換樹脂
を充填してなるカラムに溶血血液を添加し、しかる後、
前記第１の溶離液を加えてＨｂＡ＿１＿（＿ａ＿＋＿ｂ
＿）を溶出させ、次いで前記第２の溶離液を加えてＨｂ
Ａ＿１＿ｃを溶出させ、それぞれの溶出操作によって得
られる溶出液について吸光度を測定してヘモグロビン濃
度を求めることを特徴とする血中糖化ヘモグロビンの簡
易精密分画測定法。(1) A buffer solution containing an acidic salt of phosphate and a basic salt, adjusted to pH 7.2 to 7.5 according to their concentration ratio, and having a cation concentration of 0.016M to 0.027M. It contains the eluent of 1 and an acidic salt and a basic salt which are also phosphates, the pH is adjusted to 6.7 by the concentration ratio thereof, and the phosphate concentration is 0.030M to 0.050M,
The second eluent consists of a buffer solution with a cation concentration of 0.10M to 0.11M by adding salt to adjust the ion concentration, and the second eluent consists of the same phosphate as these eluents, and the pH is 6 depending on the concentration ratio. Prepare a column adjustment solution adjusted to .5 to 6.6 and have a cation concentration of 0.08M to 0.12M, and fill with a weakly acidic cation exchange resin equilibrated with the column adjustment solution. Add hemolysed blood to the column, then
Add the first eluent to obtain HbA_1_(_a_+_b
), and then add the second eluent to remove Hb.
A simple and precise fractional measurement method for blood glycated hemoglobin, which comprises eluating A_1_c and measuring the absorbance of the eluate obtained by each elution operation to determine the hemoglobin concentration.
濃度比によりｐＨ７．２〜７．５に調整され、かつ陽イ
オン濃度が０．０１６Ｍ〜０．０２７Ｍの緩衝液からな
る第１の溶離液と、同じくリン酸塩でなる酸性塩及び塩
基性塩を含み、その濃度比によりｐＨ６．７に調整され
、かつリン酸塩濃度が０．０３０Ｍ〜０．０５０Ｍで、
それに食塩を加えイオン濃度を調整して陽イオン濃度が
０．１０Ｍ〜０．１１Ｍの緩衝液からなる第２の溶離液
と、これらの溶離液と同じリン酸塩でなり、その濃度比
によりｐＨ６．５〜６．６に調整され、かつ陽イオン濃
度が０．０８Ｍ〜０．１２Ｍのカラム調整液とを用意し
、該カラム調整液で平衡化した弱酸性陽イオン交換樹脂
を充填してなるカラムに溶血血液を添加し、しかる後、
前記第１の溶離液を加えてＨｂＡ＿１＿（＿ａ＿＋＿ｂ
＿）などを除去する予備洗浄を行ない、次いで前記第２
の溶離液を加えてＨｂＡ＿１＿ｃを溶出させ、該第２の
溶離液による溶出操作によって得られる溶出液について
吸光度を測定してヘモグロビンＡ＿１＿ｃ濃度を求める
ことを特徴とする血中糖化ヘモグロビンの簡易精密分画
測定法。(2) A buffer solution containing an acidic salt of phosphate and a basic salt, whose pH is adjusted to 7.2 to 7.5 according to their concentration ratio, and whose cation concentration is 0.016M to 0.027M. It contains the eluent of 1 and an acidic salt and a basic salt which are also phosphates, the pH is adjusted to 6.7 by the concentration ratio thereof, and the phosphate concentration is 0.030M to 0.050M,
The second eluent consists of a buffer solution with a cation concentration of 0.10M to 0.11M by adding salt to adjust the ion concentration, and the second eluent consists of the same phosphate as these eluents, and the pH is 6 depending on the concentration ratio. Prepare a column adjustment solution adjusted to .5 to 6.6 and have a cation concentration of 0.08M to 0.12M, and fill with a weakly acidic cation exchange resin equilibrated with the column adjustment solution. Add hemolysed blood to the column, then
Add the first eluent to obtain HbA_1_(_a_+_b
_), etc., and then the second
Simple and precise fractionation of blood glycated hemoglobin, characterized in that HbA_1_c is eluted by adding an eluent, and the absorbance of the eluate obtained by the elution operation with the second eluent is measured to determine the concentration of hemoglobin A_1_c. Measurement method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2451288A JPH01199166A (en) | 1988-02-04 | 1988-02-04 | Method for simple and precise differential measurement of saccharified hemoglobin in blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2451288A JPH01199166A (en) | 1988-02-04 | 1988-02-04 | Method for simple and precise differential measurement of saccharified hemoglobin in blood |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01199166A true JPH01199166A (en) | 1989-08-10 |
Family
ID=12140225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2451288A Pending JPH01199166A (en) | 1988-02-04 | 1988-02-04 | Method for simple and precise differential measurement of saccharified hemoglobin in blood |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01199166A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55101050A (en) * | 1979-01-26 | 1980-08-01 | Sogo Seibutsu Igaku Kenkyusho:Kk | Simple dispensation measuring method for hemoglobin glucoside in blood |
| JPS5698658A (en) * | 1980-01-11 | 1981-08-08 | Sogo Seibutsu Igaku Kenkyusho:Kk | Method of simple fractional determination of glucosidated hemoglobin in blood |
| JPS5872055A (en) * | 1981-10-26 | 1983-04-28 | Sogo Seibutsu Igaku Kenkyusho:Kk | Simple measuring method for hemoglobin a1 in blood |
-
1988
- 1988-02-04 JP JP2451288A patent/JPH01199166A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55101050A (en) * | 1979-01-26 | 1980-08-01 | Sogo Seibutsu Igaku Kenkyusho:Kk | Simple dispensation measuring method for hemoglobin glucoside in blood |
| JPS5698658A (en) * | 1980-01-11 | 1981-08-08 | Sogo Seibutsu Igaku Kenkyusho:Kk | Method of simple fractional determination of glucosidated hemoglobin in blood |
| JPS5872055A (en) * | 1981-10-26 | 1983-04-28 | Sogo Seibutsu Igaku Kenkyusho:Kk | Simple measuring method for hemoglobin a1 in blood |
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