Nitric Oxide (NO) has been identified as an intrinsic mediator to play key roles in physiological and pathological conditions in human body. Recently, the relationships between NO and diseases are attracted much attention.
Due to its short half-life, the amount of NO is generally estimated from the concentrations of NO metabolites, i.e. nitrite (NO₂⁻) and nitrate (NO₃⁻). The colorimetric kit (Griess method) is widely used for determination of NO₂⁻ and NO₃⁻; however, this method is relatively complicated and time-consuming because of the intricate chemical reactions. The other methods, such as high performance liquid chromatography, are also complicated and time-consuming.
Recently, in order to obtain high resolution and high speed separation of NO metabolites, some studies using capillary electrophoresis (CE) have been reported. Since, microchip CE (MCE) has an advantage in high-throughput assay, we have developed a high-throughput NO assay using MCE with UV detection.
In this paper, we described the development of a novel running buffer for determination of NO₂⁻ and NO₃⁻ in human fluids, the achievement of complete MCE separation by controlling the applied voltages and the investigation of on-chip preconcentration using transient isotachophoresis. As a result, a novel running buffer based on the ionic composition of human serum was developed for CE, and then the MCE separation of NO₂⁻ and NO₃⁻ in a spiked human serum was achieved within 6.5 seconds.

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A brief review of interfaces for on-line coupling of capillary electrophoresis (CE) and mass spectrometry (MS) is presented. To enable the CE-MS system, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were introduced as MS interfaces for connecting with CE instruments, while the partial-filling method was developed to suppress the introduction of non-volatile components or additives in CE buffers as well as ionic species such as ionic micelles into the MS interface. Strategy for the coupling of micellar electrokinetic chromatography (MEKC) and MS is also discussed. Examples of CE-MS are briefly shown.

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キャピラリー電気泳動によるヒト・ゲノムDNA解析および疾患の遺伝子診断の新規方法論を確立した. キャピラリー電気泳動装置を用いて, ヒト・ゲノムDNAの遺伝子診断を高速化するために, DNAの検出条件および分離条件を最適化した. その結果, 極めて短時間のうちに高い精度でDNAの遺伝子多型解析が可能になることを明らかにした. さらに, キャピラリー電気泳動法が, 実際のヒトの心臓病の遺伝子診断に有効であることを実証した.

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Carbohydrate analysis absolutely depends on high-resolution separation, because oligosaccharides derived from glycoconjugates are usually composed of an extremely complex mixture of carbohydrates including isomers. Capillary electrophoresis (CE) is one of the most powerful techniques in terms of resolving power, and a combination of CE and laser-induced fluorescence (LIF) detection enables to detect carbohydrates at fmol (10⁻¹⁵ mol) to amol (10⁻¹⁸ mol). CE is currently applied to the analysis of various carbohydrates derived from glycoconjugates. This review describes the recent development in high sensitive analysis of carbohydrates using LIF-CE, and the techniques for profiling of carbohydrates using capillary affinity electrophoresis (CAE).

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This review summarizes the mechanism and applications of electrokinetic supercharging (EKS), an online preconcentration technique utilizing electrokinetic injection (EKI) coupled with transient isotachophoresis (tITP) in capillary electrophoresis and microchip electrophoresis. When the sample components (targets) are electrokinetically injected in the EKS procedure, the electrolyte filled in the separation capillary must (at least partly) contain a leading electrolyte (LE) that contains a co-ion with greater mobility than that of the targets to fulfill the condition of the ITP preconcentration. To complete preconcentration, further introduction of terminating electrolyte (TE) is useful. Obviously, the concentration factors of EKS strongly depend on essential EKI and tITP stages, thus two sections of this review are concentrated on the introduction of EKI and tITP. For EKI, several factors affecting the injected amount are discussed and concluded here. For tITP, different introduction modes for the sample, the optimization of the LE and TE to enhance preconcentration are also mentioned. So far, several high-sensitivity applications to metal ions, drugs, peptides, DNA fragments, and SDS-proteins have illustrated that EKS was an effective and promising stacking technique.

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Capillary electrophoresis (CE) is a highly efficient separation method and used for separation of many compounds. We developed a fast and sensitive analytical method for β-amyloid (Aβ) aggregates by the combination of CE-laser-induced fluorescence and a fluorescence reagent, thioflavine T. The developed method is also applicable to a high-throughput screening of Aβ aggregation inhibitors, which are expected to be an effective therapeutic agent candidates of Alzheimer’s disease.

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PCR-based analysis of genetic alterations has been applied to the diagnosis of various diseases, however, for this technique to become widely accepted as a tool for clinical DNA diagnosis, the development of automated analytical system is essential. Capillary electrophoresis (CE) is a promising alternative to gel electrophoresis for high-speed and reproducible separation of DNA fragments, and its use is feasible for automated DNA analysis. Therefore, CE is applied for DNA sequencing and the detection of genetic changes including microsatellite instability and mutations. Recently, capillary array electrophoresis for the simultaneous analyses of many samples is applied for the automated high-throughput sequencing. Heteroduplex analysis has been reported to be a promising technique for detecting mutated sequences because of its higher sensitivity than that of SSCP analysis. In this analysis, heteroduplex DNA containing mutated sequences is separated by denaturing gradient gel electrophoresis (DGGE). However, in DGGE, PCR products joined to a GC clamp are necessary and the gel preparation is also complicated. Therefore, it is difficult to automate. Denaturing high-performance liquid chromatography (DHPLC) has been reported to be a novel automated high-throughput technique for separating heteroduplex and homoduplex DNA fragments amplified with primers without a GC clamp under controlled temperature conditions. Hetroduplex analysis involving DHPLC enable more accurate, more rapid and easier heterozygous mutation detection than SSCP analysis. Hence, it is suggested that CE is preferable for DNA sizing and sequencing analyses, and DHPLC is effective for screening of genetic mutations and polymorphisms.

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キャピラリー電気泳動を用いて血漿 nitrite, nitrate 濃度を測定した. キャピラリーは65cm×75μmの fused-silica 型のものを用い, 電圧は20kV, 検出波長は214nmとした. 泳動バッファーは5%NICE-Pak OFM Anion-BTを含む750mM NaClとした. 血液は, 採血後1000g, 5分間遠心分離して, 血漿を-80℃で保存した. 血漿は測定前にフィルターし, 濾液の20μlを使用した.
nitrite 濃度と泳動ピークの面積は正の相関を示し, その回帰式はy=1425+8261xでr²=0.999であった. nitrate 濃度と泳動ピークの面積も同様に正の相関を示し, その回帰式はy=1417+8416xでr²=0.999であった. 41名の健康成人の静脈血 nitrite, nitrate 濃度はそれぞれ, 0.15±0.07mg/l, 3.20±1.60mg/lであった. 敗血症, 亜硝酸ナトリウム投与, クモ膜下出血後の3症例の経過中に nitrite, nitrate を測定し, 病態により血漿中 nitrate が変化することがわかった. 以上より, キャピラリー電気泳動による血漿中 nitrite, nitrate 測定は, 集中治療患者のモニターとして有用と思われる.

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ヒトゲノム解析プロジェクトの目的は, ヒトの設計図であるヒトゲノムの全配列を解明することである. 現在, ヒトゲノムのマッピングがほぼ終了し, ヒトのcDNA解析やモデル生物のゲノム解析が著しい速度で進められている. しかし, ヒトゲノムは約30億塩基という膨大な配列を有するので, 西暦2005年終了という目的を達成するには技術的なブレイクスルーが不可欠であるといわれ, 様々な新しい遺伝子配列決定法が提案されている. しかし, 現在までのところ従来からの方法であるジデオキシ法を凌ぐ方法は開発されていない. ジデオキシ法による解析の高速化には種々の課題があるが, それらの課題の多くを解決する方法としてキャピラリー電気泳動が期待されている. しかし, 残念ながら現在までのところ, キャピラリー電気泳動はゲノム解析にほとんど貢献していない. ゲノム解析研究者とキャピラリー電気泳動研究者が協力し, できるだけ早い時期にキャピラリー電気泳動を用いたシークエンサーを実用化し, ゲノム解析に貢献することが期待されている.

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リポソームを使った免疫分析は, Kinsky らの報告以来盛んに研究されている. リポソームからシグナル発生物質が漏れ出る分析モードは, リポソーム膜の不安定化に基づいている. 漏れ出たシグナル発生物質の量は被検体の濃度に比例し, そのモニタリングは通常吸収または蛍光法で行われてきた. 我々は, 標識剤としての色素包括リポソームを化学発光検出する免疫分析法を提案した. これはキャピラリー電気泳動-化学発光検出法の免疫分析へのはじめての応用である. この方法で, ごく少ない試料を用いて, 簡便かつ迅速に高感度分析ができた. ここでは, 血清分析に関する一部新しいデータを付け加えて, 色素包括リポソームを用いた免疫分析法を開発の背景とともに簡単に紹介する.

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Serum protein fractionation using capillary zone electrophoresis and the application is described. There is 20-30 major components in human serum protein, even though the several hundred proteins are exist. However, in serum protein fractionation by cellulose acetate membrane electrophoresis applied to the clinically, it is only five fractions which intermingle many proteins ingredients with one fraction. Clinically, combining change of the 5 fractions has been made to be the assistance of the diagnosis. In the meantime, capillary zone electrophoresis for excellent separation can be consequentially divided the serum proteins into 10 fractions. However, it becomes a result of being inconvenient on the contrary. The clinical significance equal to conventional electrophoresis of protein would be found when divided protein fractions are specially integrated for 5 fractions. The purpose was achieved by the CZE 2000 instrument (Beckman-Coulter Co., USA) and was obtained by the preparation of the software which arranges the serum protein fractions are originally divided into 10 peaks for 5 fractionation. Therefore, it is possible that can fundamentally follow clinical significance by cellulose acetate membrane electrophoresis and moreover, the data of 10 fractionation is also taken out. And, for the identification of regarded M-protein in cases of multiple myeloma and MGUS (monoclonal gammopathy undetermined significance), it is conveniently possible by subtraction method using the capillary electrophoresis. There is dissociation of α₁ fraction value between cellulose acetate membrane electrophoresis and capillary electrophoresis. From our detailed experimental results, it is proved that is due to destaining of α₁-acid glycoprotein.

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Carbohydrates are the least inappropriate group of compounds to be analyzed by capillary electrophoresis (CE), because normally they have no electric charge. However, they can be analyzed by this method by adding various devices. There are a number of methods for conversion of carbohydrates to positively or negatively charged derivatives, which can be analyzed directly by zone electrophoresis. In-situ conversion with certain kinds of oxyacid ions, such as the borate ion, and metal ions, such as alkaline earth metal ions, also form ionic complexes from carbohydrates and derivatives. Furthermore, introduction of hydrophobic groups allows separation based on solubilization of the derivatives into ionic micells, thus permitting micellar electrokinetic chromatography. This paper describes not only such theoretical aspect of CE but its application to the analysis of oligosaccharides in glycoproteins. It gives a number of examples of successful application.

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キャピラリー電気泳動法により血中アラントイン濃度を測定した. キャピラリー電気泳動はフューズドシリカ (75μm×50cm) をキャピラリー管に用い, 泳動溶液には50mM Na₂B₄O₇-HCl緩衝液pH9.0を用いて, 25kVで泳動し, 検出は214nmで行った. アラントイン濃度2～400μMでのピーク面積との相関係数は0.999であった. また, CVは10%以下であり, 添加回収率は92～113%であった. 安定性の検討では, 光照射によるアラントイン濃度の増加が認められた. 健常者においてはアラントインは1例を除いて検出されなかったが, リウマチ患者においては健常者と比較して有意に高値を示した. キャピラリー電気泳動法は血中アラントインの測定法として有用と考えられる.

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Recently the development and the practical application of monoclonal antibodies for clinical use are rapidly expanding in pharmaceutical business trend. Because of the heterogeneities of antibodies as glycoproteins, a new approach to test the products is required. Capillary electrophoresis (CE) is one of the important candidates that meet this demand.
CE separation is based on the various modes involving capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF). All of them are used for the analysis and test of monoclonal antibodies for clinical use. Especially with CGE, a non-glycosylated heavy chain is separated from a glycosylated one and detected. CIEF is an important technology to analyze the heterogeneity of antibodies mainly based on the N-linked oligosaccharide composition.
N-linked oligosaccharide structure is also important as it effects on the bio-activity of the antibodies like antibody-dependent cellular cytotoxity. The fluorescence and charge insertion into oligosaccharides by chemical labeling is a valuable strategy to make CE useful for the analysis of them which released by enzyme digestion of antibodies.
CZE is also useful not only for peptide mapping but also as the base of the CE-mass spectrometry approach to analyze the structure of peptides and N-linked oligosaccharide chains.
These applications are discussed with examples.

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未処理フューズドシリカキャピラリーを用いたキャピラリー電気泳動法によって, 11種のモデルタンパク質 (MW 6～75K_d: pI4.5～11.2) 及びヒト血清タンパク質の分離分析を試みた. 電解液, 添加剤, キャピラリー洗浄法, 試料注入法について, タンパク質の移動時間,ピーク面積の再現性を指標として検討した. 電解液として100mMホウ酸ナトリウム (pH10.0), 洗浄液として0.1Mリン酸水溶液 (pH2.5), 泳動ごとに洗浄液と電解液を交互に5分間送液してキャピラリーの初期化を行い, 再現性の良い分離分析が達成できた. 使用したモデルタンパク質は, チトクロムcを除き, 等電点と相関する移動時間を示した. ヒト血清タンパク質は, 8分以内で分離分析が可能であった. さらに改善すべき点はあるが, 未処理フューズドシリカキャピラリーを用いた電気泳動法は, 微量, 高分離, 迅速, 簡便なタンパク質分析手法として有用であることを確認した.

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キャピラリー電気泳動と先端分析を組み合わせることにより塩基性薬物の非タンパク結合型濃度を解析するための新規超微量分析法を開発した. 電気浸透流が発生しない条件下で, 薬物-血漿タンパク質混合試料をキャピラリーに注入する. 先端分析の原理に基づいて, 試料ゾーンから非結合型薬物ゾーンが移動し, 台形状ピークとして観測される. このプラトー部分の高さから非結合型薬物濃度を求めることができる. 泳動緩衝液中にキラルセレクターを添加して光学活性な薬物のゾーンを光学分割することにより各異性体ごとの非結合型濃度を測定できる. 本分析法の試料注入量は約200nlであり, 従来法の500分の1である. 本分析法を用いて, モデル塩基性薬物とα₁-酸性糖タンパク質(AGP) との結合に及ぼすシアル酸残基の影響を立体選択的かつ定量的に検討した. シアル酸残基はプロプラノロールとAGPとの結合における立体選択性の原因となるが, ベラパミルとの結合に影響しないことが判明した.

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