利用固定化酶柱同时测定胆汁酸

胆汁酸在其steroidal backbone的3a位置具有一个共同的羟基。3a HSD（3a-Hydroxysteroid Dehydrogenase）是一种在辅酶NAD（Nicotinamide Adenine Dinucleotide）存在下使该羟基选择性氧化的酶。在该反应中，当胆汁酸分子被氧化为3-ketosteroid时，产生一个NADH分子（NAD的还原形式），并且其具有强烈的荧光（Ex=340nm，Em=470nm）。在该方法中，使用柱后衍生法测量胆汁酸的标准混合物，以检测通过将含有NAD的反应溶液与柱洗脱液连续混合并使该混合溶液通过3a-HSD酶固定化柱而产生的NADH。Application Note 2/4Simultaneous Determination of Bile Acids Utilizing an ImmobilizedEnzyme Column SimultaneousDetermination of Bile Acid s Uti l izing an lmmobilized Enzyme Column Bi l e acids have a common hydroxyl group located at the 3a position of their steroidal backbone. 3a-HSD (3a -Hydroxysteroid Dehydrogenase) i s an enzyme that causes this hydroxyl group to be selectively oxidized in the presence of the co-enzy m e NAD (Nicotinam i de Adenine Dinucleot i de). In this reaction， when a molecule of b i le acid is oxidized to 3-ketosteroid one NADH molecule (the reduced form of NAD) is generated and it has i ntense f luorescence (Ex=340 nm，Em=470 nm). In this method a standard mixture of bile acids was measured us i ng post-co l umn derivatization for detecting NADH generated by continuously mixing the reaction solution containing NAD with the column eluent and passing t his mixed solution through a 3a-HSD enzyme-immobilized column. JASCO HPLC System www .jas co in c .com 28600 Mary's Court， Easton， MD 21601 USA Conditions Column： Bilepak-II (4.6 mml.D.x125 mmL， 5 um) Enzyme Column： Enzymepak 30-HSD (4.0mml.D. x20 mmL) Eluent A： 30 mM Ammonium acetatebuffer (pH 6.8)/Acetonitrile/Methanol (60/20/20) Eluent B： 30 mM Ammonium acetatebuffer (pH 6.8)/Acetonitrile/Methanol (40/30/30) Gradient Condition： (A/B)， 0 min (100/0) 32 min (0/100)60 min (0/100)60.1 min (100/0) 1 cycle；80 min Flow Rate： 1.0 mL/min Reagent： 0.3 mM NAD，1mMEDTA-2Na， 0.05% 2-mercaptoethanol， 10 mM potassium dihydrogenphosphate， pH7.8 (adjusted with potassium hydroxide) Reagent Flow Rate： 1.0mL/min Column Temp.： 25℃ Wavelength： Ex. 345 nm， Em. 470 nm，Gain 100x Injection Volume： 10 uL Standard Sample： 15 Bile acids (50 umol/mLeach) Application Note Fig. 1 shows the enzyme reaction for the oxidation of bile acid s and reduc t ion of NAD. F i g. 2 shows a f low diagram for the system used to analyze the bile acids. Fig . 1. Mechanism of Enzyme Reaction Fig. 2. Flow Diagram No.1： E Eluent No.2： F PU-2089No.3： C Cooled Autosampler (AS-2057)No.4： C Column oven (CO-2060)No.5： C Column (Bilepak Il)No.6：E Enzyme colu m n (Enzymepak 3a-HSD) No.7：7Reag e nt No.8：3 Reagent pump (PU-2080) No.9：：Fluorescence Detector (FP-2020) No.10： Backpressure coil Results Fig. 3 shows the chromatogram of a standard mixture of 15 bile acids and an i n ternal standard (I .S.)， which were well separated i n under 50 minutes. Fi g. 3. Chromat og ram of the Standard Sam p le of Mixed Bile Acids JASCO INC. Application Note Table 1. Shows the retention t ime and peak area reproducibi l ity of each bile acid when a 0.5 nmol standard mixture of bile ac i ds (injection volume： 10 pL) (n =10). The %RSD of retention t i m e and peak area for each component obtained was 0.2 %~0.34% and 0.8%~2.13% respectively. Table 1. Reproducibility (n =10) Bile acid %RSD Retention time Peak area GUDCA 0.34 1.43 TUDCA 0.33 1.25 UDCA 0.28 1.47 GCA 0.33 1.36 TCA 0.33 1.16 CA 0.26 1.5 GCDCA 0.24 2.04 TCDCA 0.23 2.13 GDCA 0.24 1.8 TDCA 0.22 1.17 CDCA 0.2 2.11 DCA 0.2 1.16 GLCA 0.22 0.8 TLCA 0.23 0.89 LCA 0.29 1.38