Simultaneous Determination of Bile Acids Utilizing an Immobilized Enzyme Column
January 5, 2024
Introduction
![JASCO HPLC System](https://jascoinc.com/wp-content/uploads/2013/10/LPgradient.jpg)
Bile acids have a common hydroxyl group located at the 3α position of their steroidal backbone. 3α-HSD (3α-Hydroxysteroid Dehydrogenase) is an enzyme that causes this hydroxyl group to be selectively oxidized in the presence of the co-enzyme NAD (Nicotinamide Adenine Dinucleotide). In this reaction, when a molecule of bile acid is oxidized to 3-ketosteroid one NADH molecule (the reduced form of NAD) is generated and it has intense fluorescence (Ex=340 nm, Em=470 nm). In this method a standard mixture of bile acids was measured using post-column derivatization for detecting NADH generated by continuously mixing the reaction solution containing NAD with the column eluent and passing this mixed solution through a 3α-HSD enzyme-immobilized column.
Experimental
Equipment
Eluent Pump: | PU-2089 |
Reagent Pump: | PU-2080 |
Column Oven: | CO-2060 |
Autosampler: | AS-2057 |
Detector: | FP-2020 |
Conditions
Column: | Bilepak-II (4.6 mmI.D. x 125 mmL, 5 µm) |
Enzyme Column: | Enzymepak 3?-HSD (4.0 mmI.D. x 20 mmL) |
Eluent A: | 30 mM Ammonium acetate buffer (pH 6.8)/Acetonitrile/Methanol (60/20/20) |
Eluent B: | 30 mM Ammonium acetate buffer (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, 1 mM EDTA-2Na, 0.05% 2-mercaptoethanol, 10 mM potassium dihydrogenphosphate, pH 7.8 (adjusted with potassium hydroxide) |
Reagent Flow Rate: | 1.0 mL/min |
Column Temp.: | 25 ºC |
Wavelength: | Ex. 345 nm, Em. 470 nm, Gain 100x |
Injection Volume: | 10 µL |
Standard Sample: | 15 Bile acids (50 µmol/mL each) |
Fig. 1 shows the enzyme reaction for the oxidation of bile acids and reduction of NAD
Fig. 2 shows a flow diagram for the system used to analyze the bile acids.
![](https://jascoinc.com/wp-content/uploads/2013/10/Simultaneous-determination-of-bile-acids-fig-1.jpg)
![](https://jascoinc.com/wp-content/uploads/2013/10/Simultaneous-determination-of-bile-acids-fig-2.jpg)
No.1: Eluent | No.5: Column (Bilepak II) |
No.2: PU-2089 | No.6: Enzyme column (Enzymepak 3a-HSD ) |
No.3: Cooled Autosampler (AS-2057) | No.7: Reagent |
No.4: Column oven (CO-2060) | No.8: Reagent pump (PU-2080) |
No.9: Fluorescence detector (FP-2020) | |
No.10: Backpressure coil |
Keywords
220052HRE, Bile acids, NAD, NADH, Enzymepak 3α-HSD, Bilepak-II, Fluorescence detector
Results
Fig. 3 shows the chromatogram of a standard mixture of 15 bile acids and an internal standard (I.S.), which were well separated in under 50 minutes.
![](https://jascoinc.com/wp-content/uploads/2013/10/Simultaneous-determination-of-bile-acids-fig-3.jpg)
Table 1. Shows the retention time and peak area reproducibility of each bile acid when a 0.5 nmol standard mixture of bile acids (injection volume: 10 µL) (n=10). The %RSD of retention time and peak area for each component obtained was 0.2 %~ 0.34% and 0.8% ~ 2.13% respectively.
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![JASCO Application Note](https://jascoinc.com/wp-content/uploads/2023/10/app-pdf-header-2.png)
Simultaneous Determination of Bile Acids Utilizing an Immobilized Enzyme Column
Introduction
![JASCO HPLC System](https://jascoinc.com/wp-content/uploads/2013/10/LPgradient.jpg)
Bile acids have a common hydroxyl group located at the 3α position of their steroidal backbone. 3α-HSD (3α-Hydroxysteroid Dehydrogenase) is an enzyme that causes this hydroxyl group to be selectively oxidized in the presence of the co-enzyme NAD (Nicotinamide Adenine Dinucleotide). In this reaction, when a molecule of bile acid is oxidized to 3-ketosteroid one NADH molecule (the reduced form of NAD) is generated and it has intense fluorescence (Ex=340 nm, Em=470 nm). In this method a standard mixture of bile acids was measured using post-column derivatization for detecting NADH generated by continuously mixing the reaction solution containing NAD with the column eluent and passing this mixed solution through a 3α-HSD enzyme-immobilized column.
Experimental
Equipment
Eluent Pump: | PU-2089 |
Reagent Pump: | PU-2080 |
Column Oven: | CO-2060 |
Autosampler: | AS-2057 |
Detector: | FP-2020 |
Conditions
Column: | Bilepak-II (4.6 mmI.D. x 125 mmL, 5 µm) |
Enzyme Column: | Enzymepak 3?-HSD (4.0 mmI.D. x 20 mmL) |
Eluent A: | 30 mM Ammonium acetate buffer (pH 6.8)/Acetonitrile/Methanol (60/20/20) |
Eluent B: | 30 mM Ammonium acetate buffer (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, 1 mM EDTA-2Na, 0.05% 2-mercaptoethanol, 10 mM potassium dihydrogenphosphate, pH 7.8 (adjusted with potassium hydroxide) |
Reagent Flow Rate: | 1.0 mL/min |
Column Temp.: | 25 ºC |
Wavelength: | Ex. 345 nm, Em. 470 nm, Gain 100x |
Injection Volume: | 10 µL |
Standard Sample: | 15 Bile acids (50 µmol/mL each) |
Fig. 1 shows the enzyme reaction for the oxidation of bile acids and reduction of NAD
Fig. 2 shows a flow diagram for the system used to analyze the bile acids.
![](https://jascoinc.com/wp-content/uploads/2013/10/Simultaneous-determination-of-bile-acids-fig-1.jpg)
![](https://jascoinc.com/wp-content/uploads/2013/10/Simultaneous-determination-of-bile-acids-fig-2.jpg)
No.1: Eluent | No.5: Column (Bilepak II) |
No.2: PU-2089 | No.6: Enzyme column (Enzymepak 3a-HSD ) |
No.3: Cooled Autosampler (AS-2057) | No.7: Reagent |
No.4: Column oven (CO-2060) | No.8: Reagent pump (PU-2080) |
No.9: Fluorescence detector (FP-2020) | |
No.10: Backpressure coil |
Keywords
220052HRE, Bile acids, NAD, NADH, Enzymepak 3α-HSD, Bilepak-II, Fluorescence detector
Results
Fig. 3 shows the chromatogram of a standard mixture of 15 bile acids and an internal standard (I.S.), which were well separated in under 50 minutes.
![](https://jascoinc.com/wp-content/uploads/2013/10/Simultaneous-determination-of-bile-acids-fig-3.jpg)
Table 1. Shows the retention time and peak area reproducibility of each bile acid when a 0.5 nmol standard mixture of bile acids (injection volume: 10 µL) (n=10). The %RSD of retention time and peak area for each component obtained was 0.2 %~ 0.34% and 0.8% ~ 2.13% respectively.