Intended Use
The Immuchrom Bile Acids Assay Kit is photometric test that enables easy, rapid and precise quantitative determination of free bile acids in human stool samples. For research use only.
Description
Bile acids play a significant role in the absorption of lipids and fat-soluble vitamins in the small intestine, as well as in the excretion of cholesterol. Furthermore, they contribute to the stimulation of intestinal motility.
Bile acids function, among other things, in the digestion of fats, are formed in the liver and released into the duodenum with the bile. In the last section of the small intestine, about 95-97% of the bile acids are reabsorbed (enterohepatic circulation).
If the absorption of bile acids in the small intestine is impaired, they are increasingly excreted in the stool, which leads to irritation of the colon and causes severe diarrhea, resulting in severe fluid loss as well as vitamin and mineral deficiencies. This is called bile acid malabsorption syndrome or chologenic diarrhea.
The ImmuChrom bile acids kit allows an easy, rapid and precise quantitative determination of bile acids in stool samples. The kit includes all reagents ready to use for preparation of the samples.
Type
Photometric assay
Other names
steroid carboxylic acids, cholic acid, chenodeoxycholic acid
Principle of Method
The quantitative determination of bile acids is carried out via their enzymatic oxidation to 3-keto steroids with the formation of thio-NADH. The resulting change in absorbance (ΔOD) at 405 nm is determined photometrically. The concentration can be calculated from the standard curve.
Sample Types
Stool
Sample Volume
Extraction in stool extraction vials
The bile acids are extracted by the extraction buffer out of the stool sample. Extraction in stool extraction vials In a stool sample extraction vial mix 15 mg stool with 1.5 ml EXT, then vortex it until the mixture is homogenous. Transfer the resulting slurry to a plastic vial and centrifuge it for 10 min at 3000xg.
The supernatant is directly pipetted into the microtiter plate wells with no further dilution.
Standard Curve
Calibration Range: 9 - 280 µmol/L
Limit of Detection
1.91 µmol/L
For the determination, the zero-standard was measured 12 times. The 3-fold standard deviation was added to the mean value. The respective concentration was read from the standard curve.
Precision and reproducibility
Intra-Assay (Within-Run) CV:
- 2.5 % (508 μmol/l) [n = 10]
- 4.7 % (2248 μmol/l) [n = 10]
- 10.7 % (6263 μmol/l) [n = 10]
Inter-Assay (Run-to-Run) CV:
- 7.6 % (466 μmol/l) [n = 10]
- 9.9 % (1787 μmol/l) [n = 10]
- 13.6 % (6281 μmol/l) [n = 10]
Dilution Linearity
| Test sample | Dilution |
Expected Concentration (μmol/l) |
Measured Concentration (μmol/l) |
Recovery (%) |
| 1 |
- | - |
17480 |
- |
| 1 | 1:1.3 |
13539 |
10760 |
79.5 |
| 1 | 1:1.5 |
11787 |
8983 |
76.2 |
| 1 | 1:1.7 |
10448 |
7466 |
71.5 |
| 1 |
1:2 | 8941 |
5625 |
62.9 |
| 1 |
1:3 |
6095 |
3683 |
60.4 |
| 1 |
1:4 |
4671 |
2838 |
60.8 |
| 2 | - |
- |
5235 |
- |
| 2 | 1:1.3 | 4120 | 3358 | 81.5 |
| 2 | 1:1.5 | 3624 | 2709 | 74.8 |
| 2 | 1:1.7 | 3245 | 2410 | 74.3 |
| 2 | 1:2 | 2818 | 1961 | 69.6 |
| 2 | 1:3 | 2013 | 1353 | 67.2 |
| 2 | 1:4 | 1610 | 1063 | 66.0 |
| 3 | - | - | 1043 | - |
| 3 | 1:1.3 | 895 | 783 | 87.5 |
| 3 | 1:1.5 | 829 | 754 | 90.9 |
| 3 | 1:1.7 | 779 | 700 | 89.8 |
| 3 | 1:2 | 723 | 675 | 93.5 |
| 3 | 1:3 | 616 | 605 | 98.3 |
| 3 | 1:4 | 562 | 577 | 103.0 |
Spiking Recovery
| Test sample | Endogenous (μmol/l) | Spike (μmol/l) |
Expected Concentration (μmol/l) |
Measured Concentration (μmol/l) |
Recovery (%) |
| 1 |
744 | 734 |
1478 |
1282 |
86.7 |
| 1 | 744 | 3670 |
4415 |
4619 | 105 |
| 1 | 744 | 7341 |
8085 |
7208 |
89.2 |
| 2 |
2223 | 1043 |
3266 |
3027 |
92.7 |
| 2 | 2223 | 5215 | 7438 | 6528 | 87.8 |
| 2 | 2223 | 10430 | 12653 | 11460 | 90.6 |
| 3 | 6175 | 1043 | 7218 | 6591 | 91.3 |
| 3 | 6175 | 5215 | 11390 | 10800 | 94.8 |
| 3 | 6175 | 10430 | 16605 | 15730 | 94.7 |
Storage
Store the kit refrigerated at 2-8 °C.
Intended Use
This assay is intended for research use only and is not for use in diagnostic procedures.
Resources
References to Summary
- Neumeister, B.: Klinikleitfaden Labordiagnostik. Elsevier Urban & Fischer, München 2009
- Pschyrembel. Klinisches Wörterbuch. 266. Auflage. De Gruyter, Berlin 2014
- Stephen D. Turley and John M. Dietschy: Re-evaluation of the 3α-hydroxysteroid dehydrogenase assay for total bile acids in bile; Journal of Lipid Research Volume 19 Notes on Methodology 1978: 924-928
- Priya Vijayvargiya, Michael Camilleri, Andrea Shin and Amy Saenger: Methods for Diagnosis of Bile Acid Malabsorption in Clinical Practice; CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2013; Vol. 11: 1232–1239
- Jack L. Porter, John S. Fordtran, Carol A. Santa Ana, Michael Emmett, Lee R.Hagey, Edith A. MacDonald, and Alan F. Hofmann: Accurate enzymatic measurement of fecal bile acids in patients with malabsorption; JOURNAL OF LABORATORY AND CLINICAL MEDICINE, July 2003 (https://www.researchgate.net/publication/10696339
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Ilex Life Sciences LLC is an official distributor of Immuchrom GmbH.