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Letters to the Editor |
-Hydroxycholest-4-en-3-one, a Marker of Bile Acid Malabsorption
1 Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Czech Republic
2 4th Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague, Czech Republic
3 Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
4 IBD Clinical and Research Center, ISCARE I.V.F. Lighthouse, Prague, Czech Republic
aAddress correspondence to this author at: Laboratory of Hepatology, UKBLD, U Nemocnice 2, 12808 Prague 2, Czech Republic, Fax +420-22496-2532, e-mail mleni{at}centrum.cz
To the Editor:
Serum concentrations of 7
-hydroxycholest-4-en-3-one (cholesten, bile acid synthesis intermediate) have been shown to correlate with the severity of bile acid malabsorption(1)(2). Current techniques for holster quantification require sophisticated instrumentation(3) or solid-phase extraction (SPE) at 64 °C(4). We developed and validated a method for measuring serum cholesten concentrations using routinely available HPLC instrumentation, focusing on the optimization of the SPE step.
Cholesten was purchased from Steraloids, and used to prepare serum calibrators containing 0–1000 µg/L. We added 30 ng of internal standard (7β-hydroxycholest-4-en-3-one, Steraloids) dissolved in 80 µL of methanol, to 1 mL of serum. Then 5 mL of chloroform:methanol (2:1, vol/vol, analytical grade, Penta) was added; the mixture was vortex-mixed vigorously, and centrifuged (2000g, 3 min, ambient temperature). The upper phase was discarded, and 2 mL of 125 mmol/L NaCl in 50% methanol (vol/vol) was added to the sample, vortex-mixed, and centrifuged as above. The lower phase was transferred to another tube and dried at 60 °C under nitrogen, dissolved in 1 mL of toluene (analytical grade, Penta), and loaded onto a Phenomenex Strata SI-1 100-mg silica precolumn that had been prewashed with 1 mL of isopropanol (Chromasolv, Merck), and equilibrated with 1 mL of hexane (Uvasol, Merck). After a washing step with 1 mL of hexane and 15 mL of isopropanol:hexane (0.4:99.6 vol/vol), cholesten was eluted with 1 mL of isopropanol, dried under nitrogen at 60 °C, and dissolved in 170 µL of acetonitrile:water (95:5 vol/vol). Then 150 µL of the sample was injected into the Agilent HP1100 HPLC system. The chromatographic parameters were: Tessek SGX C18 column (4 x 250 mm, 4 µm), acetonitrile:water (95:5, vol/vol) mobile phase, flow rate 1 mL/min, temperature 20 °C, detection/reference wavelength 241/360 nm(4).
The method yielded a linear response (13 calibration points in triplicate) up to 1000 µg/L (y = 0.9957x, R2 = 0.9979). A typical chromatogram is shown in Fig. 1
. The detection limit, calculated as a concentration corresponding to a signal 3 SD above the mean for a calibrator free of analyte (n = 10), was 1.2 µg/L when 1 mL of serum was processed. Intraassay imprecision values (CV for 15 measurements of 3 specimens) were 2.8%, 3.2%, and 2.2% for samples with mean (SD) cholesten concentrations of 18.1 (0.5), 136.8 (4.3), and 237.7 (5.3) µg/L, respectively. We determined interassay imprecision by assaying 3 specimens 20 times (1 measurement per day) over a 3-month period. CVs were 5.1%, 4.3%, and 4.1% for samples with mean (SD) cholesten concentrations of 18.0 (0.9), 139.2 (6.0), and 244.4 (9.9) µg/L, respectively.
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The average recovery, calculated as [(measured concentration – initial concentration)/added concentration], was 93%. The effects of hemoglobin, bilirubin, cholesterol, and triglycerides were estimated by the recovery of a known amount of analyte added to a serum sample with the interferent being tested. Recoveries (means of triplicates) were 97%, 90%, 108%, and 105% with added hemoglobin (5 g/L), bilirubin (96.2 µmol/L), cholesterol (9.1 mmol/L), or triglycerides (10.7 mmol/L), respectively.
To demonstrate the utility of this method, we measured serum cholesten concentrations in 2 groups: healthy volunteers [20 males, 30 females, mean (SD) age 39.6 (9.4) years] and patients who had undergone resection of terminal ileum, for whom malabsorption of bile acids would be expected owing to the loss of ileal bile acid transporter [22 males, 28 females, mean (SD) age 41.7 (13.5) years]. The study was approved by the local ethics committee. Compared to the healthy controls, median concentrations of cholesten (interquartile range) in patients with ileal resection were significantly higher [87.8 µg/L (range 42.1–150.5 µg/L) vs 11.9 µg/L (range 9.2–16.9 µg/L), P < 0.001, Mann–Whitney Rank-Sum test].
This method retains the advantages of other HPLC-based methods, while eliminating the need for temperature-controlled SPE. Silica cartridges offer higher binding capacity and the possibility of analyte extraction at ambient temperature, which, compared to previously used C8 cartridges, results in a wider linear range (up to 1000 µg/L vs 200 µg/L)(4). Except for the chloroform:methanol extraction, the analysis can theoretically be automated. Additionally, the initial chloroform:methanol extraction ensures quantitative extraction of both cholesten and the internal standard and thus potential imprecision caused by either internal standard precipitation or incomplete extraction of protein-bound cholesten can be avoided.
The increased availability of a laboratory diagnosis of bile acid malabsorption is of considerable importance, especially for patients with chronic diarrhea and irritable bowel syndrome. These disorders belong to the most common gastrointestinal conditions, and it is estimated that bile acid malabsorption might be present in about half of these patients(5). Because the majority of patients wit bile acid malabsorption respond to bile acid sequestrants(5), targeted therapy, based on serum cholesten concentrations, should both improve outcomes and lower treatment costs.
Acknowledgments
Grant/Funding Support: This work was supported by a grant NR 8963-3 from the Czech Ministry of Health.
Financial Disclosures: None declared.
Acknowledgments: We give our thanks to Iva Subhanova, for helpful advice and assistance in the validation procedure.
References
The following articles in journals at HighWire Press have cited this article:
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  M. Lenicek, V. Komarek, M. Zimolova, J. Kovar, M. Jirsa, M. Lukas, and L. Vitek CYP7A1 promoter polymorphism -203A>C affects bile salt synthesis rate in patients after ileal resection J. Lipid Res., December 1, 2008; 49(12): 2664 - 2667. [Abstract] [Full Text] [PDF]
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