Aug 11, 1989 - Berens KL, Vadiei K, Brunner U, Wasan KM, Luke DR. Influence of lovastatin on ..... Perry B, Doumas BT, Buffone G, Glick M, Ou CN, Ryder K.
tics, 4th ed. Baltimore: Williams & Wilkins, 1988:229-42. 2. Huang WY, Lipsey Al, Cheng MH. Comparison of cyclosporine determinations in whole blood by three different methods. Am J Clin Pathol 1987;87:528-32.
3. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345-53. 4. Brunner U, Vadiei K, Luke DR. Cyclosporine disposition in the hyperlipidemic rat model. Res Commun Chem Pathol Pharmacol 1988;59:339-48.
5. Berens KL, Vadiei K, Brunner U, Wasan KM, Luke DR. Influence of lovastatin on the pharmacokinetics, toxicity, and immunologic response of cyclosporine in the obese Zucker rat. Biopharm Drug Dispos, in press. 6. Sgoutas D, MacMahon W, Love A, et a!. Interaction of cyclosporin A with human lipoproteins. J Pharm Pharmacol 1986;38:583-8.
7. LeMaire M, TillementJP. Role of lipoproteins and erythrocytes in the in vitro binding and distribution of cyclosporin A in the blood. J Pharm Pharinacol 1982;34:715-8.
CLIN. CHEM. 36/2, 331-333 (1990)
Simple Enzymatic Assay for Determining Cholesterol Concentrations in Bile CIndIeM. Luhman,Stewart1. Galloway,and DonaldC. Beltz1 We use bilirubin oxidase (EC 1.3.3.5) to remove interference by bilirubin in the assay of cholesterol concentration in bile by standard enzymatic methods. Samples are treated for 10 mm with nonlimiting amounts of biliru bin oxidase to form biliverdin from bilirubin before the reagent for cholesterol is added. The relatively small interference by biliverdin is easily eliminated by use of sample blanks. The method is simple, convenient, and not hampered
by the “chromogen
oxidase”
activity
(the
inherent ability of bilirubin oxidase to oxidize some chromogens) that plagues other assays of thistype. Using this assay, we have accurately and precisely determined the concentration of cholesterol in bile. Such elimination of bihrubin will also be useful in assays of other biliary constituents or constituents of urine or icteric plasma. AddItIonal Keyphrases: analytical error bilirubin oxidase
.
enzymatic methods
bilia,y cholesterol
Bile salts, phospholipids (primarily phosphatidylcholine), cholesterol, and proteins are the major organic solutes in bile (1). The bilirubin also present gives bile its characteristic color. Unfortunately, this color interferes with several spectrophotometric assays used to quantify bile constituents. Moreover, bilirubin can serve as a competitor in the peroxidase reaction by reacting with hydrogen peroxide; as a result, formation of the chromogens is incomplete, resulting in errors in the assay. Although the primary mechanism for disposal of cholesterol from the body is conversion to bile acids, cholesterol in bile also represents an excretory route for excess cholesterol in the body via the feces (2). The concentration of cholesterol in bile is an important determinant of the proportion of cholesterol dissolved in bile. The tendency of cholesterol gallstones either toward growth or dissolution depends largely on the amount of dissolved cholesterol in bile: as the concentration of cholesterol in bile increases, formation of cholesterol gallstones is more likely if the concentration of other bile components
Nutritional Physiology Group, Department of Animal Science, Iowa State University, Ames, LA 50011. 1 Author for correspondence. Received August 11, 1989; accepted November 13, 1989.
remains unchanged (3). Biliary cholesterol also has been implicated in a protective mechanism for the gall bladder, preventing damage induced by bile salts to the mucosal lining of the gall bladder, the common, hepatic, and cystic bile ducts, and the small intestine (4). Analytical techniques currently used for determining concentrations of cholesterol in bile are primarily gasliquid chromatographic procedures (5), laborious and time consuming. Bleaching with ultraviolet light for 24 h is also commonly used to remove bilirubin from bile or other compounds. This obviously time-consuming method also includes risks of sample contamination, evaporation, or spillage. We report here an enzymatic assay, similar to that reported by Fromni et al. (6) but more convenient and time saving, for determining biliary cholesterol. Interference by bilirubin in peroxidase-coupled enzymatic procedures involving spectrophotometric measurement of hydrogen peroxide is common (7,8) and can cause erroneous results. Our procedure eliminates interference from biirubin, and it is potentially applicable to other analyses, such as quantifying compounds other than cholesterol in bile, urine, and icteric plasma.
Materials and Methods Samples. Hepatic bile was obtained from pigs in which common bile duct and duodenal catheters had been implanted. Samples were stored at -20 #{176}C in screw-top vials and thawed at room temperature just before use. Chemicals and enzymes. All chemical reagents were analytical grade. Bilirubin oxidase (from Myrothecium species; EC 1.3.3.5), lauryl sulfate (sodium salt, 99% pure), L-ascorbic acid, Diagnostic Kit 352 for total serum cholesterol, cholesterol standard (Accutrol calibrator A2034), and cholesterol calibrators (C0534) were all from Sigma Chemical Co., St. Louis, MO. Apparatus. A Model 2600 spectrophotometer (Gilford Instrument Laboratory, Inc., Oberlin, OH) was used for all spectrophotometric analyses. Reagent preparation. Bilirubin oxidase, diluted in distilled, de-ionized water to give a final concentration of 223 kUIL, was stable for at least 10 h in an ice bath. The pH 7.0 reaction buffer used in the assay was 100 mmol/L phos-
CLINICAL CHEMISTRY, Vol. 36, No. 2, 1990 331
phate buffer containing 10 mmol of lauryl sulfate per liter. Stored at 4 #{176}C, this buffer was stable for at least two weeks. The cholesterol reagent was prepared according to the manufacturer’s directions by adding distilled, de-ionized water. To obtain blank values, we added ascorbic acid to w duplicate samples to inhibit cholesterol oxidase in the 0 z reagent mix. The ascorbic acid was dissolved in 0.5 mol/L NaCl, and the pH of the solution was adjusted to 7.0. 0 A standard curve was prepared with Sigma cholesterol Cl) calibrators. Adding bilirubin oxidase to these standards produced no deleterious effects; the standard curves were similar to those obtained when bilirubin oxidase was not added. We measured the cholesterol in porcine bile 20 mm after pretreatment with bilirubin oxidase. Procedure. Pipet 30 L of bile or 10 jiL of standard into four 12 x 75 mm test tubes. Add 20 L of reaction buffer WAVELENGTH (nm) and 30 jL of bilirubin oxidase solution (final concentration, 7 U per tube) to all tubes. Vortex-mix and incubate at 37#{176}C Fig. 1. Spectra of porcine hepatlc bile samples with (A)and without ( treatment with bilirubin oxidase for 10 mm. After incubating, add 50 .&L of the ascorbate solution to two of the tubes (these will be the blanks). Then add 1 mL of cholesterol reagent to all tubes, vortex-mix, used in assays that measure concentrations of biliary and incubate for 10 mm at 37 #{176}C. Determine the absorconstituents in bile; thus, our procedure is unique. bance of the mixture at 505 nm vs water as the blank. Table 1 shows data for the linear regression analysis of a Determine the net absorbance values of the sample (substandard curve from cholesterol calibrators (four standards tract the blank absorbance values from the sample absorin duplicate) and similar curves developed from cholesterol bance values) and read the concentration of cholesterol calibrator plus 10 and 20 p.L of porcine bile. Curves from from the standard curve. calibrator plus bile parallel the standard curve from 0 to 4 g/L and are linear over the same range of cholesterol Results and DiscussIon concentrations. Regression coefficients (r) and coefficients of variation (CV) for the curves also are shown in Table 1. This use of bilirubin oxidase to pretreat samples for Regression coefficients in the range of 0.998 substantiate biliary cholesterol assay is based on tue method of Allain et linearity, and slopes of 0.015 to 0.016 indicate that the lines al. (9), with modifications by Sigma Diagnostics, and with are parallel. Thus, the assay has good repeatability. The use of Sigma Diagnostics’ cholesterol reagent after removal assay was linear through sample sizes ranging from 0 to 40 of interference from bilirubin. The cholesterol assay rez.L of bile (concentration of cholesterol in bile from 0 to 3.6 agent includes cholesterol esterase, cholesterol oxidase, mg/dL) with a slope of 0.009 and a regression coefficient of and peroxidase (9). The chromophores 4-aminoantipyrine 0.997. and p-hydroxybenzenesulfonate in the presence of hydroOur laboratory has measured cholesterol concentration gen peroxide and peroxidase yield a quinoneimine dye that in bile from 66 pigs in two different trials (13,14).The pigs has an absorbance maximum at 505 nm. The intensity of were fed diets varying in type and amount of fat and in the color produced is directly proportional to the total amount of calcium and vitamin D3. The mean concentraconcentration of cholesterol in the sample. tion of biliary cholesterol was 0.47 gIL, and it did not vary Bilirubin has a peak absorbance at 440 nm but also significantly with diet. absorbs some at 505 nm. This so interferes with the assay The precision of the method was determined by assaying that use of blank samples is insufficient, not only because of a commercial cholesterol calibrator that contained 1.64 g of its color but also because bilirubin interferes competitively cholesterol per liter (Accutrol, Sigma Chemical Co.). We in peroxidase-coupled reactions. We used bilirubin oxidase used results from 21 separate runs, performed during seven to remove this interference by facilitating the conversion of days, to determine the results shown in Table 2. The mean bilirubin to biliverdin, a compound that absorbs at a value for all samples was 1.64 g/L. Means for all runs maximum of 320 nm and thus does not interfere at 505 nm during a day ranged from 1.77to 1.53g/L. Per-run means to the extent that bilirubin does. Figure 1 illustrates the spectra of hepatic porcine bile, either treated or not treated with bilirubin oxidase. The sample’s absorbance in the 505-nm range is substantially decreased and is readily corrected with a sample blank. Bilirubin oxidase has been used to measure bilirubin directly (10, 11) and to remove interference caused by bilirubin when creatinine is measured in serum and urine (12). Artiss et al. (12) noted that pretreatment with bilirubin oxidase resulted in somewhat lower concentrations of apparent creatinine than did the common methods of measurement, because results by commonly used methods are affected by bilirubin interference. Therefore, results obtained by using pretreatment with bilirubin oxidase in spectrophotometric assays may be more nearly accurate. Bilirubin oxidase pretreatment, however, has not been 332 CLINICAL CHEMISTRY, Vol. 36, No. 2, 1990
Table 1. LInear RegressIon Analyses of the Standard Curve and Standards plus Bile for Samples Pretreated wIth BIlIrubIn Oxldase Slop. x 105
Sample
y.lnt.rc.pt Msan
± SEM
r
CV,%
0.998 4.93 0.5 0.007 ± 0.10 10 16 ± 0.3 0.10 ± 0.008 0.999 2.70 20 15 ± 0.6 0.18 ± 0.013 0.997 3.83 a Std.: standard curve (tour standards ranging in cholesterol concentration from0 to 4 g/L);10:standardsplus 10 p1.of addedbile; 20: standards plus20 Std.
15 ±
pL of added bile; n
=
3
each.
Table 2. PrecisIon of Assay with Bile Samplesa Pretreated with Bilirubin Oxldase
Journal Paper No. J-13663 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA (Project No. 2735).
Category
SD, g/L
References
Total Within-run Between-run Within-day Between-day
0.09 0.05 0.07 0.02 0.06
1. Coleman
a
Mean = 1.64 g/L for42 samples in 21 runs over seven days.
ranged from 1.79 to 1.49 g/L. Use of bilirubin oxidase to remove interference of bilirubin in certain chromogen systems has been hampered by the inherent ability of bilirubin oxidase to oxidize the chromogen and thus destroy the ability to absorb spectrophotometrically. Chromogens and chromophores affected by bilirubin oxidase were 4-aminophenazone and tribromohydroxybenzoic acid (15). The chromophore used in our procedure, 4-aminoantipyrine, may be less susceptible to oxidation than are other color-producing compounds. The dye complex was stable for at least 30 mm, so no further precautions were necessary if absorbance was determined within that time frame. Absorbance values were 0.2955 at 0 mm, 0.2958 at 15 mm, and 0.2967 at 30 mm. Our procedure eliminates the need for ferrocyanide (16), 4dimethylammnoantipyrmne (17), or other compounds that have been incorporated into peroxidase-coupled reagent mixtures to obviate oxidation of chromogen complexes that can occur with bilirubin oxidase. Bilirubin interferes in spectrophotometric procedures for quantifying several biliary constituents in bile, including biliary cholesterol. In our procedure, bilirubin oxidase removes this interference by changing bilirubin to biiverdin, which absorbs maximally at a shorter wavelength than does bilirubin and therefore interferes less. In addition, pretreatment with bilirubin oxidase removes interference of bilirubin with hydrogen-peroxide-coupled reactions. The small amount of interference caused by biliverdin can easily be eliminated by use of sample blanks. Our procedure is easy, convenient, and time-efficient and may be readily adaptable for colorimetry of other biliary constituents. This study was partly funded by the National Dairy Board and the Iowa Beef Industry Council. It was administered in cooperation with the National Dairy Council.
R. Biochemistry
of bile
secretion.
Biochem
J
1987;244:249-.61. 2. Sabine JR. Metabolism and excretion. Chap 8 in: Cholesterol. New York: Marcel Dekker, Inc., 1977. 3. Carey MC, Small DM. The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man. J Clin Invest 1978;61:998-1026. 4. Jacyna MR. RossPE, Bouchier LAD. Biliary cholesterol-friend or foe? Q J Med 1986;61:991-.6. 5. Bailey E, Brooks AGF. Improved methods for the determination of the major neutral steroids and unconjugated bile acids in human faeces using capillary gas chromatography. J Chromatogr 1987;421:21-31. 6. Fromm H, Amin P, Klein H, Kupke I. Use of a simple enzymatic assay for cholesterol analysis in human bile. J Lipid Res 1980; 21:259-61. 7. Witte DL, Brown LF, Feld RD. Effectsof bilirubin on detection of hydrogen peroxide by use of peroxidase. Clin Chem 1978;
24:1778-82. 8. Glick MR, Ryder KW, Jackson SA. Comparisons of clinical chemistry: response to interfering substancesas shown by analytespecific “interferograms” [Abstract]. Clin Chem 1985;31:1015-6. 9. Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol.Cliii Chem 1974; 20:470-5. 10. Perry B, Doumas BT, Buffone G, Glick M, Ou CN, Ryder K. Measurement of total bilirubin by use of bilirubin oxidase. Clin Chem 1986;32:329-32. 11. Mullon CJP, Langer R. Determination of conjugated and total bilirubin in serum of neonates, with use of bilirubin oxidase. Clin Chem 1987;33:1822-5. 12. Artiss J, McEnrow RJ, Zak B. Bilirubin interference in a peroxidase-coupled procedure for creatinine eliminated by bilirubin oxidase. Clin Chem 1984;30:1389-92. 13. Luhman CM, Faidley TD, Galloway ST, Foley MK, Beit.z DC. Influence of dietary fat on biliary cholesterol and bile acid secretion in swine [Abstract]. FASEB J 1989;3:A957. 14. Galloway ST, Foley MK, Luhman CM, Faidley TD, Beitz DC. Effects of dietary calcium (Ca) and vitamin D3 (vit D) on bile in pigs [Abstract]. FASEB J 1989;3:A957. 15. Maguire GA. Elimination of the “chromogen oxidase” activity of bilirubin oxidase added to obviate bilirubin interference in hydrogen peroxide/peroxidase detecting systems. Clin Chem 1985;31:2007-8. 16. Spain MA, Wu AHB. Bilirubin interference with determination of uric acid, cholesterol,and triglycerides in commercial peroxidase-coupled assays, and the effect of ferrocyanide. Clin Chem 1986;32:518-21. 17. Zoppi F, DeLuco U, Manso G, Fenili D. Suppression of chemical interference of bilirubin in Barham-Trinder reaction for direct uric acid determination in serum. Lab J Res Med 1981;8:481-6.
CLINICAL CHEMISTRY, Vol. 36, No. 2, 1990 333
