Discrete analysis of bile acid in serum and bile with 3α-hydroxysteroid dehydrogenase and diaphorase immobilized onto alkylamine glass beads

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1 Indian Journal of Biochemistry & Biophysics Vol. 43, April 2006, pp Discrete analysis of bile acid in serum and bile with 3α-hydroxysteroid dehydrogenase and diaphorase immobilized onto alkylamine glass beads Kirti Rani, P Garg and C S Pundir* *Biochemistry Research Laboratory, Department of Bio-Sciences, M.D. University, Rohtak , Haryana Department of Surgery, Pt BDS P.G.I.M.S., Rohtak , Haryana Received 18 July 2005; revised 31 January α-Hydroxysteroid dehydrogenase (3α-HSD) from Pseudomonas testosteronei and diaphorase (lipoyl dehydrogenase) from Clostridium spp were immobilized individually onto alkylamine glass beads through glutaraldehyde coupling. A costeffective enzymic colorimetric method for determination of bile acid in the serum and bile was developed employing mixture of the immobilized enzymes. The method was based upon measurement of NADH generated from NAD + during oxidation of bile acid by immobilized 3α-HSD with a color reagent consisting of nitrobluetetrazolium (NBT) chloride salt and immobilized diaphorase in M sodium phosphate buffer (ph 7.0). The minimum detection limit of the method was 4.8 µmol/l in the serum and 19.5 µmol/l in bile. The per cent recovery of added bile acid in the serum and bile was 89.1 and 95.0, respectively. Within and between batch coefficients of variation (CV) for bile acid determination were <1.0% and <0.2% in the serum and <0.2% and <0.6% in bile, respectively. A good correlation for bile acid in the serum (r 1= 0.95) and in bile (r 2 = 0.93) was obtained by a standard chemical method (a commonly used method in India) and the present method. The mixture of immobilized 3α-HSD and diaphorase lost 30% of its initial activity after 4 months of regular use. The cost of bile acid determination for 100 the serum and bile samples by the present method was found to be lower than by a commercially available method (Sigma kit 450-A) Keywords: Bile acid, 3α-hydroxysteroid dehydrogenase, diaphorase, immobilization, alkylamine glass beads, serum, bile, gallstone Bile acids, the C 24 steriods with 3α-hydroxyl group are synthesized from cholesterol in the liver. Their increased level in serum, under fasting and postprandial state is a specific indicator of liver disease, while decreased level indicates bile acid malabsoption 1. The patients with gallstones have shown significantly high level of bile acids in their bile and serum 2. A number of methods have been used to measure bile acids, such as chemical method 3, direct spectrophotometry 4, gas liquid chromatography (GLC) 5, HPLC 6, fluorimetry 7, radio-immunoassay 8, bioluminescence 9, potentiometry 10, tendem mass spectrometry 11, proton magnetic resonance 12 and electro-kinetic chromatography 13. The most commonly used method for clinical purpose is the enzymic-colourimetric method 14 based on following reactions: (i) 3-α-Hydroxy bile acid + NAD + *Corresponding author Phone: (01262) (R) E mail: pundircs@rediffmail.com 3α ΗSD 3-α-keto bile acid + NADH +H + Diaphorase (ii) NADH+ H + + Chromogen (NBT) Chromophore (Formazon) +NAD + λ max = 540 nm 3-α-HSD = 3α-Hydroxy steroid dehydrogenase; NBT = Nitrobluetetrazolium Although the method is popular and its commercial kit (Sigma) is available, it involves considerable amount of expensive enzymes. Immobilization of the enzymes onto insoluble support combines high selectivity and an increased stability. Besides, immobilized enzymes can be reused, making the method economical for analysis of a large number of clinical samples. 3α-HSD has been immobilized on cellulose beads 15 and co-immobilized alongwith diaphorase onto Sepharose 4B through CNBr, for determination of bile acids in a continuous flow system and HPLC, respectively 16. The enzymes bound to such organic supports are generally less stable and susceptible to fungal attack. However, the enzymes immobilized covalently onto inorganic supports, such as arylamine and alkylamine glass beads have been found comparatively more stable, and resistant to microbial attack

2 RANI et al: DISCRETE ANALYSIS OF BILE ACID IN SERUM AND BILE 99 3α-HSD and diaphorase have been immobilized onto arylamine glass beads for quantitative analysis of bile acid in the serum and bile 18. However, alkylamine glass bound enzymes are easier to prepare and have shown higher retention, conjugation yield and a longer working life span than arylamine glass bound enzymes 20. Earlier, alkylamine glass bound 3α-HSD and diaphorase were used in an HPLC system for determination of serum bile acids 6. However, this method was an expensive method, as it required costly equipment and trained person to operate. In the present report, we describe a cost-effective method for determination of bile acid in the serum and bile using 3α-HSD and diaphorase immobilized onto alkylamine glass beads. Materials and Methods Materials Zirconia-coated alkylamine glass beads (pore diam 55 nm were a gift from Prof H H Weetall, (Environment Protection Group, Las Vegas, USA). Glutaraldehyde (25%), 3α-HSD from Pseudomonas testosteroni (10 U/ 0.75 mg) and diaphorase from Clostridium sp. (33 U/ mg), taurodeoxycholic acid (Sigma Chemical Co., USA), nitrobluetetrazolium chloride salt, NAD + and NADH (SISCO Research Lab., Mumbai) were used. All other chemicals were of analytical reagent grade. Preparation of color reagents for 3α-HSD and diaphorase For preparation of color reagent for 3α-HSD, NAD +, g, NBT, g and 10 U diaphorase per 20 ml of M sodium phosphate buffer (ph 7.0) were mixed and diluted to 45 ml with distilled water at the time of use. For preparation of color reagent for diaphorase, NADH, g and NBT, g were mixed in 20 ml of M sodium phosphate buffer (ph 7.0) and diluted to 25 ml with distilled water at the time of use. The color reagents were stored at 4 C in an amber-colored bottle and prepared fresh every day. Methods Assay of free 3α-HSD It was carried out as described 14. 3α-HSD (0.75 mg containing 10 U) was dissolved in 20 ml sodium carbonate-bicarbonate buffer (0.1 M, ph 9.0) and stored at 4 C. The assay was carried out in a 15 ml conical flask wrapped with a black paper. The reaction mixture contained 0.1 ml dissolved 3α-HSD and 0.5 ml color reagent. The reaction was started by adding 0.2 ml bile acid solution (taurodeoxycholic acid, 200 µmol/l goat serum) and after incubation at 25 C for 15 min, reaction was stopped by adding 0.5 ml stop solution (Triton-X-100; 75 g/l 38% HCl). The control was run in the same manner, except that it contained heated enzyme. A 540 of reaction mixture was read against control in a spectrophotometer (Spectronic-20D, Milton & Roy, USA). Although NADH generated in reaction (i) was consumed in reaction (ii), A 540 of the dye produced in reaction (ii) was directly proportional to NADH concentration. Immobilization of 3α-HSD onto alkylamine glass beads and its assay It was carried out as using described previously 21. To alkylamine glass beads (100 mg) in a 15 ml conical flask, 1.0 ml of 2.5% glutaraldehyde solution in M sodium phosphate buffer (ph 7.0) was added and kept for 2 hr at room temperature (30 C) with occasional stirring. Excess of glutaraldehyde was removed from the flask carefully and the beads were washed with 0.1 M sodium phosphate buffer (ph 7.0) repeatedly until the ph of washing discard was 7.0. An aliquot of 1.0 ml (1.8 Unit) of dissolved 3α-HSD was added to the activated beads and kept for 48 hr at 4 C with occasional shaking. The unbound enzyme was decanted and tested for the activity and protein. The beads were washed with 0.1 M sodium carbonatebicarbonate buffer (ph 9.5), repeatedly until no activity was detected in the washing discard. The beads bound to enzyme were tested for activity and stored in M sodium phosphate buffer (ph 7.0) at 4 C when not in use. The enzyme bound to beads was estimated by determining the loss of protein from the enzyme solution during its immobilization using Lowry method 22. The per cent retention of the enzyme activity was determined as follows: % Retention = Specific activity of immobilized enzyme 100 Specific activity of free enzyme The assay of immobilized 3α-HSD was carried out as described for free enzyme, except that free/ dissolved enzyme was replaced with immobilized enzyme (100 mg alkylamine glass beads bound to 3α- HSD) and reaction mixture was kept under continuous stirring during incubation at 25 C for 15 min. After completion of assay, coloured reaction mixture was withdrawn from the flask with help of an Eppendorff

3 100 INDIAN J. BIOCHEM. BIOPHYS. VOL. 43, APRIL 2006 pipette, avoiding the loss of beads and transferred to a cuvette. A 540 was read. The content of NADH generated in the reaction was determined. One unit of immobilized enzyme was defined as the amount of enzyme bound to glass beads, which generated 1 µmole NADH per min under standard assay conditions. Assay of native/free diaphorase It was carried out as described 14. Briefly, diaphorase (1 mg containing 33 U) was dissolved in 2.0 ml sodium carbonate and bicarbonate buffer (0.1 M, ph 9.0) and the assay was carried out in a 15 ml conical flask, wrapped with black paper. The reaction mixture contained 0.5 ml colour reagent and 0.1 ml diaphorase (0.4 U) was added to start the reaction. The control received the same, except heated diaphorase in boiling water bath for 2 min. After incubation at 25 C for 15 min, 0.5 ml stop solution (prepared by adding 7.5 g Triton-X-100 and 8.3 ml conc. HCl into 50 ml distilled water and final volume made up to 100 ml with distilled water at the time of use) was added. A 540 of reaction mixture was read and NADH utilized in the assay was determined. One unit of diaphorase was defined as the amount of enzyme bound to glass beads, which utilized 1 µmole NADH per min under standard assay conditions. Immobilization of diaphorase onto alkylamine glass beads and its assay Dissolved diaphorase (1 ml containing 3.99 U) was immobilized onto alkylamine glass beads (100 mg) through glutaraldehyde coupling as described for immobilization of 3α-HSD. The enzyme bound to glass beads was estimated by determining the loss of protein from the enzyme solution during immobilization 22. The assay was carried out as described for their native diaphorase, except that free enzyme was replaced by immobilized enzyme (100 mg alkylamine glass beads bound to diaphorase) and reaction mixture was kept under continuous stirring during incubation at 25 C for 15 min. After completion of the assay, reaction mixture was withdrawn from the flask carefully using Eppendorff pipette, to avoid loss of beads and transferred to a cuvette. A 540 was read. Assay of mixture of immobilized 3α-HSD and diaphorase 3α-HSD and diaphorase bound to 100 and 200 mg alkylamine glass beads, respectively were mixed in a 15 ml conical flask and 0.5 ml colour reagent of 3α- HSD, followed by 0.2 ml bile acid solution (200 µmol/l), were added. The rest of procedure was same as described for assay of immobilized 3α-HSD. Kinetic properties of immobilized 3α-HSD and diaphorase The kinetic properties, such as effect of ph, incubation temperature, time of incubation, effect of substrate concentration and calculation of K m and V max from Lineweaver Burk plot, of immobilized 3α-HSD and diaphorase, both individually, as well as in mixture were studied. Preparation of standard curve for bile acid with mixture of immobilized 3α-HSD and diaphorase A standard curve of the bile acid was prepared by varying concentrations (6.25 to 800 µmol/l) of taurodeoxycholic acid (TC), dissolved in the goat serum and the assay was carried out under the optimal assay conditions (for 15 min at 20 C). A plot extrapolated between TC conc. vs A 540 (Fig. 1). Determination of bile acid in serum by mixture of immobilized 3α-HSD and diaphorase Apparently healthy individuals with no gallstones, as confirmed by ultrasound and gallstone patients after cholecystectomy admitted at PGIMS Hospital, Rohtak were chosen in the following age groups: Group I: children below 20 yr; group II: adults between yr; and group III: aged persons >50 yr. The serum level of cholesterol, triglyceride and glucose in these healthy persons was also checked at the hospital. Aliquot of 1.0 ml blood was withdrawn intravenously from the apparently healthy persons and gallstone patients with a sterilized needle and syringe and transferred to a glass vial. After keeping at room temperature for 1 hr, it was centrifuged at 5000 rpm for 10 min. The supernatant (serum) was collected and stored at 4 C until use. The bile acid content in serum was measured in the similar manner as described for standard curve of bile acid, expect that TC solution was replaced by the serum. The concentration of bile acid in serum was extrapolated from standard curve between TC concentration vs A 540 (Fig. 1). Determination of bile acid in bile with alkylamine glass bound 3α-HSD and diaphorase Bile samples from gallstone patients were collected in glass vials during their laproscopic cholecystectomy at local PGIMS Hospital, Rohtak and sterilized and stored at 4 C until use. The content of bile acid in bile was measured as described for serum.

4 RANI et al: DISCRETE ANALYSIS OF BILE ACID IN SERUM AND BILE 101 Reuse and storage of immobilized enzymes To reuse the immobilized enzymes, glass beads were washed with reaction buffer (0.065 M sodium phosphate buffer, ph 7.0) 4-5 times and stored in reaction buffer at 4 C, when not in use. Evaluation of method for bile acid determination Analytical recovery To determine reliability of the method, two concentrations of TC (50 and 200 µmol/l) were added to the serum samples and bile acid content was determined, before and after addition of TC. The per cent recovery of added TC was calculated. Correlation To determine accuracy of the method, bile acid in 20 serum and 20 bile samples was determined by the chemical method 3 (a commonly used method in India) with modification (x) and present method (y). The values obtained by two methods were correlated using the regression equation. Interference study To study interference by various serum compounds, 0.2 ml aqueous solution of bilirubin, glucose, cholesterol, triglycerides, acetone, urea, uric acid, citric acid, L-ascorbic acid, citrate, pyruvic acid, haemoglobin, γ globulin, sodium pyruvate, NaCl, KCl and EDTA, each at their physiological concentration, was added in reaction mixture individually, before starting the reaction. The control contained 0.2 ml distilled water. A 540 was read in both the cases and the value of A 540, with no addition was considered as 100%. Results and Discussion Commercially available 3α-HSD from Pseudomonas testosteroni and diaphorase from Clostridium sp. were immobilized through glutaraldehyde coupling onto alkylamine glass beads. A conjugation yield of 1.2 and 0.19 mg/g with 99 and 70% retention of initial activity of native enzyme, was obtained respectively, which was higher than immobilized onto arylamine glass beads (Table 1). The kinetic properties of immobilized enzymes and their mixture and of free enzymes are given in Table 2. After immobilization, optimum ph of both enzymes increased, whereas optimum temperature remained unchanged for 3α-HSD, but decreased for diaphorase and mixture of enzymes. The activation energy (E a ) increased for 3α-HSD, but decreased for diaphorase and mixture of enzymes. V max decreased for 3α-HSD and mixture of enzymes, but remained unchanged for diaphorase. K m of 3α-HSD for bile acid decreased, but K m of diaphorase for NADH increased, after immobilization. Earlier, an increase in K m of 3α-HSD and diaphorase, after immobilization on cellulose beads through CNBr, was reported 15. Table 1 Immobilization of 3α-HSD and diaphorase on alkylamine glass beads (pore diam 55 nm) through glutaraldehyde coupling Enzyme added (mg protein) Conjugation yield (mg/g) Retention of specific activity (%) [The data are the mean of three observations] On alkylamine On arylamine glass beads glass beads 18 Present method 3α- Dia- 3α- Dia- HSD phorase HSD phorase Table 2 Kinetic properties of free and immobilized 3α-HSD and diaphorase onto alkylamine glass beads (pore diam 55 nm) individually and their mixture Kinetic property 3α-HSD Diaphorase 3α-HSD + Diaphorase Free Immobilized Free Immobilized Free Immobilized Optimum ph Optimum temperature E a (Kcal/mol) Incubation time for linearity (min) K m (mm) V max (µmol/nadh/min)

5 102 INDIAN J. BIOCHEM. BIOPHYS. VOL. 43, APRIL 2006 A simple, sensitive, specific and cost-effective enzymic colorimetric method was developed for determination of bile acid in serum and bile, employing mixture of alkylamine glass bound 3α- HSD and diaphorase in 1:2 ratio. The assay was based on measurement of NADH generated from NAD + during oxidation of bile acid by immobilized 3α- HSD. The NADH produces a yellow color dye (formazan) with chromogen (NBT) and the reaction is catalyzed by immobilized diaphorase. A 540 of the dye was directly proportional to the bile acid concentration. The following parameters were studied to evaluate the method. Linearity Using a mixture of immobilized 3α-HSD and diaphorase, linearity was observed between A 540 and bile acid concentrations (6.25 to 150 µmol/l) (Fig. 1). It was similar to the method, employing arylamine glass bound enzymes 18, but was not better than spectrophotometric method (1.0 to 1000 µmol/l) 4. The minimum detection limit (4.8 µmol/l) of the method was higher than employing free 3α-HSD and diaphorase (3 µmol/l) 14. Analytical recovery The recovery of added solid bile acid in the serum and bile samples of gallstone patients (50 and 200 µmol/l) was ± 4.64 and ± 2.32 respectively (Table 3), which was comparable to the methods such as direct spectrophotometry (96.2%) 4, HPLC (96.75%) 6, GLC (80% in serum and 100% in bile) 5, and fluorimetry (80%) 7, and enzymic colorimetric method employing arylamine glass bound enzymes (95.5 and 88.4%) 18. Precision To assess reproducibility and reliability of the method, bile acid content was determined in six serum and six bile samples, 6 times on the same day (within day) and after 1-week storage at 20 C (between days). The coefficient of variation (CV) of within batch and between batch for bile acid determination in serum and bile samples were <1.0 and <0.2% and <0.2 and <0.6%, respectively (Table 4). These results were comparable to that by arylamine glass bound enzymes (0.2% and 0.2%) 18, and were better than those obtained by other methods, such as TLC 15, HPLC with evaporating light scattering mass detection 23, HPLC with electrochemical detector 6, fluorimetric 7 and colorimetric method 4. Correlation To study accuracy of the method, bile acid concentration in 20 serum and bile samples of Table 3 Analytical recovery of added bile acid in serum and bile as measured by mixture of alkylamine glass bound 3α-HSD and diaphorase Bile acid added % Recovery (Mean ±SD, n= 6) (mol/l) In serum In bile None ± ± ± ± 8.95 Table 4 Within and between day determination of bile acid in serum and bile by mixture of alkylamine glass bound 3α HSD and diaphorase (n= 6) Within assay In serum Between assay* Within assay In bile Between assay* Fig. 1 Standard curve for bile acid concentration using mixture of alkylamine glass bound 3α-HSD from (Pseudomonas testosteroni) and diaphorase from (Clostridium sp.) [Taurodeoxycholic acid was used as bile acid] Mean (µmol/l) S.D CV (%) *Samples were assayed after 1-week storage at 20 C and 2-4 C SD, Standard deviation; CV, coefficient of variation

6 RANI et al: DISCRETE ANALYSIS OF BILE ACID IN SERUM AND BILE 103 Table 5 Bile acid values of apparently healthy and gallstone patients as measured by mixture of alkylamine glass bound 3α-HSD and diaphorase [Values represent mean ± S.D] Age group Sex Serum bile acid Bile acid in bile (n=20) Healthy Diseased (µmol/l) (µmol/l) (µmol/l) Children F 5.74±3.6 (Below 20 yr) M 8.68±3.66 Adult (21-50 yr) F 5.00± ± 2.47* ± M 10.15± ± 3.17* ± Old-aged F 16.9± ±2.35* ± (Above 50 yr) M 12.01± ± 3.45* ± *P < 0.001, F, female; M, male gallstone patients was determined by the standard chemical method 3 (x) and present method (y). The results showed a good correlation with r 1 = 0.95 for serum and r 2 = 0.93 for bile samples of gallstone patients. Determination of bile acid in serum and bile The level of serum bile acids in apparently healthy children (<20 yr), adults (20-50 yr) and aged persons (>50 yr) of both sexes and gallstone-formers under fasting condition as measured by the present method is given in Table 5. Our results were in good agreement with those obtained by fluorimetric method (6.8 µmol/l for female and 4.66 µmol/l for male) 7, enzymic colorimetric method using free enzymes (6.2 µmol/l) 14 and using arylamine conjugated enzymes ( µmol/l) for both male and female 18. The level of bile acid in bile of gallstoneformers in different age groups as determined by the present method is given in Table 5. Interference study Among various metabolites tested at their physiological concentration, only bilirubin, urea, L- ascorbic acid and KCl caused 20% inhibition, while NaCl and CaCl 2 caused 40% inhibition of the immobilized enzymes. Rest had no effect. Storage stability and reusability The mixture of immobilized 3α-HSD and diaphorase lost 30% of its initial activity, after 4 months of regular use (300 times), when stored in M sodium phosphate reaction buffer (ph 7.0) at 4 C, while the mixture of dissolved free 3α-HSD and diaphorase was stable for 1-week under the similar conditions. These results indicated an increase in storage stability of enzyme after immobilization (Fig. 2). Fig. 2 Storage stability of mixture of free and alkylamine glass bound 3α-HSD and diaphorase [Standard assay conditions were used for each assay] Cost-effectiveness The method is economical as the cost of 100 serum bile acid assays by the present method is $ 93.62, which is about 3.45 times lower than Sigma enzo-kit (No. 450-A, cost for 100 assays $ , for year ), based on same enzymic colorimetric method using free enzymes. The method is also more economical than employing arlyamine glass bound enzymes (3.14 times economical than Sigma) 18. References 1 Islam S, Poupon R E, Barbare J C, Chretein Y, Denis F & Poupon R (1985) J Hepatol 1, Castelden W M, Detchon P & Misso N L A (1989) Gut 30, Carey J B (1958) J Clin Invest 17, Mashige F, Tanaka N, Maki A, Kamel S & Yamanaka M (1981) Clin Chem 27, Van Berge Henegeuwen G P, Ruben A & Brandt K H (1994) Clin Chim Acta 54, Kamada S, Maeda M & Tsuji A (1982) J Chromatogr 239, Murphy G M, Billing H B & Baton D N (1970) J Clin Pathol 23,

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