Effect of dietary garlic and onion on biliary proteins and lipid peroxidation which influence cholesterol nucleation in bile

Steroids 75 (2010) 272–281

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Effect of dietary garlic and onion on biliary proteins and lipid peroxidation which influence cholesterol nucleation in bile Satyakumar Vidyashankar, Kari Sambaiah, Krishnapura Srinivasan ∗ Department of Biochemistry and Nutrition, Central Food Technological Research Institute, CSIR, Mysore 570 020, India

a r t i c l e

i n f o

Article history: Received 2 August 2009 Received in revised form 27 December 2009 Accepted 6 January 2010 Available online 14 January 2010 Keywords: Cholesterol gallstones Cholesterol nucleation factors Biliary proteins Dietary garlic and onion Lipid peroxidation Model bile system

a b s t r a c t Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile. Dietary garlic and onion have been recently observed to possess anti-lithogenic potential in experimental mice. In this investigation, the role of biliary proteins from rats fed lithogenic diet or garlic/onion-containing diet in the formation of cholesterol gallstones in model bile was studied. Cholesterol nucleation time of the bile from lithogenic diet group was prolonged when mixed with bile from garlic or onion groups. High molecular weight proteins of bile from garlic and onion groups delayed cholesterol crystal growth in model bile. Low molecular weight (LMW) proteins from the bile of lithogenic diet group promoted cholesterol crystal growth in model bile, while LMW protein fraction isolated from the bile of garlic and onion groups delayed the same. Biliary LMW protein fraction was subjected to affinity chromatography using Con-A and the lectin-bound and unbound fractions were studied for their influence on cholesterol nucleation time in model bile. Major portion of biliary LMW proteins in lithogenic diet group was bound to Con-A, and this protein fraction promoted cholesterol nucleation time and increased cholesterol crystal growth rate, whereas Con-A unbound fraction delayed the onset of cholesterol crystallization. Biliary protein from garlic/onion group delayed the crystallization and interfered with pronucleating activity of Con-A bound protein fraction. These data suggest that apart from the beneficial modulation of biliary cholesterol saturation index, these Allium spices also influence cholesterol nucleating and antinucleating protein factors that contribute to their anti-lithogenic potential. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Cholesterol gallstones (CGS) occur primarily due to supersaturation of cholesterol in bile, leading to its precipitation (nucleation) in bile. Cholesterol precipitation is controlled by various factors such as mucin glycoprotein, calcium ions, bilirubin and low molecular weight proteins present in the bile [1,2]. Biliary proteins play a pivotal role in both promoting and inhibiting cholesterol precipitation. Biliary cholesterol supersaturation is frequently reported in patients with gallstones [3]. A rapid cholesterol nucleation time and an increased cholesterol crystal growth rate are the two most important indices that influence CGS formation and they are affected by cholesterol supersaturation [4]. Apart from biliary lipid components, biliary proteins can influence cholesterol nucleation time and cholesterol crystal growth rate [5–8]. Proteins that enhance the nucleation of cholesterol are called pronucleating and those which inhibit or prevent the nucleation of cholesterol, are called antinucleating factors. Both pronucleation and antinu-

∗ Corresponding author. Tel.: +91 821 2514876; fax: +91 821 2517233. E-mail address: [email protected] (K. Srinivasan). 0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2010.01.003

cleation factors are found in human bile as well as in the bile of experimental animals such as mice, rats, gerbils and Prairie dogs. Many proteins that bind to the lectin Conconavalin-A such as ␣acid glycoprotein [9,10], haptoglobin [11] and aminopeptidase-N9 [12] have been shown to promote cholesterol nucleation and crystal growth. Protein binding to the Helix pomatia lectin [13] and a recently introduced family of cholesterol crystal associated proteins [14] appear to act as inhibitors of cholesterol crystallization. However, with the sole exception of apolipoproteins, no inhibitor protein has so far been identified and fully characterized. In contrast to the assumed direct interaction of glycoproteins during cholesterol crystal growth process, another biliary inhibitor protein, apo A-I has been reported either to partially shift the distribution of cholesterol in bile from vesicles towards the more stable mixed micellar form [15] or to stabilize cholesterol carriers such as phospholipids lamellae [16]. This increases the stability and appears to be based on its amphiphillic properties, allowing integration of apo A-I in to lipid aggregates. These studies indicate the complexity of the system in terms of the large number of factors, which may affect cholesterol precipitation in bile. Apart from these factors, yet another factor such as lipid peroxides in bile may play a crucial role in the cholesterol crystallization [17]. Since an

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increase in the fraction of peroxidized lipids in the aged population and the increasing frequency of gallbladder stone formation in the elderly support this hypothesis. We have recently shown that dietary Allium spices garlic and onion are effective in reducing the formation of CGS under lithogenic conditions [18] and also in the regression of the preformed CGS [19]. Hence, in order to understand the mechanism of cholesterol crystal nucleation and the probable effect of proteins present in biles of rats fed these spices, they were tested with supersaturated model biles for cholesterol nucleation. The role of lipid peroxidation on the onset of cholesterol nucleation in rat biles fed these two test spices was also studied in normal and CGS-induced conditions.

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experimental procedures and with due clearance from the Institutional Animal Ethics Committee. Male albino rats [OUT-Wistar, IND-cft (2c)] weighing about 140 g obtained from Experimental Animal Production Facility of this institute were used. The animals were placed in individual cages in an approved animal house facility with 12 h light and dark cycles with temperature 25 ± 2 ◦ C and fed fresh diets daily. The animals had free access to food and water throughout the study. The food intake and growth of the animals were monitored at regular intervals. The rats were grouped into 6 groups with 8 animals/group and fed with different experimental diets for 8 weeks. At the end of the experimental period, bile was collected after cannulating the bile duct as described below. 2.4. Bile duct cannulation

2. Experimental 2.1. Materials Acrylamide, bovine serum albumin, bile acids (kit), bile salts (kit), commassie brilliant blue, dipalmitoyl phosphatidyl choline, EDTA, HEPES, 3␣-hydroxysteroid dehydrogenase, heparin, hydrazine hydrate, hydrogen peroxide, low molecular weight protein standard kit for electrophoresis, manganese chloride, NADPH, N,N-methylene-bis-acrylamide, sodium dodecyl sulfate, sodium metaperiodate, triglyceride purifier, 1,1,3,3-tetraethoxypropane, TEMED, thiobarbituric acid, tripalmitin, Tris–HCl, ethyl urethane, xanthine, xanthine oxidase and Sephadex G-100 were purchased from M/s Sigma Chemical Co. (St. Louis, USA). Polyethylene tubing (PE-10) was purchased from M/s Thomas Scientific Co. (New Jersey, USA). Con-A sepharose was obtained from M/s Bangalore Genei (Bangalore, India). dl-Alanine, choline chloride, dinitrophenyl hydrazine, l-aspartate, ␣-cellulose, mercaptoethanol, l-methionine and sodium pyruvate were purchased from HiMedia Laboratories (Mumbai, India). Casein was purchased from M/s Nimesh Corporation (Mumbai, India). All other chemicals used were of finest quality available and solvents were distilled before use. Dehydrated onion (Allium cepa) and garlic (Allium sativum) powders used in the experiment were a generous gift from Indo Nissin Foods Ltd. (Bangalore, India). 2.2. Animal diets The animals were fed with AIN-76 semi-purified diet. The basal control diet consisted of: sucrose, 60%; casein, 20%; cellulose, 5%; AIN-76 mineral mix, 3.5%; AIN-76 vitamin mix, 1%; dl-methionine, 0.3%; choline chloride, 0.2% and refined peanut oil, 10%. Lithogenic diet was prepared by supplementing 0.5% cholesterol and 0.25% bile salts (1:1 mixture of sodium cholate and sodium deoxycholate) to the AIN-76 basal diet. Various test diets were prepared by adding garlic powder (0.6%) or onion powder (2%) to the AIN-76 basal diet. The diets were made isocaloric by varying the sucrose content. Heat processing of garlic and onion was done by adding the respective freeze-dried powder to the boiling water and boiled for 15 min with constant stirring as practiced normally in the Indian culinary and used as heat-processed spices in these experiments. The diets were made isocaloric by varying sucrose concentration. All these diets were prepared by mixing the ingredients in a mechanical mixer and pellets were prepared using hand-operated pelletizer. Diets were stored at 4 ◦ C in air-tight containers. 2.3. Animals Animal experiment was carried out taking appropriate measures to minimize pain or discomfort in accordance with the guidelines laid down regarding the care and use of animals for

Rats were anaesthetized with ethyl urethane (1.2 g/kg body weight) by intra-peritoneal injection. Laparatomy was performed and common bile duct was cannulated with intramedic PE-10 polyethylene tubing (Thomas Scientific Co., USA). Bile was collected for 3 h in sterile tubes during which the body temperature of the animals was maintained at 37 ◦ C using incandescent lamps. The volume of the collected bile was measured and stored at −20 ◦ C until further analysis. 2.5. Biliary lipids and cholesterol saturation index (CSI) Biliary lipids were extracted by the method of Bligh and Dyer [20]. The upper methanolic layer was used for bile acid and uronic acid analysis and lower chloroform layer was used for cholesterol and phospholipid estimation. Total bile acids in methanolic layer were estimated by using 3∝-hydroxysteroid dehydrogenase [21]. Cholesterol was estimated by the method of Searcy and Bergquist [22]. Phospholipids were quantitated by ferrous ammonium thiocyanate method using dipalmitoyl phosphatidyl choline as standard [23]. Cholesterol saturation index (CSI) in the bile was calculated by using the cholesterol, phospholipids and bile acids as input data using Windows 98, Microsoft Excel statistical program. The coefficient for the fifth degree polynomial equation was used in the program taken from Carey’s table [24]. 2.6. Total protein and glycoprotein Total proteins were measured according to Lowry et al. [25] with BSA as a reference standard. Glycoprotein was estimated according to Mantle and Allen [26] using mucin as a reference standard. 2.7. Biliary lipid peroxidation induced by hydrogen peroxide Hepatic bile was incubated in two sets with hydrogen peroxide (0.1 mM), ferrous sulfate (0.2 mM) and ascorbic acid (0.25 mM) for 30 min for the induction of lipid peroxidation. One set was used for total lipid extraction and used for measuring lipid peroxidation. The other set was used to study the nucleation of cholesterol at different time intervals by incubating at 37 ◦ C. 2.8. Evaluation of cholesterol nucleation time in bile samples Rat hepatic bile from lithogenic diet group and various other dietary groups was mixed in various ratios of 90:10, 75:25 and 50:50, and incubated with 0.04% sodium azide in sterile tubes with air-tight caps for 21 days at 37 ◦ C. An aliquot of the bile was withdrawn at different time intervals during this incubation and observed under polarized microscope for precipitation and appearance of cholesterol monohydrate crystals. The earliest day on which any crystal appeared was considered as the cholesterol nucleation time of that particular bile sample. At the end of the incubation

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period, all the bile samples were extracted and lipid composition was determined. CSI was also calculated from the lipid composition for the individual as well as mixed bile samples. 2.9. Preparation of model bile Model biles of predetermined CSI were prepared according to the procedure of Kibe et al. [27]. Sodium taurocholate in methanol was mixed with phosphatidylcholine and cholesterol in chloroform. The mixture was dried at 45 ◦ C under a mild stream of nitrogen and subsequently for 24 h under reduced pressure. The lipid film was reconstituted in 10 mM Tris, 150 mM sodium chloride, and 3 mM sodium azide, and incubated for 16 h at 56 ◦ C and subsequently stored at 37 ◦ C in dark under nitrogen. 2.10. Preparation of cholesterol monohydrate crystals (seed crystals) Cholesterol monohydrate crystals were prepared according to Igimi and Carey [28]. Five grams of cholesterol was dissolved in 400 mL hot ethanol (95%, v/v) and the solution was slowly cooled to room temperature and kept at 4 ◦ C for 3 days. Large cholesterol crystals were harvested by filtration (0.45 ␮m), washed with 100 mL water and resuspended in 50 mL water containing 3 mM sodium azide. The suspension was sonicated for 60 s and centrifuged at 1000 × g for 5 min. The supernatant was collected and the pellet was resuspended, re-sonicated and centrifuged. The procedure was repeated until about 200 mL of supernatant was obtained. For the preparation of seed crystals, the pooled cloudy supernatants were stored at 4 ◦ C for 4 days. During this period, a small amount of sediment formed. The supernatant was decanted and filtered through 0.8 ␮m filter. The filtrate was passed through a 0.22 ␮m filter and the concentrate was then resuspended in water containing 3 mM sodium azide thus producing a seed crystal suspension with a crystal size ranging between 0.22 and 0.8 ␮m. Cholesterol concentration in the final seed crystal suspension was determined [29]. 2.11. Cholesterol crystal growth assay Cholesterol crystal growth assay was carried out separately in seeded and unseeded sets to study the effect of different biliary protein fractions from rats of different diet groups on nucleation of cholesterol crystals and other parameters. The crystal growth of seeded set was initiated by the addition of 10 ␮g crystalline cholesterol per mL of model bile. Aliquots of solutions of proteins of interest were placed in screw-capped vials and filtered model bile was added to these vials. Both model bile and the vials with proteins were pre-equilibrated at 37 ◦ C before the two were mixed. An aliquot of model bile mixture was withdrawn at different time intervals and diluted with Tris buffered saline. The absorbance at 900 nm was measured [29]. The crystal growth parameters were calculated from the crystal growth curves [29]; (1) maximum growth rate [crystal growth index (Ig )] = maximal slope of experimental curve/maximal slope of control, (2) final crystal concentration [crystal index (Ic )] = final crystal concentration of experimental/final crystal concentration of control and (3) onset time of crystal detection [time index (It )] = onset time of experimental/onset time of control. 2.12. Separation of vesicles and micelles from model bile and cholesterol nucleation of vesicles and micelles Supersaturated model bile (CSI = 1.5) was incubated with protein of interest at 37 ◦ C for 48 h and chromatographed on Sephadex

G-100 column (75 cm × 1 cm). Protein and lipids in the peak fractions were determined using standard procedures [30]. The model biles were subjected to gel filtration chromatography to separate vesicles and micelles. The vesicles and micelles were incubated with Concanavalin-A bound biliary protein (200 ␮g) separated from the rat bile of different diet groups to study the effect of dietary Allium spices on the nucleation of vesicles and micelles. 2.13. Separation of biliary proteins by gel filtration and affinity chromatography Concentrated rat bile was chromatographed on Sephadex G100 column (75 cm × 1 cm). The elution buffer contained 10 mM Tris, 150 mM NaCl [29]. For the separation of low molecular weight (LMW) glycoprotein by lectin affinity chromatography, the lyophilized LMW protein fraction obtained by gel filtration was solubilized in the eluting buffer and chromatographed on affinity column [29]. 2.14. Electrophoresis Mini slab gel electrophoresis was performed according to Lammelli’s procedure to determine the purity and approximate molecular weight of the isolated biliary proteins [31]. Running gels were made of 10% acrylamide in 0.375 M Tris–HCl, 0.1% SDS at pH 8.8. Stacking gels were made of 4% acrylamide in 0.125 M Tris–HCl, 0.1% SDS at pH 7.8. Protein samples were mixed with an equal volume of sample buffer (0.1 M Tris–HCl, pH 6.8, 2% SDS, 8% 2-mercaptoethanol, and 0.005% bromophenol blue) and then boiled for 10 min before loading on the gel. Low molecular weight protein markers were co-electrophoresed. Electrophoresis of the proteins was performed at a constant current of 40 mA for 5 h. Staining was done with Coommassie blue and destained in acetic acid:methanol:water (10:25:65, v/v) overnight. Approximate molecular weight of Concanavalin-A bound protein fraction was determined. 2.15. Statistical analysis Statistical analysis was carried out using Windows 98 Microsoft excel statistical software and Prism Graphpad statistical software. Results were analyzed and the significance level was calculated using Tukey–Kramer multiple comparison test and results are considered significant at P < 0.05. 3. Results 3.1. Cholesterol nucleation time in hepatic bile from different diet groups and their mixtures Cholesterol nucleation time of hepatic bile of rats from various diet groups is presented in Table 1. Bile of lithogenic diet group having a CSI of 2.16 nucleated on the 5th day. On the other hand, nucleation of cholesterol in the bile of rats fed with garlic (raw), garlic (heat-processed), onion (raw) and onion (heat-processed) occurred on 18th, 16th, 19th and 20th day, respectively. Their CSI was also significantly lower compared to that of LG diet group. Cholesterol nucleation time of mixtures of bile from different diet groups is presented in Table 2. The mixture containing the bile of lithogenic group and the bile of garlic or onion group in the ratio of 90:10 nucleated between 7th and 8th days of incubation. Nucleation time for other bile mixtures containing the bile of lithogenic group and the bile of Allium spice group in the ratio of 75:25 and 50:50 was increased as the content of the bile from spice group increased in the mixture (Table 2). When the bile of lithogenic diet group was mixed with the bile of other diet groups in the ratio of

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Table 1 Lipid composition, Cholesterol Saturation Index, cholesterol nucleation time in the bile from rats fed various diets. Dietary group

Lipids (mM) Bile acid

Basal control (C) Lithogenic (LG) C + garlic (raw) C + garlic (heat processed) C + onion (raw) C + onion (heat processed)

182.6 140.1 173.3 152.5 151.0 175.8

± ± ± ± ± ±

Total lipid (g/dL) Phospholipid

12.1 7.86a 6.00b 15.7b 12.4b 11.0b

20.3 25.6 20.4 17.7 21.4 19.3

± ± ± ± ± ±

1.23 2.05a 1.04b 1.01b 1.84b 1.00b

CSI

Cholesterol nucleation time (days)

Cholesterol 4.40 32.4 15.9 20.5 16.8 12.5

± ± ± ± ± ±

0.33 2.34a 2.01b 2.88b 1.76b 1.80b

10.7 10.1 10.7 9.65 9.72 10.6

± ± ± ± ± ±

0.51 0.43 0.28 0.75 0.55 0.58

0.53 2.16 1.06 1.30 1.15 0.95

± ± ± ± ± ±

0.09 0.23a 0.07b 0.08b 0.15b 0.13b

ND 5 18 16 19 20

Each value is the mean ± SD of 6 samples. ND: crystals not detected even on 21 days. a Statistically significant when compared to basal control group at P < 0.01. b Statistically significant when compared to LG group at P < 0.01.

50:50, the cholesterol nucleation time of the same was significantly prolonged to 15th (raw garlic), 14th (heat-processed garlic), 15th (raw onion), and 16th day (heat-processed onion) (Table 2). 3.2. Effect of dietary garlic and onion on lipid peroxidation in bile and cholesterol nucleation time Lipid peroxidation may promote the cholesterol nucleation time in bile leading to pathogenesis of CGS. Hence, hepatic bile from rats of different diet groups was analyzed for the extent of lipid peroxidation and cholesterol nucleation time in normal and induced conditions (Table 3). The lipid peroxides were 80 and 57% higher in the lithogenic diet group in normal and induced conditions, respectively, as compared to basal control group. Lipid peroxidation was lowered by 28, 24, 26, and 33% in raw garlic, heat-processed garlic, raw onion, and heat-processed onion groups, respectively, compared to lithogenic diet group in normal conditions. Similarly, the level of lipid peroxidation was lowered by 20, 12, 16, and 26% in raw garlic, heat-processed garlic, raw onion, and heat-processed onion groups, respectively, compared to lithogenic diet group under induced conditions. The nucleation of cholesterol in normal bile was observed at 20 days compared to 6 days in lithogenic diet group. Whereas nucleation of cholesterol was observed on 15th, 13th, 16th, and 17th day in raw garlic, heat-processed garlic, raw onion, and heat-processed onion groups, respectively. Similarly, under induced conditions the nucleation of cholesterol in the bile of basal control group was observed at 16 days compared to 5 days in lithogenic diet group. Nucleation of cholesterol in the bile under induced conditions was observed on 12th, 10th, 11th, and 15th day in raw garlic, heat-processed garlic, raw onion, and heat-processed onion groups, respectively. These results suggested that nucleation times were shortened in bile samples where the lipid peroxides were higher. The present results suggest that the extent of lipid peroxidation in bile is inversely related to the cholesterol nucleation time. Bile from lithogenic diet group is more prone for peroxidation in both normal and induced condition compared to basal control group and hence the observed reduction in cholesterol nucleation time (Fig. 1). The extent of lipid peroxidation in bile under normal

and induced conditions was lower in raw garlic, heat-processed garlic, raw onion, and heat-processed onion groups compared to the lithogenic diet group as shown in Fig. 1, and it is also evident that the two Allium spices effectively reduced lipid peroxidation in bile and concomitantly increased the cholesterol nucleation time in bile. 3.3. Effect of biliary protein fractions from dietary garlic and onion groups on supersaturated model bile 3.3.1. Effect of high molecular weight fraction The cholesterol crystal growth assay was carried out in two separate sets of model bile, namely, seeded and unseeded. The seeded model bile set is the one to which 10 ␮g of cholesterol crystal/mL model bile was added. Addition of cholesterol crystals abolishes the event of nucleation. Data presented in Fig. 2 show that the addition of HMW fraction obtained from the lithogenic diet group shortened the cholesterol nucleation time, and increased the crystal growth rate and final cholesterol crystal concentration of the unseeded model bile. The onset time of crystal detection (It ), growth index (Ig ) and crystal index (Ic ) of the model bile to which 250 ␮g of HMW biliary protein was added were 1.0, 1.54 and 1.64, respectively (Table 4). The cholesterol crystal detection time was 10 h in the bile system having biliary HMW fraction from lithogenic diet group. The effect of HMW fractions from the groups fed with Allium spices was opposite to that of lithogenic diet group. The nucleation time was prolonged and it was between 15 and 20 h in garlic or onion fed groups as shown in Fig. 2. The It values for the raw garlic, heat-processed garlic, raw onion, and heat-processed onion was 2.5, 1.5, 2.0, and 2.5, respectively. The Ic values for the raw garlic, heat-processed garlic, raw onion, and

Table 2 Nucleation of cholesterol crystals in the bile of lithogenic rats mixed with bile of rats fed Allium spices. Diet group

Lithogenic Lithogenic Lithogenic Lithogenic Lithogenic

Nucleation time (days)

Diet group

100:0

90:10

75:25

50:50

5 5 5 5 5

6 7 7 7 8

8 11 9 10 11

10 15 14 15 16

ND: cholesterol crystals not detected even at 21 days. Each value is the mean of 6 samples.

Basal control (C) Garlic (raw) Garlic (heat processed) Onion (raw) Onion (heat processed) Fig. 1. Relationship between biliary lipid peroxidation and cholesterol nucleation time.

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Table 3 Effect of dietary Allium spices on lipid peroxidation induced by hydrogen peroxide in rat bile and on cholesterol nucleation time. Diet group

Basal control Lithogenic Garlic (raw) Garlic (heat processed) Onion (raw) Onion (heat processed)

Lipid peroxides as MDA (nmol/dL)

Cholesterol nucleation time (days)

Uninduced

Uninduced

Induced with hydrogen peroxide

20 6 15 13 16 17

16 5 12 10 11 15

110.4 198.9 143.3 151.7 147.3 132.5

± ± ± ± ± ±

3.89 5.77a 8.34b 9.44b 6.21b 3.29b

Induced with hydrogen peroxide 168.9 265.3 210.9 234.4 221.7 196.8

± ± ± ± ± ±

3.45 7.89a 8.78b 6.44b 11.4b 7.47b

Values are mean ± SD of 6 samples/group. a Statistically significant when compared to basal control group at P < 0.01. b Statistically significant when compared to LG group at P < 0.01.

heat-processed onion were 1.33, 1.57, 1.47, and 1.15, respectively. The Ig values for the raw garlic, heat-processed garlic, raw onion, and heat-processed onion were 1.31, 1.5, 1.42, and 1.18, respectively (Table 4). In the seeded set of the model bile, addition of HMW protein from lithogenic diet group increased the crystal growth rate and final crystal concentration. But dietary groups of garlic or onion brought down the crystal growth rate and final crystal concentration (Fig. 2). Thus, feeding of Allium spices produced marked

Table 4 Influence of HMW protein fraction from bile of rats fed Allium spices on cholesterol crystal growth parameters tested in model bile. Diet group

Protein (␮g)

Basal control Lithogenic Garlic (raw) Garlic (heat processed) Onion (raw) Onion (heat processed)

250 250 250 250 250 250

Seeded

Unseeded

Ig

Ic

Ig

Ic

It

1.05 1.23 1.15 1.19 1.17 1.09

1.04 1.35 1.20 1.29 1.25 1.09

1.08 1.54 1.31 1.50 1.42 1.18

1.08 1.64 1.33 1.57 1.47 1.15

2.5 1.0 2.5 1.5 2.0 2.5

Each value is the mean of 4 samples. Ig —growth index; Ic —crystal index; It —onset time of crystal detection. Data calculated from Fig. 2.

reduction in It , Ig and Ic values in the model supersaturated bile system (Table 4). 3.3.2. Effect of low molecular weight fraction Data presented in Fig. 3 show that the addition of LMW fraction obtained from the lithogenic diet group shortened the cholesterol nucleation time, increased the crystal growth rate and final cholesterol crystal concentration of the unseeded model bile. The Ig , Ic , and It of the model bile to which 250 ␮g of LMW biliary protein was added were 1.36, 1.58 and 1.0, respectively (Table 5). The cholesterol crystal detection time was 2 h in the model bile system having biliary LMW fraction from lithogenic group. The effect of LMW fractions from the groups fed with Allium spices was opposite to that from lithogenic diet group. Cholesterol nucleation time was prolonged in garlic and onion fed groups as shown in Fig. 3. The It values for the raw garlic, heat-processed garlic, raw onion, and heat-processed onion were 3.0, 2.0, 2.5, and 3.41, respectively, compared to 1.0 in lithogenic diet group. The Ic values for the raw garlic, heat-processed garlic, raw onion, and heat-processed onion were 1.36, 1.50, 1.44, and 1.22, respectively, compared to 1.58 in lithogenic diet group. The Ig values for the raw garlic, heatTable 5 Influence of LMW protein fraction from bile of rats fed different diets on crystal growth parameters tested in model bile. Diet group

Basal control Lithogenic Garlic (raw) Garlic (heat processed) Onion (raw) Onion (heat processed)

Fig. 2. Effect of high molecular weight biliary proteins on crystal growth in (A) unseeded and (B) seeded model bile sets.

Protein (␮g)

250 250 250 250 250 250

Seeded

Unseeded

Ig

Ic

Ig

Ic

It

1.16 1.19 1.17 1.16 1.15 1.21

1.15 1.66 1.44 1.62 1.51 1.33

1.13 1.36 1.27 1.33 1.31 1.20

1.14 1.58 1.36 1.50 1.44 1.22

2.0 1.0 3.0 2.0 2.5 3.41

Each value is the mean of 4 samples. Ig —growth index; Ic —crystal index; It —onset time of crystal detection. Data calculated from Fig. 3.

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Fig. 4. (A and B) Affinity lectin column (Con-A) chromatography of low molecular weight protein fractions of bile obtained from different dietary groups.

Fig. 3. Effect of low molecular weight biliary proteins on crystal growth in (A) unseeded and (B) seeded model bile sets.

processed garlic, raw onion, and heat-processed onion were 1.27, 1.33, 1.31, and 1.20, respectively, compared to 1.36 in lithogenic diet group (Table 5). In the seeded set of model bile, addition of biliary LMW protein from lithogenic diet group increased the cholesterol crystal growth rate and final cholesterol crystal concentration. But dietary garlic and onion brought down the cholesterol crystal growth rate and final crystal concentration (Fig. 3). Thus, feeding of Allium spices produced marked reduction in It , Ig and Ic values in the model supersaturated bile system (Table 5).

terol nucleation time, increased the cholesterol crystal growth rate and final cholesterol crystal concentration (Fig. 5). When 100 ␮g of Con-A bound biliary protein fraction from garlic and onion groups was added to the model bile system, the cholesterol nucleation time was 10–15 h, and the cholesterol crystal growth rate and final cholesterol crystal concentration were lesser than that of the lithogenic group. On the other hand, addition of Con-A bound protein fraction from lithogenic group to the seeded model bile system had a higher cholesterol crystal growth rate and final crystal concentration (Fig. 5). But the addition of Con-A bound fraction from garlic and onion groups reduced the cholesterol crystal growth rate and final crystal concentration. The data indicated that biliary proteins from lithogenic group accelerated the nucleation of cholesterol crystal and crystal growth, whereas the proteins isolated from spice groups inhibited the cholesterol nucleation and crystal growth rates.

3.4. Effect of biliary glycoprotein fractions separated by affinity lectin chromatography on cholesterol nucleation and crystal growth rates in model bile system

3.5. Effect of Conconavalin-A bound biliary glycoprotein fraction on cholesterol nucleation time of vesicles and micelles

The biliary LMW protein fraction obtained from different dietary groups on gel filtration was further purified on Con-A lectin affinity column to separate sugar-specific glycoprotein (Fig. 4). The concentration of the protein bound to Con-A column was higher in lithogenic group compared to those from Allium spice groups. Addition of Con-A bound biliary protein fraction from lithogenic diet group to unseeded supersaturated model bile shortened the choles-

Cholesterol is highly insoluble in aqueous medium of the bile and it is transported in the form of cholesterol–phospholipid structure called vesicles and cholesterol–phospholipid–bile salt structure called micelles. Cholesterol crystal nucleation occurs from vesicles. In this context, the effect of Con-A bound glycoprotein fraction isolated from the bile of rats fed spices on cholesterol crystal nucleation was studied in vesicles and micelles from model

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Fig. 6. SDS-gel electrophoresis of Conconavalin-A bound biliary protein fractions separated from different spice fed groups. (1) Basal control; (2) lithogenic; (3) garlicR; (4) garlic-H; (5) onion-R; (6) onion-H.

was added to vesicles, cholesterol nucleation time was prolonged and it was nucleated on 9th, 7th, 8th, 12th and 9th day in raw garlic, heat-processed garlic, raw onion, and heat-processed onion groups, respectively, compared to 5th day in lithogenic diet group. The micellar fraction nucleated on 19th, 15th, 16th, and 20th day in raw garlic, heat-processed garlic, raw onion, and heat-processed onion groups, respectively, compared to 10th day in lithogenic diet group (Table 6). 3.6. Effect of garlic and onion on Concanavalin-A bound protein fraction separated on SDS-gel electrophoresis

Fig. 5. Effect of Con-A bound biliary protein fraction on crystal growth in (A) unseeded and (B) seeded model bile set.

bile separated by gel filtration. Vesicles and micelles from the model bile were incubated in sterile vials and observed for cholesterol crystal nucleation under polarized microscope. Initially, no crystals were seen in either vesicles or micelles. Vesicles and micelles separated from the control model bile nucleated on 12th and 18th day, respectively (Table 6). When 200 ␮g Con-A bound biliary protein fraction isolated from lithogenic group was added to vesicles or micelles, cholesterol nucleation occurred on 5th day and 10th day, respectively. Similarly, when protein fraction from spice groups Table 6 Effect of Con-A bound protein fraction from the bile of rats fed various diet on the nucleation time on vesicles and micelles separated from model bile. Diet group

Model bile Basal control Lithogenic Garlic (raw) Garlic (heat processed) Onion (raw) Onion (heat processed)

Protein (␮g)

– 200 200 200 200 200 200

Each value is the mean of 4 samples.

The LMW fraction obtained from the bile of rats from different dietary groups resolved into several bands on SDS-gel electrophoresis (Fig. 6). There was a distinct difference between gel patterns between the groups. The Con-A bound LMW fraction resolved on the gel showed several bands and there was a prominent wide band in the protein region of molecular weight 24 kDa in the lithogenic diet group, suggesting an increased concentration of proteins in the region of 24 kDa in Con-A bound protein fraction of lithogenic group. The intensity of this protein band was highly diffused in the two spice fed groups suggesting expression of these proteins in much lesser concentration. This indicated that proteins in the molecular weight region of 24 kDa might be responsible for the pronucleating activity when fed lithogenic diet. Feeding of garlic and onion may be playing an inhibitory role in the secretion of these proteins into bile. 4. Discussion

Nucleation time (days) Vesicles

Micelles

12 16 5 9 7 8 12

18 ND 10 19 15 16 20

Health beneficial anti-lithogenic potential of dietary garlic and onion in experimental mice in terms of preventing the incidence of CGS and regression of preformed CGS has been recently observed by us [18,19]. Such an influence although has been attributable primarily to their influence on cholesterol metabolism so as to relieve supersaturation of biliary cholesterol, additional influences on cholesterol nucleation and antinucleation factors present at the site of CGS cannot be ruled out. Cholesterol crystal nucleation in bile is reported to be influenced by biliary proteins but the crystalliza-

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tion sequence due to proteins is not completely clear. Pronucleating proteins such as mucin [32] and other glycoproteins [32,33] are thought to provide a nucleating matrix for cholesterol crystal formation. IgM and IgA can serve as pronucleating agents [6]. The antinucleation proteins such as apolipoproteins (Apo-AI, Apo-AII) are also found in bile. Bile samples from gallbladders of gallstone patients contain cholesterol crystals [34,35], which cannot be completely removed with the result that reliable crystal growth assays cannot be performed. Further, procedures used for the removal of cholesterol crystal could also remove other critical components such as vesicles or proteins such as mucin and some LMW glycoproteins, which are also important in the nucleation process [36]. Unlike native bile, model bile system is a simpler and clear system containing three components, viz., cholesterol, phospholipids and bile acids. Their association and dissociation during cholesterol crystal nucleation process can be very well studied in model bile system, and the influence of any exogenous factor on the kinetics of cholesterol crystal nucleation can also be evaluated. In the present study, hepatic biliary proteins from garlic and onion fed rats were examined for their influence on cholesterol crystal nucleation. Rat hepatic bile which is essentially free of any cholesterol crystals; and being very dilute and hence do not nucleate, was concentrated to simulate gallbladder bile. During concentration, there was no precipitation of any biliary components. Concentration of hepatic bile by the gallbladder leads to lipid concentration. Upon concentrating the bile samples, there is an increase in vesicular cholesterol:phospholipid ratio and an increased propensity to nucleate cholesterol crystals [37]. Higher concentrations of biliary components may lead to vesicular aggregation, which precedes cholesterol nucleation [38]. In the present study, high cholesterol diet caused a near saturated condition in bile, which was made supersaturated by dehydration. Earlier studies have shown a high cholesterol secretion in the bile of rats fed a high cholesterol diet and feeding garlic reduced the biliary cholesterol and increased bile secretion in rats [39]. Addition of bile from basal control group to that of lithogenic group had only little effect on the nucleation time although the CSI of the bile mixture (50:50) was >1. On the other hand, addition of bile from garlic or onion fed groups even at the lowest proportion markedly prolonged the nucleation time of the bile from lithogenic diet group. These differences in the nucleation time can be attributed to the presence of factors in the bile apart from cholesterol supersaturation. These factors may be proteins as suggested by earlier reports [32,33]. It is suggested that lithogenic diet fed rats may secrete proteins in to bile which act as promoters of cholesterol crystal nucleation, whereas rats fed Allium spices secrete biliary proteins which act as antinucleators. An increase in the nucleation time in the bile of lithogenic diet group upon addition of small quantity of bile from dietary garlic and onion groups suggests that the antinucleating factor present in these spice groups probably neutralizes the pronucleating factor of the lithogenic bile and hence prolongs the cholesterol nucleation time in the bile of lithogenic diet group. The bile of rats fed lithogenic diet probably contains large quantity of pronucleating activity, but this pronucleating activity decreased upon feeding Allium spices. Thus, feeding garlic and onion decreased the pronucleating activity which, suppressed the pronucleating activity. Both high molecular weight and low molecular weight protein fractions from the bile of lithogenic group shortened the cholesterol nucleation time in model bile indicating the presence of pronucleating factors. In contrast, both high molecular weight and low molecular weight protein fractions from the bile of dietary garlic and onion groups showed the presence of antinuleating factors which, exerted a dose-dependent effect in model bile system. Earlier, it has been reported that LMW protein fraction of hepatic bile

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of rats from lithogenic diet group obtained by sepharose-4B gel filtration chromatography shortened the cholesterol nucleation time and increased the crystal growth rate and final crystal concentration in supersaturated model bile [40]. But with the LMW protein fractions from the hepatic bile of rats on lithogenic diet group supplemented with curcumin and capsaicin, the nucleation times were prolonged and crystal growth rates and final crystal concentrations were decreased [40]. Biliary proteins were purified on affinity lectin column to examine whether sugar specificity of glycoproteins present in the bile of spice fed groups would have any role in cholesterol nucleation. A higher concentration of Con-A bound activity was isolated from the bile of lithogenic diet group. This glucose or mannose specific activity was shown to be a potent promoter of cholesterol nucleation. The SDS-PAGE profile indicated that this activity is associated with 24 kDa protein. On the other hand, a 130 kDa protein has been shown to have cholesterol nucleation promoting activity in humans [32]. Our present observation is in agreement with the earlier report that a higher proportion of LMW proteins from the hepatic bile of lithogenic group was bound to Con-A, and this Con-A bound fraction showed a pronucleating effect [40]. It is possible that the Con-A binding protein or its sugar moiety may interact with biliary lipids resulting in the formation of lipoprotein or glycolipid complexes [41]. Such interaction may cause destabilization of vesicles and micelles, the two major cholesterol carriers in the bile. A higher proportion of LMW proteins from the hepatic bile of rats fed curcumin and capsaicin are found to, respectively, bind to wheat germ agglutinin and H. pomatia lectin, and these WGA-bound or H. pomatia lectin-bound protein fractions showed a potent antinucleating activity in model bile [40]. The present study is the first to report such a novel antinucleating activity present in the hepatic bile of rats fed garlic and onion. This study provides evidence that the anti-lithogenicity of Allium spices is not only controlled by reducing hepatic and biliary cholesterol but perhaps also by causing the secretion of antinucleating proteins in the bile and by suppressing the pronucleating activity. There is no evidence that biliary proteins are affected by dietary components except indirectly by the fact that some bile acids stimulate secretion of mucin [42] and a high cholesterol diet feeding leads to hypersecretion of mucin in Prairie dogs [43]. In all the dietary groups, both cholesterol crystal promoting and inhibiting activities can be seen except for the differences in their concentration. The concentration of cholesterol nucleation promoting activity is higher in the lithogenic diet group than its inhibiting activity. This study has evidenced that dietary components have a profound effect on cholesterol nucleation inhibiting or promoting activity. One possibility is that garlic and onion feeding may reduce the promoting activity produced in response to the dietary cholesterol and may increase the synthesis or secretion of inhibiting activity. The present study has clearly demonstrated that the liver or bile duct of rats secretes factors influencing the nucleation of cholesterol crystal. Feeding cholesterol in the diet increases the pronucleating factors. This release of pronucleating factors may be the result of the pathogenic effect of high cholesterol load on the liver. Feeding garlic or onion increases the antinucleation factor, which is the result of an antagonistic effect of these Allium spices. In this context, it is evident that feeding garlic and onion prevents CGS not only by reducing cholesterol levels in bile and increasing bile acids, but also by increasing the antinucleation factors. Lipid peroxides may also play a vital role in CGS pathogenesis. Bile is a complex mixture of phospholipids, bile acids and cholesterol. But, little is known about the redox status of bile contributing to the pathogenesis of CGS. Conceivably, biliary phospholipid may undergo peroxidation in conditions associated with oxidant stress. Peroxidation products may be factors responsible for the cholesterol nucleation and thus leading to the formation of CGS. Despite

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increasing evidence of an important role of oxidant stress in various hepatobiliary diseases such as cholestasis [44] and cholangiocarcinoma [45], no attempt has been made to assess oxidant stress in bile. Earlier, Eder et al. [46] have reported that generation of lipid peroxidation products in model bile significantly reduced cholesterol crystal formation time indicating that lipid peroxidation may play a role in CGS pathogenesis. In vivo study also reports increased levels of MDA in the bile samples of patients with cholesterol gallstones as compared to stone free patients [17]. Our results are in agreement with the earlier reports, where the bile of lithogenic diet group showed higher levels of MDA in normal and peroxidationinduced conditions. But Allium spices could effectively reduce the levels of MDA formation in normal and peroxidation-induced conditions leading to the delayed onset of nucleation of cholesterol in hepatic bile. These observations indicated that dietary garlic and onion have a profound influence of promoting the antinucleating factors and inhibition of peroxidation in the bile. These factors help in the stabilization of cholesterol carriers and keeping the same in solubilized form, which is thermodynamically feasible from inhibiting cholesterol from nucleating. Thus, proteins of different sugar specificities coexist in bile; some of them have cholesterol nucleation promoting activity, while others have cholesterol nucleation inhibiting activity. The dominance of one over the other determines the net cholesterol nucleation behaviour. Our study has demonstrated that factor(s) influencing the nucleation of cholesterol crystal is secreted by the liver or bile duct of rats. The present report is the first one showing the presence of antinucleating proteins in hepatic bile of rats fed garlic or onion. It is inferred that dietary garlic or onion leads to a change in the profile of hepatobiliary proteins in such a way that antinucleating activity was manifested unlike in the lithogenic diet group where pronucleating activity was manifest. Hence the suggestion that the anti-lithogenicity of these dietary Allium spices is due not merely to their ability to lower cholesterol saturation index [18,19], but also to their beneficial influence on the biliary protein profile. Acknowledgements The first author (SV) is grateful to Indian Council of Medical Research, New Delhi, India for the award of senior research fellowship. This work was supported by grant-in-aid from the Department of Science and Technology, Government of India, New Delhi. References [1] Gallinger S, Harvey PR, Petrunka CN, Taylor RD, Strasberg SM. Effect of binding of ionised calcium on the in vitro nucleation of cholesterol and calcium bilirubinate in human gall bladder bile. Gut 1986;27: 1382–6. [2] Harvey PR, Rupar CA, Gallinger S, Petrunka CN, Strasberg SM. Quantitative and qualitative comparison of gall bladder mucus glycoprotein from patients with and without gall stones. Gut 1986;27:374–81. [3] Holzbach RT, Marsh M, Olszewski M, Holan K. Cholesterol solubility in bile. Evidence that supersaturated bile is frequent in healthy man. J Clin Invest 1973;52:1467–79. [4] Holan K, Holzbach RT, Hermann RE, Cooperman AM, Claffey WJ. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979;77:611–7. [5] Groen AK, Stout JP, Draper JA, Hoek FJ, Grijm R, Tygat GN. Cholesterol nucleation-influencing activity in T-tube bile. Hepatology 1988;8: 347–52. [6] Harvey PR, Upadhya AG, Strasberg SM. Immunoglobulins as nucleating proteins in the gallbladder bile of patients with cholesterol gallstones. J Biol Chem 1991;266:13996–4003. [7] Harvey PR, Strasberg SM. Will the real cholesterol nucleating and antinucleating proteins please stand up? Gastroenterology 1993;104:646–50. [8] Holzbach RT, Kibe A, Thiel E, Howell JH, Marsh M, Hermann RL. Biliary proteins, unique inhibitors of cholesterol crystal nucleation in human gallbladder bile. J Clin Invest 1984;73:35–45.

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