Analysis of Bile and Gallstone

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Cody Phelps
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1 준대한쉐담도연구회심포지엄 Analysis of Bile and Gallstone 한양대학교의과대학내과학교실 함 T - 1. BILE SAMPLING 1. Condition 1) appropriate donors 2) no containation by blood, tissue or bacteria 3) no deterioration(teperatiure change etc) 4) functioning gallbladder 5) no coplications of cholelithiasis 2. Technique 1) duodenal intubation(string test, stiulation of gallbladder contraction) 2) ERCP, nasobiliary tube 3) percutaneous gallbladder puncture 4) operation(tie: at the beginning of operation) 3. Maintenance 1) frozen at 200C(standard) repeated thawing and freezing should be avoided 2) diluted with 9 vol. of isopropanol,(centrifuged), ade into an appropriate volue and stored in nitrogen-flushed tubes fitted with Teflon-lined c10sures 3) dilated with 9 vol. of CHCl3 MeoH(2:1) and stored 4) for proteins, add proteolytic inhibitors or rapidly freeze the bile(-700c) 5) for nu c1eation tie studies, kept at 370C in a sterile environrnent. ANALYSIS OF BILE 1. Routine easureent 3 ajor constituents(cholesterol, phospholipid, bile acid), bilirubin, calciu salts and proteins

2 Bile Total Bilirubin Ethanol extraction chlorolor-ethanol-water partition Total lipids cholic acid, and organic phosphorus Ether-heptane-ethanol-water Methanol-water Chlorolor layer partition Ether-heptane layer 1 Ethanol-water-Iayer Saponilication Silicic acid colun chroatography Chlorolor and ether Chlorolor-ethanol(3:2) eluate eluate Extraction 01 Iree bile acids Methylation Methylation BF3-ethanol Transethylation TMSi ethers TLC* on silica gel-g-silver nitrate plate TMSi ethers* Bile acids Cholesterol Phospholipid TMSi ethers, Triethylsily ethers; TLC, Thin-Iayer chroatography Fig. 1. Flow sheet for clinical analysis of bile 1 2. Preparation stepwise solvent extraction (Figure 1, 2) 3. Cholesterol 1) enzyatic ethod 2) spectrophotoetric ethod 3) GLC (Gas-liquid chroatography) 4. Phospholipid 1) Technique for easureent lnorgenic phosphorus a. enzyatic ethod

3 Bile CHC13-MeOH(CM, 2:1) extraction (20 vol of bile fluid) Centηtrifuge ppt Re-extraction with CM(2 ties) ppt Add 4 volue of water Dry by evaporation Methanol water layer (Discard) Silica gel colue chroatography Chlorofor :PE Cholesterol n u l I 메- g Stepwise elution Methanol Phospholipid PE ayer Dissolve in Ethanol Add water: PE Vigorous Shaking 70% etha n 이 layer Hydrolysis (NaOH, C 7h) 빼 4 t 0n Bile acid Fig. 2. Flow sheet for clinical analysis of bile II b. hydroquinone c. ANS(l-aino-2-naphthol-4-sulfonic acid) deterined at OD 660n 2) Class separation HPLC(High-perforance liquid chroatogrphy)

4 Gallstone Extract with ethanol:ether(3: 1) for 18h at 4 0 C C 때-p 히떼P) 때샤F ι i u - f e Add 0.05M Tris- 10% sodiu deoxycholate ω I discard Shake(8h, 4 0 C) Centrifuge at 12,000 rp, for 20 in precipitate Add Tris- sodiu deoxycholate Extract with ethanol:ether(3: 1) for 18h, at 4 0 C Shake(18h, 4 0 C) ~dis c ard pre 때 tate Shpernatant Extract with ethanol: ether for 18h at 4 0 C discard Repeat extraction step Repea Centrifuge at 12,000 rp, 20 in Precipitate Wash with ether Dry Add deionized water precipitate precipitate Protein deterination SDS-PAGE Aino acid analysis Pretein deterination SDS- PAGE Aino acid analysis Fig. 3. Suary of preparation for protein and aino acid analysis

5 5. Bile acids requires extraction with organic solvent or anion exchange chroatography before deterination 1) GLC standard ethod 2) HPLC 3) Enzyatic activity a. spectrophotoetric b. fluoroetric 4) RIA(Radioiunoassay) * ajor bile acids priary cholic acid, chenodeoxycholic acid secondary deoxycholic acid, lithocholic acid tertiary ursodeoxycholic acid 6. Bilirubin 1) Malloy-Evelyn, Michaelson ethods, easured at OD 560n 2) Bilirubinoeter(OD454n) 3) HPLC 4) Bilirubin oxidase ethod 7. Fatty acids palitate, stearate colorietric deterination(od 550n -페때Gallstone Powder F 바뎌재 P 야시 n ] Ca Deterlnation Dry weight i 뻐Residue Bilirubin Addition of Water P 밍r 타k l 꺼0 ) 1 n % U 타 9 p 때 u n W/ > Methanol-Water Layer P ] - 때I p ---t 뼈떼n 이티Chlorofor Layer 때-않C t 아Silica Gel Colun Chroatography MethanoI Elute Phospho!ipid Petr. Ether Layer 70% Ethanol Layer Hydrolysis Methylation TMSi Ethers Bile Acids Fig. 4. Flow sheet for c1inical analysis of gallstones

6 째빼때1 u 뼈歐뼈이때 y }뼈빼뻐ν+ 비C (8. Proteins and aino acids (Fig. 3) 1) total protein concentration Lowry & Bensadaun ethod 2) lndividual glycoproteins SDS P AGE (Sodiu dodecyl sulphate-polyacrylaide gel electrophoresis) 3) aino acid analysis autoatic ainoacid analyzer 9. Ca sa1ts 1) X-ray diffraction Stone Acidify Chlorofor-Methanol(ACM 2: 1) Extraction)g/di) Centrifuge I Re-extraction with ACM(2 ties) 빼ppt 0 에뼈g chlorofor layer Silica gel colun chroatography 뻐I 않때Stepwise elution Methanol Phospholipid PE layer Dry by evaporation Dissolve in Ethanol Add water:pe ~ 70% ethanol layer Hydrolysis (NaOH, 120 o C, 7h) Methylation Bile acid Fig. 5. Flow -sheet for clinical analysis of gallstone II

7 2) IR spectroscopy 3) NMR spectroscopy 1. ANALYSIS OF STONES 1. Sapling 1) rinse stones several ties with distilled water and let dry (store over anhydrous ca1ciu sulfate in the dark) 2) powder(pulverize) stones finely usign otar and pestle, put in vials and dessicate for at least a week 2. Preparation stepwise solvent extraction (Figure 4, 5) 3. Methods 1) X-ray diffraction analysis 2) Infrared spectrophotoeter 3) TLC, GLC 4. Analysis of each constituents sae as analysis of bile 니F 넉

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