대한쉐담도연구회 심포지엄

Analysis of Bile and Gallstone

한양대학교 의과대학 내과학교실

함 준

-‘ T

1. BILE SAMPLING

1. Condition

1) appropriate donors

2) no contamination by blood, tissue or bacteria

3) no deterioration(temperatiure change etc)

4) functioning gallbladder

5) no complications of cholelithiasis

2. Technique

1) duodenal intubation(String test, stimulation of gallbladder contraction)

2) ERCP, nasobiliary tube

3) percutaneous gallbladder puncture

4) operation(time: 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), made into an appropriate volume 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 nuc1eation time studies, kept at 370C in a sterile environrnent

11. ANALYSIS OF BILE

1. Routine measurement 3 major constituents(cholesterol, phospholipid, bile acid),

bilirubin, calcium salts and proteins

- 4 5-

Total Bilirubin

Bile

Ethanol extraction chlorolorm-methanol-water partition

Total lipids cholic acid, and organic

phosphorus

Ether-heptane-ethanol-water Methanol-water Chlorolorm layer

partition (discard)

1 Ethanol-water-Iayer

Silicic acid column chromatography

Ether-heptane layer

(discard)

Chlorolorm and ether Chlorolorm-methanol(3:2)

Saponilication

Extraction 01 Iree

bile acids

Methylation

TMSi ethers*

eluate eluate

Methylation BF3-methanol

Transmethylation TMSi ethers

Cholesterol

TLC* on silica gel-G-silver

nitrate plate

Phospholipid

Bile acids

TMSi ethers, Trimethylsily ethers; TLC, Thin-Iayer chromatography

Fig. 1. Flow sheet for clinical analysis of bile 1

2. Preparation stepwise solvent extraction (Figure 1, 2)

3. Cholesterol

1) enzymatic method

2) spectrophotometric method

3) GLC (Gas-liquid chromatography)

4. Phospholipid

1) Technique for measurement

lnorgenic phosphorus a. enzymatic method

- 4 6-

Bile

CHC13-MeOH(CM, 2:1) extraction

(20 vol of bile fluid)

Vigorous shaking

Centηtrifuge

supernatant ppt

Re-extraction with CM(2 times)

supernatant ppt

Silica gel colume chromatography

Stepwise elution

Methanol

Dry by evaporation

Dissolve in Ethanol

Add water: PE

Vigorous Shaking

PE |ayer 70% etha|n이 layer

Hydrolysis

(NaOH, 1200C 7h)

빼|4t|0n Bile acid

Add 4 volume of water

Vigorous shaking

Methanol water layer

(Discard)

g m 메

-m

l I | m n u

m

Chloroform:PE

Cholesterol Phospholipid

Fig. 2. Flow sheet for cl inical analysis of bile II

b. hydroquinone

c. ANS(l -amino-2-naphthol-4-sulfonic acid)

determined at OD 660nm

2) Class separation HPLC(High-performance liquid chromatogrphy)

- 4 7 -

Gallstone

Extract with ethanol:ether(3: 1) for 18h at 40 C

m mω

m f m

e

p

히

--

”u

m i

m

mι

mF

샤m

P)

때

떼 때

C

I supernatant

Add 0.05M Tris- 10% sodium deoxycholate

Shake(8h , 40 C)

discard

precipitate

Centrifuge at 12,000 rpm , for 20 min

supernatant

Extract with ethanol :ether(3: 1) for 18h, at 40 C Add Tris- sodium deoxycholate

Shake(18h, 40C)

~discard pre때tate Shpernatant

Repeat extraction step Extract with ethanol: ether for 18h at 40 C

Repea

Centrifuge at 12,000 rpm , 20 min

1----------- discard supernatant

Precipitate

11 Wash with ether

11 Dry

11 Add deionized water

11

precipitate supernatant precipitate supernatant

Protein determination Pretein determination

SDS- PAGE

Amino acid analysis

SDS-PAGE

Amino acid analysis

Fig. 3. Summary of preparation for protein and amino acid analysis

- 4 8 -

5. Bile acids

requires extraction with organic solvent or anion exchange chromatography before determination

1) GLC standard method

2) HPLC

3) Enzymatic activity a. spectrophotometric

b. fluorometric

4) RIA(Radioimmunoassay)

* major bile acids

primary cholic acid, chenodeoxycholic acid

secondary deoxycholic acid, lithocholic acid

tertiary ursodeoxycholic acid

6. Bilirubin

1) Malloy-Evelyn, Michaelson methods, measured at OD 560nm

2) Bilirubinometer(OD454nm)

3) HPLC

4) Bilirubin oxidase method

7. Fatty acids palmitate, stearate

colorimetric determination(OD 550nm

Gallstone Powder

n ” m 때

야 시

P

」-페

i 뻐

“ 재

뎌

바

F ] Ca Determlnation Dry weight

Residue Bilirubin Addition of Water

Chloroform Layer

n ” m ” u 때

p m 9 “ ‘ ”

1

타

% n U 꺼

0 )

k ” l ’ ’

타

r 밍

P

Methanol-Water Layer

(discard)

Petr. Ether Layer

(discard)

70% Ethanol Layer

。〕떼

티 뼈

때

--t

t

아

C

I p

--않

n

이

-”“ 때

P]>

W/ m

Silica Gel Column Chromatography

MethanoI Elute

Hydrolysis

Methylation

TMSi Ethers

Phospho!ipid

Bile Acids

Fig. 4. Flow sheet for c1 inical analysis of gallstones 1

- 4 9-

8. Proteins and amino acids (Fig. 3)

1) total protein concentration Lowry & Bensadaun method

2) lndividual glycoproteins SDS P AGE

(Sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

3) amino acid analysis automatic aminoacid analyzer

9. Ca sa1ts

1) X-ray diffraction

Stone

Acidify Chloroform-Methanol(ACM 2: 1) Extraction)mg/d i)

Vigorous shaking

Centrifuge

I supernatant

Re-extraction with ACM(2 times)

ppt

mg

뼈

my

이

때 ”

뼈 |빼}빼

뼈

째 m

뼈 歐

1

。

“u

때 빼 뻐

Dry by evaporation

Dissolve in Ethanol

chloroform layer Add water:PE

Silica gel column chromatography

Stepwise elution

PE layer

Vigorous shaking

~ 70% ethanol layer

Hydrolysis

(NaOH, 120oC, 7h)

Methylation

뻐 때

I m

|않

m

에

0

+”

비

C

(ν

Methanol

Phospholipid

Bile acid

Fig. 5. Flow-sheet for clinical analysis of gallstone II

- 5 0-

2) IR spectroscopy

3) NMR spectroscopy

111. ANALYSIS OF STONES

1. Sampling 1) rinse stones several times with distilled water and let dry (store over anhydrous

ca1cium sulfate in the dark)

2) powder(pulverize) stones finely usign motar 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 spectrophotometer

3) TLC, GLC

4. Analysis of each constituents same as analysis of bile

F

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