Fransisca Putri, Nuri Dyah Indrasari

REVIEW ARTICLE

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Fransisca Putri*, Nuri Dyah Indrasari**

• Faculty of Medicine, Universitas Indonesia/Dr Cipto Mangunkusumo General National Hospital, Jakarta **Department of Clinical Pathology, Faculty of Medicine, Universitas Indonesia Dr. Cipto Mangunkusumo General National Hospital, Jakarta

  Corresponding author:

Nuri Dyah Indrasari. Department of Clinical Pathology, Dr. Cipto Mangunkusumo General National Hospital.

  

Jl. Diponegoro No.71 Jakarta Indonesia. Phone/facsimile: +62-21-3147713. E-mail: nuridyahkusmardi@yahoo.com

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  cholesterol stone, pigment stone (black and brown), and mixed stone. Mechanism which underlies the formation

  of cholesterol or pigment gallstone is different. Information on chemical component of the stone will assist the

  management and prevention of its recurrence. Analysis of gallstone component can be performed by colorimetry

  method or even gas liquid chromatography (GLC). Chemical component analysis of gallstone by colorimetry

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  cholesterol content is > 80%, pigment stone if cholesterol content is < 20%, and mixed stone if cholesterol

  content is 25-80%. Gallstone analysis by GLC method is conducted by separation of fatty acid chain and

  evaluation of fatty acid quantity in the methylester derivatives form, which is fatty acid methyl estered. Fatty

  acid content in cholesterol stone (310.09 + 49.7 mg/gram) is higher compared to pigment stone (55.59 +7.71

  mg/gram). Saturated to unsaturated fatty acid (S/U) ration in cholesterol stone (8.6 + 3.1) is higher compared

  to pigment stone (4.8 + 1.5).

  Keywords: gallstone, colorimetry, gas liquid chromatography $%675$.

  Batu empedu adalah deposit kristal yang terbentuk dalam kandung empedu atau saluran empedu. Batu

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yang mendasari pembentukan batu empedu kolesterol maupun pigmen berbeda. Informasi tentang komponen

kimia batu akan membantu dalam penatalaksanaan dan pencegahan kekambuhannya. Analisis komponen

batu empedu dapat dilakukan dengan metode kolorimetri ataupun gas liquid chromatography (GLC). Analisis

komponen kimia batu empedu dengan metode kolorimetri meliputi pemeriksaan kolesterol, bilirubin dan kalsium.

%DWX GLNODVL¿NDVLNDQ VHEDJDL EDWX NROHVWHURO ELOD NDQGXQJDQ NROHVWHURO ! EDWX SLJPHQ ELOD NDQGXQJDQ

kolesterol < 20% dan batu campuran bila kandungan kolesterol 25-80%. Analisa batu empedu dengan metode

GLC dilakukan dengan pemisahan rantai asam lemak dan menilai kuantitas asam lemak dalam bentuk derivat

methylester yaitu fatty acid methyl estered. Kandungan asam lemak pada batu kolesterol (310.09 + 49.7 mg/

gram) lebih tinggi dibandingkan batu pigmen (55.59 +7.71 mg/gram). Ratio saturated to unsaturated fatty acid

(S/U) pada batu kolesterol (8.6 + 3.1) lebih tinggi dibandingkan batu pigmen (4.8 + 1.5).

  Kata kunci: batu empedu, kolorimetri, gas liquid chromatography

  Gallstone Analysis ,1752'8&7,21

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  Hyposecretion of bile acid or phospholipid also may contribute to this condition. Second mechanism is nucleation of monohydrate cholesterol in bile acid

  Long parental nutrition, fasting Gall bladder hypomotility Pregnancy Progesterone inhibit gall bladder contractility

  Bile salt hyposecretion which originates from enterohepatic cycle

Female Sex hormones Estrogen stimulates hepatic lipoprotein receptor, thus causing increase in the uptake of dietary

cholesterol and secretion of biliary cholesterol Progesterone causes decreased contraction of gall bladder

  0XWDWLRQ LQ &\S $ JHQH FDXVLQJ Į 2+DVH GH¿FLHQF\ WKXV ODWHU GHFUHDVH V\QWKHVLV RI ELOH DFLG MDR3 mutation which cause defect in phospholipid secretion Age Increased secretion of biliary cholesterol Decreased secretion of bile salt Obesity, metabolic syndrome Increased biliary cholesterol Decreased cholecystokinin response

Body weight loss Breakdown of fat during not eating period and fast body weight loss causes liver to secrete extra

cholesterol to bile

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  Formation of cholesterol gallstone involve several PHFKDQLVPV LQFOXGLQJ VXSHUVDWXUDWLRQ RI ELOH DFLG ZLWK FKROHVWHURO PRQRK\GUDWH FKROHVWHURO QXFOHDWLRQ and gallbladder hypomotility. Excessive cholesterol level compared to phospholipid and bile acid causes vesicle rich in cholesterol to aggregate and cholesterol crystal to precipitate. Hypersecretion of cholesterol in REHVLW\ FRQGLWLRQ KLJK FDORULH GLHW KLJK FKROHVWHURO diet may cause bile supersaturation with cholesterol.

  Gallstone is a crystal deposit which is formed in the gallbladder or bile duct. Gallstone is a disease with multifactorial causes which are associated with GLHWDU\ KDELWV ORVV RI ERG\ ZHLJKW VH[ DQG IDPLO\ history. Its chemical composition can be associated ZLWK QXWULWLRQDO VRFLDO HFRQRPLFDO DQG JHQHWLF factors.

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  0RVW JDOOVWRQH FDVHV LQ :HVWHUQ FRXQWULHV DUH FKROHVWHURO JDOOVWRQH ZKLOH LQ (DVW $VLD PRVWO\ DUH brown pigment stone.

  Gallstone is formed due to abnormal composition of bile acid. This stone can be located in gallbladder or even bile duct as shown in Figure 1. Gallstone is FODVVL¿HG LQWR FKROHVWHURO VWRQH SLJPHQW VWRQH EODFN DQG EURZQ DQG PL[HG VWRQH &KROHVWHURO VWRQH FRQWDLQV ! PRQRK\GUDWH FKROHVWHURO ZLWK RWKHU FRPSRQHQWV LQFOXGLQJ FDOFLXP VDOW ELOH SLJPHQW SURWHLQ DQG IDWW\ DFLG 3LJPHQW VWRQH SDUWLFXODUO\ consists of bilirubinate calcium with cholesterol FRQWHQW RI 0L[HG VWRQH KDV FKROHVWHURO content between cholesterol and pigment stone.

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  DEGRPLQDO [ UD\ UDGLRLVRWRSH FRPSXWHG WRPRJUDSK\ PDJQHWLF UHVRQDQFH FKRODQJLRSDQFUHDWRJUDSK\ endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous transhepatic cholangiogram. Gallstone analysis can give information regarding chemical composition of the stone. Information regarding chemical composition of stone will help in the management and recurrence prevention.

  Laboratory examination in gallbladder disease is QRW VSHFL¿F FRQVLVWLQJ RI KDHPDWRORJ\ H[DPLQDWLRQ FRPSOHWH EORRG FRXQW OLYHU IXQFWLRQ WHVW DP\ODVH DQG lipase. Other supporting examination for gallbladder and

  WKDW HPHUJH LQ \HDUV DIWHU GLDJQRVLV *DOOVWRQH WKDW passes through gallbladder to the intestine is potential to cause ileus and require surgical procedure.

  Gallstone disease is frequently found inadvertently through abdominal ultrasonography (USG) examination

  7KH PRUWDOLW\ UDWH LV DSSUR[LPDWHO\ ,QFLGHQFH of gallstone disease in Indonesia is thought to be of no difference with Asian.

  ,QFLGHQFH RI JDOOVWRQH GLVHDVH LV LQ (XURSHDQ SRSXODWLRQ DQG LQ $VLDQ DQG $IULFDQ population. Incidence of gallstone disease in American ,QGLDQ SRSXODWLRQ UHDFKHV XS WR LQ IHPDOH DGXOWV

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  Cirrhosis Hypersplenism and the presence of changes in the composition of erythrocyte lipid membrane which leads to haemolysis

  ,Q FKROHVWHURO DQDO\VLV RI JDOOVWRQH WKHUH DUH WZR SURFHVVHV ZKLFK DUH VDPSOH H[WUDFWLRQ DQG WKH SURFHVV of cholesterol level measurement using colorimetry SULQFLSOH 7KH ¿UVW SURFHVV LV VDPSOH H[WUDFWLRQ SURFHVV ZKLFK FDQ EH FRQGXFWHG LQ ZD\V 7KH ¿UVW PHWKRG LV DV IROORZ *DOOVWRQH LV ZDVKHG DQG GULHG IURP ZKLFK 0.1 gram is taken. Stone is trimmed in the mortar and PJ RI WKH VWRQH SRZGHU LV WUDQVIHUUHG WR D WXEH ([WUDFWLRQ LV SHUIRUPHG E\ DGGLQJ P/ HWKDQRO ZKLFK LV ODWHU LQFXEDWHG DW C for 10 minutes. It is then left standing in room temperature for 10 minutes

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  Colorimetry method is a technique to determine concentration of particular substance by measuring the absorption in sample in the form of coloured solution. The colour can be produced from the substance itself or the coloured product formed through a chemical reaction which is then measured with particular wavelength. Gallstone component chemical analysis ZLWK FRORULPHWU\ PHWKRG LQFOXGHV FKROHVWHURO ELOLUXELQ DQG FDOFLXP H[DPLQDWLRQ 7KH SHUFHQWDJH of cholesterol in the stone determines the type of JDOOVWRQH 6WRQH LV FODVVL¿HG DV FKROHVWHURO VWRQH LI WKH FKROHVWHURO FRQWHQW LV ! SLJPHQW VWRQH LI FKROHVWHURO FRQWHQW LV DQG PL[HG VWRQH LI WKH FKROHVWHURO FRQWHQW LV

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  %DFWHULD RU SDUDVLWH LQGXFH ȕ glucuronidase which deconjugated saturated bilirubin and cause increased of unconjugated bilirubin which can form complex with calcium Ileal disease, ileal resection/ bypass Malabsorption of bile acid in the ileum which increase bile acid in the colon and saturate unconjugated bilirubin and increase its passive absorption in the colon (pathological enterohepatic recirculation of bilirubin).

  Pernicious anaemia Ineffective erythropoiesis due to YLWDPLQ % RU IRODWH GH¿FLHQF\ Chronic infection of biliary tract

  0HFKDQLVP Demography (Asia) Genetic factor Chronic haemolysis Increased of biliary unconjugated bilirubin which can precipitate with calcium

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  3LJPHQW JDOOVWRQH LV FODVVL¿HG LQWR EODFN DQG EURZQ SLJPHQW VWRQH %ODFN VWRQH LV VPDOO LQ VL]H LUUHJXODU and formed in the gallbladder. Black pigment stone is formed from pure bilirubinate calcium or polymer like complex with calcium and glycoprotein mucin. Black pigment stone is associated with chronic haemolysis or cirrhosis. Brown pigment stone is formed in the bile duct. Components of brown pigment stone consist of calcium salt and unconjugated bilirubin with varied number of cholesterol and protein. Brown pigment stone is associated with bacterial or parasitic infection in the bile duct. Picture of black and brown pigment

  ,Q QRUPDO FRQGLWLRQ KHPRJORELQ EUHDNGRZQ SURGXFW WKH ELOLUXELQ ZLOO XQGHUJR FRQMXJDWLRQ LQ WKH liver into conjugated bilirubin which is more soluble in water. Conjugated bilirubin will be hydrolysed by beta glucoronidase bacteria in the colon into unconjugated bilirubin which can precipitate as calcium salt or even catabolised by bacteria into urobilinogen. Pigment stone is formed due to the increase of unconjugated bilirubin in the bile. Predisposition factors and mechanism of SLJPHQW VWRQH IRUPDWLRQ FDQ EH VHHQ LQ 7DEOH

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  VXFK DV PXFLQ DQG LPPXQRJOREXOLQ RU GH¿FLHQF\ of anti-nucleation factors such as apolipoprotein AI and AII. The third mechanism is the presence of gallbladder hypomotility. Gallbladder emptying disorder which contains crystal or bile acid which undergo supersaturation with cholesterol supports gallstone formation. Factors that support cholesterol gallstone formation can be seen in Table 1. Picture of FKROHVWHURO JDOOVWRQH LV VKRZQ LQ )LJXUH

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  Gallstone Analysis

  DQG WXEHV DUH FHQWULIXJHG J IRU PLQXWHV DQG the supernatant is being extracted. The second one is as follow: stone is made into powder using pestle

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  VSHFWURPHWU\ XVHV VLPLODU VDPSOH SUHSDUDWLRQ ZLWK R FUHVROSKWKDOHLQ PHWKRG 6DPSOH LV KHDWHG LQ ÀDPH thus the element in the sample will dissociate from

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  RI K\GUR[\TXLQROLQH HOLPLQDWHV WKH LQWHUIHUHQFH RI magnesium and iron. Chemical reaction in calcium analysis is shown in Figure 6. Increased of absorbance in reaction is line with calcium concentration in the sample.

  WR YROXPHWULF IODVN $GG GURSV RI FRQFHQWUDWHG HCl and 1 mL of water. This mixture is used to analyse calcium. ¹⁰ Principle of examination using o-cresolphthalein complexon method is calcium ion will react with o-cresolpthalein complex which in alkaline solution will form violet coloured complex

  7KH ¿UVW PHWKRG LV R FUHVROSKWKDOHLQ FRPSOH[RQ (non-enzymatic colorimetry) with sample preparation

  Calcium analysis in gallstone with colorimetry PHWKRG FDQ EH SHUIRUPHG XVLQJ PHWKRGV ZKLFK are o-cresolphthalein complexon (non-enzymatic FRORULPHWU\ DWRPLF DEVRUSWLRQ VSHFWURPHWU\ DQG PRGL¿FDWLRQ RI R FUHVROSKWKDOHLQ FRPSOH[RQ DIWHU ashing the sample). ¹⁰

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  XQLW —PRO / %LOLUXELQ ZHLJKW FDQ EH REWDLQHG by inserting the result to the formula of cholesterol examination with colorimetry method. Bilirubin weight is then compared to total stone weight which is evaluated to obtain its percentage.

  Results from the chemical reaction is examined at

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  The second process is measuring bilirubin level by using colorimetry principles. Results of extraction from WKH ¿UVW SURFHVV LV XVHG DV WKH LQJUHGLHQW WR GHWHUPLQH total bilirubin concentration. Chemical reaction in

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  ,Q WKH DQDO\VLV RI WRWDO ELOLUXELQ LQ JDOOVWRQH WKHUH DUH SURFHVVHV WKH VDPSOH H[WUDFWLRQ DQG WKH SURFHVV of bilirubin level measurement using colorimetry principle. First process is sample extraction process from the gallstone which is performed by the following SURFHGXUH JDOOVWRQH LV EHLQJ ZDVKHG DQG GULHG IURP which 0.1 gram is taken. The stone is crushed using mortar and 10 mg of the stone powder is placed in D WXEH $GG —O GLPHWK\OVXOSKR[LGH '062 WR dissolve unconjugated bilirubin and incubate for half RI DQ KRXU $GG —O +&O PRO / WR WKH WXEH DQG incubate again for another half of an hour to separate

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  Cholesterol weight can later be compared with total weight of the stone which is examined to obtain its percentage.

  5HDFWLRQ UHVXOWV TXLQRQH LPLQ LV UHDG DW nm wavelength. Cholesterol level in molarity unit PPRO / )RUPXOD EHORZ LV XVHG WR REWDLQ FKROHVWHURO weight in stone:

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  UHVXOW ZKLFK LV REWDLQHG IURP WKH ¿UVW RU VHFRQG SURFHVV is used as the substance to determine its cholesterol level. Chemical reactions in cholesterol examination is shown in Figure 4.

  FKORURIRUP WR PJ VWRQH SRZGHU LQ D WXEH ZKLFK LV ERLOHG LQ ZDWHUEDWK IRU PLQXWHV The second process is the measurement of cholesterol level using colorimetry method. Extraction

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  7DEOH VKRZHG IDWW\ DFLG FRQWHQW in different type of gallstone.

  Fatty acid in the form of methylester derivatives (fatty acid methyl estered) is made by following PHWKRG LQWR —J IDWW\ DFLG VDPSOH ZKLFK LV PDGH EHIRUH P/ K\GURFKORULF DFLG LV EHLQJ DGGHG and heated in 60 C in boiling water for 4 hours.

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  Fatty acid chromatogram in gallstone sample can be

  The duration of component inside the column in this condition is called retention time. Retention time which is produced in each fatty acid can identify fatty DFLG LQ WKH VDPSOH 4XDQWL¿FDWLRQ LV SHUIRUPHG E\ comparing sample peak area with standard peak area.

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  Chromatography is a method to separate and identify components in a mixture. The process is as follow: sample is injected and the sample component ZLOO EH WUDQVIHUUHG E\ KHOLXP JDV ÀRZ DV PRELOH SKDVH through the column. The column contains chemically ERXQG IXVHG VLOLFD 29 SRO\VLOR[DQH LQ FURPRVUE W as its stationary phase which is used to separate fatty acids. Each component will move together with the mobile phase in different velocity which is later detected by a detector. Detector signals are then being plotted as chromatogram. Process of GLC method H[DPLQDWLRQ FDQ EH VHHQ LQ )LJXUH

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  C for 1 hour. After EHLQJ FHQWULIXJHG WKH VXSHUQDWDQW LV WDNHQ DQG WKHQ the residue is being extracted again using hexane-butyl alcohol solution. The mixture is evaporated until it is dried and fatty acid content is obtained. Myristic acid in hexane-butyl alcohol is used as internal standard.

  Fransisca Putri, Nuri Dyah Indrasari its chemical bond (atomised) and is in ground state.

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  Sample preparation in examination using GLC method is as follows: gallstone is washed using deionized distilled water until it is clean. The stone is crushed using mortar into a homogenous powder.

  VDWXUDWHG IDWW\ DFLG VXFK DV SDOPLWLF DFLG ODXULF DFLG VWHDULF DFLG PD\ SUHFLSLWDWH DQG GHFUHDVH WKH capacity of cholesterol solution which supports the formation of cholesterol gallstone. Examination using this method is to obtain the fatty acid content DQG VDWXUDWHG WR XQVDWXUDWHG IDWW\ DFLG UDWLR 6 8 LQ cholesterol or pigment gallstone. Gallstone analysis using GLC method can be performed by fatty acid chain separation and evaluation of fatty acid quantity in the form of methylester derivatives which is fatty acid methyl estered.

  Fatty acid in the gallstone may originate from phospholipid bile acid which is hydrolysed due to the presence of phospholipase. Fatty acid (particularly

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  4. Ginting S. A description characteristic risk factor of the kolelitiasis disease in the Colombia Asia Medan Hospital.

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  6DHW\ 62 HW DO 4XDQWLWDWLYH DQDO\VLV RI JDOOVWRQHV LQ /LE\DQ SDWLHQWV /LE\DQ - 0HG Erpecum KJV. Pathogenesis of cholesterol and pigmen gallstone: an update. Clin res hepatol gastroenterol

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  US Department of health and human services. Gallstone.

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