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1 J. Exp. Biomed. Sci. 13 (2007) Effects of Intravenous Administration of Taurocholic Acid on Hepatic Catechol-O-Methyltransferase Activity in Rats with Choledocho-Caval Shunt Jun-Young Do 1, Kyo-Cheol Mun, You-Hee Kim and Chun-Sik Kwak Department of Biochemistry, Keimyung University, School of Medicine, Daegu , Korea. 1 Department of Internal Medicine, Yeungnam University, College of Medicine, Daegu , Korea The possible mechanism of decreased catechol-o-methyltransferase (COMT) activity in cholestatic rat liver was studied. Hepatic and serum were determined from the experimental rats with choledocho-caval shunt (CCS). The Michaelis-Menten constants in this hepatic enzyme was also measured. The activities of cytosolic, mitochondrial and mircosomal COMT as well as their V max values were found to be decreased significantly in CCS plus taurocholic acid (TCA) injected group than in the control group, such as CCS alone groups. However, their K m values in the experimental groups did not vary. Seru4m COMT activity increased slightly in the CCS plus TCA injected group than in the control group. The above results suggest that TCA represses biosynthesis of the COMT in the liver. The elevated activity of the serum COMT is believed to be caused by the increment of membrane permeability of hepatocytes upon TCA mediated liver cell necrosis. Key Words: Catechol-O-methyltransferase, Choledocho-caval shunt, Taurocholic acid 서 간에담즙울체가야기되면간과혈청에서는많은효소들의활성도가변동되며그활성도변동에는담즙울체간에축적된 taurocholic acid (TCA) 가관여하는것 (Han and Kim, 1997; Kim and Kim, 1997; Rhee and Kwak, 2000; Kim and Shin, 2002; Rhee and Kwak, 2002; Rhee and Kwak, 2004) 으로알려져있다. 이러한연구를하기위해서는두가지동물모델을만들어실험을하는데 (Ogawa et al., 1990; Park and Kwak, 1999) 그첫째모델은쥐에게총담관결찰 (common bile duct ligation) 로담관을폐쇄시킨후 TCA를혈중에주입하는모델이며둘째모델은쥐에게총담관대정맥문합 (choledocho -caval shunt) 을시킨후 TCA를혈중에주입하는모델이다. 특히이둘째모델인총담관대정맥문합직후 TCA를상대정맥내주입한모델로는간내 TCA의저농도증가에따른 TCA 효과를알아낼수가있으며 (Ogawa et al., 1990; Park and Kwak, 1999) 이모델의특징은첫째모델보다 TCA 효 * 논문접수 : 2007년 3월 5일수정재접수 : 2007년 5월 25일 교신저자 : 곽춘식, ( 우 ) 대구광역시중구동산동 194번지, 계명대학교의과대학생화학교실 Tel: , Fax: kwak@dsmc.or.kr 론 과를더욱민감하게알아낼수가있다는것 (Choi et al., 2004) 이다. 이러한둘째모델로간에서생체이물생체변환효소 (xenobiotic biotransformation enzyme) 의활성도변동을조사한보고는많다. 즉, catalase, alcohol dehydrogenase, microsomal ethanol oxidizing system, aldehyde dehydrogenase (Kim and Shin, 2002), arylesterase (Han and Kim, 1997), arylamine N-methyltransferase (Rhee and Kwak, 2000), thiol methyltransferase (Rhee and Kwak, 2002), thiosulfate sulfurtransferase (Rhee and Kwak, 2004) 등이며 TCA가이들효소의합성을조절하여이들효소의합성속도를변동시킨다는것이다. 따라서총담관대정맥문합과함께 TCA를혈중에주입시킨동물모델의간에서그활성도가변동되는효소들에대해서는 TCA의효과를더욱분명하게알수가있을것이다. Catechol-O-methyltransferase (S-adenosyl-L-methionine: catechol -O-methyltransferase, EC , COMT) 는제 2상생체이물생체변환효소의일종이며 catechol 및 catecholamine에 S-adenosyl-L-methionine으로부터메틸기를이전받아 catechol 또는 catecholamine을메틸화시키는효소이다 (Kim, 1979; Borchardt, 1980). COMT는간세포의세포질, 미토콘드리아및내혈질세망에국재되어있다 (Borchardt, 1980; Raxworthy et al., 1982; Mun, 1996). 이효소는담즙울체간에서활성도가감소됨이 (Mun, 1996) 알려져있을뿐만아니라 TCA에의해

2 서이들효소의합성이억제된다고추정하고있다 (Do and Kwak, 2005). 이연구는 COMT의활성도가담즙울체간에서감소되는기전을분명하게알아내기위하여시행하였으며, 쥐에게총담관대정맥문합을시킨직후의담즙울체간에서효소유전자의발현율을변동시키는것으로추정하는 TCA (Ogawa et al., 1990; Han and Kim, 1997; Kim and Kim, 1997) 와간의효소합성에영향을미치지않는다는 Tauroursodeoxycholic acid (TUDCA) (Ogawa et al., 1990; Han and Kim, 1997; Kim and Kim, 1997) 를각각상대정맥내에주입한후경시적으로간과혈청에서 COMT의활성도를측정하여담즙울체간에서이효소의활성도감소기전의일부를밝혀보고자하였다. 재료및방법 1. 시약 S-(5'-adenosyl)-L-methionine iodide, 3,4-dihydroxybenzoic acid, DL-dithiothreitol, Triton X-100, catechol-o-methyltransferase (from porcine liver, C 1897), TCA (from ox bile, sodium salt, T 0750) TUDCA (sodium salt, T 0266) 및단백질표준액 (10 g/100 ml bovine albumin) 등은 Sigma사 (St. Louis, MO, USA) 제품을사용하였으며 [methyl- 3 H] S-adenosyl-L-methionine은 New England Nuclear사 (Boston, MA, USA) 의제품을, 그리고 PPO (2,5-diphenyloxazole), bis-msb [ρ-bis-(o-methylstyryl benzene)], toluene (scintillation grade) 등은 Packard사 (Downers Grove, IL, USA) 의제품을, 사용하였다. 그외시약은시판되는특급또는일급품을사용하였다. 2. 동물및처치동물은 4주이상같은조건으로사육한체중 280~320 g이되는 Sprague-Dawley종의숫쥐를사용하였으며 1군을 5마리로하여다음과같이 9개군으로나누었다. 1) 정상군 (1군). 2) 가수술 (sham operation) 군가수술후 1일및 2일에각각희생시킨군 ( 총 2군 ). 3) 총담관대정맥문합 (choledocho-caval shunt) 군총담관대정맥문합후 1일및 2일에각각희생시킨군 ( 총 2군 ). 4) 총담관대정맥문합과함께 TCA를주입한군총담관대정맥문합직후 Ogawa et al. (1990) 과 Park and Kwak (1999) 의방법에따라 TCA ( 체중 100 g당 45 µmoles) 를상대정맥내에주입한후 1일및 2일에각각희생시킨군 ( 총 2군 ). 5) 총담관대정맥문합과함께 TUDCA를주입한군총담관대정맥문합직후 Ogawa et al. (1990) 과 Park and Kwak (1999) 의방법에준하여 TUDCA ( 체중 100 g당 45 µmoles) 를상대정맥내에주입한후 1일및 2일에각각희생시킨군 ( 총 2군 ). 각실험군은개별분리수용하였으며실험전후에일정한조건으로사육하였다. 사료는삼양유지사료주식회사제품인실험동물사료를먹도록하였다. 총담관대정맥문합수술및가수술은효소활성의일중변동을고려하여쥐를오후 2시에서 5시사이에희생시킬수있도록수술시간을조절하였으며 12시간금식시킨후이터마취하에서실시하였다. 총담관대정맥문합은상대정맥과총담관을 medical grade silicon tube를사용하여연결하였으며가수술은단순개복술만시행하였다. TCA 및 TUDCA액의상대정맥내주입은 syringe pump (model 341A, Sage instruments, USA) 를사용하여 15분간주입하였다. 3. 간적출및세포분획모든실험군에서간의적출은 12시간금식시킨후이터마취하에서시행하였으며복부대동맥으로부터채혈하여쥐를실혈사시켰다. 그리고는간문맥에삽관한후 4 의 0.25 M sucrose액으로관류하여간에남아있던혈액을제거한다음간을적출하였다. 적출한간은면포로균등히압박하여간에남아있던 sucrose액을가능한한모두제거하였다. 채혈한혈액은원심분리하여혈청은얻고곧효소활성도를측정하였다. 간의세포분획은적출한간을즉시 2~4 로냉각한후잘게썰어서절편으로만들고혼합하여그중약 5 g을취하여 9배량의 0.25 M sucrose액을넣은다음 Teflon pestle glass homogenizer (chamber clearance 0.005~0.007 inches, Thomas Co., USA) 로 2~4 를유지하면서 400 rpm의속도로조심스럽게 5회왕복마쇄하여 10% (w/v) 의간조직균질액을만들었다. 이간균질액모두를취하여 sucrose density gradient 원심분리법 (Kwak and Kwak, 1986) 으로세포질, 미토콘드리아및마이크로솜분획을분리하였다. 위의세포분획법에서모든조작은 2~4 에서시행하였으며이때사용한원시분리기는 Du Pont Sorvall사 (USA) 의 RC-5B refrigerated superspeed centrifuge와 OTD-65B ultracentrifuge였다. 이때사용한 rotor 는 Du Pont Sorvall사의 SS-34 및 T865 rotor였고 sucrose linear density gradient 용액의제조는 gradient former (model 570, ISCO, USA) 를사용하였다. 4. 효소시료조제 COMT 활성도측정용효소시료의조제는분리한마이크로솜분획및미토콘드리아분획 4.5 ml에대해서 0.5 ml의 sucrose-triton X-100 (Triton X ml와 sucrose 8.56 g을증류수에녹여 100 ml로만든다 ) 액을넣어 4 에서 30분간

3 Table 1. Effects of choledocho-caval shunt (CCS) on hepatic subcellular catechol-o-methyltransferase (COMT) activities in rats Experimental groups (pmol 3-hydroxy-4-methoxybenzoic acid with 4-hydroxy-3-methoxybenzoic acid min -1 mg protein -1 ) Cytosol Mitochondria Microsome Normal 2,862± ±19 417±60 Sham 1 day 2,889± ±22 424±66 Sham 2 days 2,907± ±17 429±63 CCS 1 day 2,782±412 92±19 375±46 CCS 2 days 2,716±397 88±15 354±40 The data are expressed as mean ± SD with 5 rats in each group; Sham 1 day or Sham 2 days, sacrificed on the 1st day or 2nd day after sham operation; CCS 1 day or CCS 2 days, sacrificed on the 1st day or 2nd day after choledocho-caval shunt. 방치한후이액을이효소활성도측정용시료로사용하였으며세포질분획은아무런처치없이원액그대로사용하였다. 5. 효소활성도측정혈청과간세포질, 미토콘드리아및마이크로솜분획의 COMT 활성도측정은효소시료와함께 3, 4-dihydroxybenzoic acid와 [methyl- 3 H] S-adenosyl-L-methionine이함유된 S-(5'-adenosyl)-L-methionine iodide를기질로사용하여 37 에서 30분간반응시키는동안에생성된방사성 3-hydroxy -4-methoxybenzoic acid와 4-hydroxy-3-methoxybenzoic acid를 toluene-isoamylalcohol (7 : 3) 혼합액으로추출한후그방사능을측정하여효소의활성도를산출하는 Borchardt (1981) 의법에준하였으며, 효소활성도단위는 1 분간에 1 mg의단백질이반응하여생성한 3-hydroxy-4-methoxybenzoic acid와 4-hydroxy-3-methoxybenzoic acid의총량을 pmol로나타내었다. 이실험에서채택한효소활성도측정법의정확도를높이기위하여같은시료에대하여 2회측정하여그평균치를취하였다. 이실험에서사용한방사능계측기는 Packard Tricarb 4530, liquid scintillation spectrometer (Packard Co., Dowenrs Grove, IL, USA) 였다. 6. K m 값및 V max 값의측정수술후 2일경과한모든실험군의세포분획효소시료들과 COMT의 2종기질중 3, 4-dihydroxybenzoic acid를선택하여이기질의원액과희석액을제조한후이기질액들과 [methyl- 3 H] S-adenosyl-L-methionine이함유된 S-(5'-adenosyl) -L-methionine iodide 원액을사용하여 COMT의활성도를측정한후이들성적으로부터 1/vi값을그리고기질농도로부터 1/[S] 값을계산하여이중역수도 (double reciprocal plot) 를그린다음이것으로부터 K m 값과 V max 값을산출하였다. 7. 단백질정량효소시료중의단백질정량은 0.5 M perchloric acid와 methanol-ether 혼합액 (3 : 1) 으로단백질을정제하는 Greenberg and Rothstein (1957) 법으로효소시료중의단백질을정제한다음 biuret법 (Gornall et al., 1949) 으로정량하였다. 8. 성적검정유의성검정은 Student's t-test로하였으며유의수준은 0.05 이하로하였다. 결과 1. 쥐에서총담관대정맥문합시 TCA 또는 TUDCA 주입이간의 COMT 활성도에미치는영향쥐에게총담관대정맥문합을시킨후 1일및 2일째 ( 결과 Table 에서 CCS 1 day 및 CCS 2 days) 간의세포질액, 미토콘드리아및마이크로솜분획의 COMT 활성도는변동을나타내지않았다 (Table 1). 쥐에게총담관대정맥문합을시킨직후 TCA를주입시켜 1일및 2일 ( 결과 Table 에서 CCS 1 day + TCA 및 CCS 2 days + TCA) 경과시켰을때간의세포질액과미토콘드리아및마이크로솜분획의 COMT 활성도는대조군인총담관대정맥문합만시킨군에비해통계학적으로유의한감소를나타내었다. 즉총담관대정맥문합직후 TCA를주입시키고 1일및 2일경과시켰을때간세포질액의 COMT 활성도는대조군인총담관대정맥문합만시킨군 ( 결과 Table 에서 CCS 1 day 및 CCS 2 days) 보다각각약 20% (P<0.05) 및약 21% (P<0.05) 의감소를나타내었고간미토콘드리아분획의 COMT 활성도는대조군인총담관대정맥문합만시킨군보다각각약 30% (P<0.05) 및약 34% (P<0.01) 의감소를나타내었으며간마이크로솜분획의이효소활성도는대조군인총담관대정맥문합만시킨군보다각각약 34% (P< 0.001) 및약 39% (P<0.001) 의감소를나타내었다 (Table 2). 한편총담관대정맥문합을시킨직후 TUDCA를주입시켜 1일및 2일 ( 결과 Table에서 CCS 1 day + TUDCA 및 CCS 2 days + TUDCA) 경과시켰을때는간의 3종세포분획에서

4 Table 2. Effects of taurocholic acid (TCA), and tauroursodeoxycholic acid (TUDCA) infusions after choledocho-caval shunt (CCS) on hepatic subcellular catechol-o-methyltransferase (COMT) activities in rats Experimental groups (pmol 3-hydroxy-4-methoxybenzoic acid with 4-hydroxy-3-methoxybenzoic acid min -1 mg protein -1 ) Cytosol Mitochondria Microsome CCS 1 day 2,782±412 92±19 375±46 CCS 1 day + TCA 2,216±343 j 64±11 j 246±34 l CCS 1 day + TUDCA 2,806±425 86±17 383±51 CCS 2 days 2,716±397 88±15 354±40 CCS 2 days + TCA 2,158±358 m 58±9 n 215±32 o CCS 2 days + TUDCA 2,687±373 84±13 359±43 The data are expressed as mean ± SD with 5 rats in each group; CCS 1 day or CCS 2 days, sacrificed 1st or 2nd day after choledocho-caval shunt; One of the following bile acids, TCA or TUDCA (45 µmoles/100 g body weight) was intravenously administered through the superior vena cava. j, P<0.05 vs. CCS 1 day; l, P<0.001 vs. CCS 1 day; m, P<0.05 vs. CCS 2 days; n, P<0.01 vs. CCS 2 days; o, P<0.001 vs. CCS 2 days Table 3. Rat hepatic catechol-o-methyltransferase (COMT) kinetic parameters from 2 days after choledocho-caval shunt (CCS 2 days) determined with 3, 4-dihydroxybenzoic acid Experimental groups Cytosol Mitochondria Microsome K m V max K m V max K m V max (K m ; mm, V max ; pmol 3-hydroxy-4-methoxybenzoic acid with 4-hydroxy-3-methoxybenzoic acid min -1 mg protein -1 ) Sham 2 days 2.82±0.63 4,912± ± ± ± ±98 CCS 2 days 2.97±0.68 4,592± ± ± ± ±58 CCS 2 days + TCA 3.12±0.75 3,562±552 g,m 5.04± ±12 i,m 3.36± ±47 i,o CCS 2 days + TUDCA 2.93±0.71 4,517± ± ± ± ±61 Michaelis-Menten constants for COMT were determined using 3, 4-dihydroxybenzoic acid, S-adenosyl-L-methionine iodide and [methyl- 3 H]S-adenosyl-L-methionine from experimental rat livers at two days after CCS. The data are expressed as mean ± SD with 5 rats in each group. Experimental groups are described in Table 1, 2 and text. g, P<0.05 vs. Sham 2 days; i, P<0.001 vs. Sham 2 days; m, P<0.05 vs. CCS 2 days; o, P<0.001 vs. CCS 2 days COMT 활성도는모두대조군과통계학적으로유의한차이는없었다 (Table 2). 2. 쥐에서총담관대정맥문합후 2일경과한실험군에서간 COMT 의 K m 값및 V max 값의변동수술후 2일경과시킨모든실험군에서간세포질액과미토콘드리아및마이크로솜분획의 COMT를 3, 4-dihydroxybenzoic acid를기질로사용하여 K m 값및 V max 값을측정했을때 K m 값은모두변동이없었다. 또한수술후 2일경과시킨실험군에서간세포질액, 미토콘드리아및마이크로솜분획의 COMT의 V max 값도모두통계학적으로유의한변동은없었다 (Table 3). 쥐에게총담관대정맥문합을시킨직후 TCA를주입하고 2일 ( 결과 Table 에서 CCS 2 days + TCA) 경과시켰을때간세포질액, 미토콘드리아및마이크로솜분획의 COMT의 V max 값은가수술만시킨군 ( 결과 Table 에서 Sham 2 days) 보다는각각약 27% (P<0.05), 약 51% (P<0.001) 및약 50% (P<0.001), 총담관대정맥문합만시킨군 ( 결과 Table 에서 CCS 2 days) 보다는각각약 22% (P<0.05), 약 21% (P<0.05) 및약 41% (P<0.001) 의감소를나타내었다. 그러나총담관대정맥문합을시킨직후 TUDCA를주입하고 2일 ( 결과 Table 에서 CCS 2 days + TUDCA) 경과시켰을때는이효소의 V max 값은유의한변동을나타내지않았다 (Table 3). 3. 쥐에서총담관대정맥문합과담즙정체시간이혈청의 COMT 활성도에미치는영향정상쥐와가수술을시행한쥐의혈청에서는 COMT의활성이측정되지않았다. 그러나쥐에게총담관대정맥문합을시켰을때는혈청의 COMT 활성도가현저하게높았다. 즉혈청의 COMT 활성도는총담관대정맥문합후 1일경과시켰을때는 63±16.3 pmol 3-hydroxy-4-methoxybenzoic acid with 4-hydroxy-3-methoxybenzoic acid min -1 mg protein -1 ( 이하단위생략함 ) 이었으며총담관대정맥문합후 2일경과시켰을때는 76±19.6이었다 (Table 4). 4. 쥐에서총담관대정맥문합시 TCA 또는 TUDCA 주입이혈청 COMT 활성도에미치는영향쥐에게총담관대정맥문합을시킨직후 TCA를주입시키

5 Table 4. Effects of choledocho-caval shunt (CCS) on serum catechol-o-methyltransferase (COMT) activities in rats Experimental groups (pmol 3-hydroxy-4-methoxybenzoic acid with 4-hydroxy-3-methoxybenzoic acid min -1 mg protein -1 ) Normal Undetectable Sham 1 day Undetectable Sham 2 days Undetectable CCS 1 day 63±16.3 CCS 2 days 76±19.6 The data are expressed as mean ± SD with 5 rats in each group. Experimental groups are described in Table 1 and text. Table 5. Effects of taurocholic acid (TCA), and tauroursodeoxycholic acid (TUDCA) infusions after choledocho-caval shunt (CCS) on serum catechol-o-methyltransferase (COMT) activities in rats Experimental groups (pmol 3-hydroxy-4-methoxybenzoic acid with 4-hydroxy-3-methoxybenzoic acid min -1 mg protein -1 ) CCS 1 day 63±16.3 CCS 1 day + TCA 89±24.8 CCS 1 day + TUDCA 65±17.5 CCS 2 days 76±19.6 CCS 2 days + TCA 104±23.4 CCS 2 days + TUDCA 72±22.7 The data are expressed as mean ± SD with 5 rats in each group; Experimental groups are described in Table 2 and text. 고 1일및 2일경과시켰을때의혈청 COMT의활성도는대조군인총담관대정맥문합만시킨군에비해증가되었으나통계학적유의성은없었다. 그리고총담관대정맥문합직후 TUDCA를주입시켜 1일및 2일경과시켰을때혈청의 COMT 활성도는대조군과별차이가없었다 (Table 5). 고 쥐에서담즙울체를야기시켜간이손상을받으면생체이물생체변화효소인간의 COMT 활성도가감소되는것 (Mun, 1996) 은알려져있으나그기전은분명하게밝혀져있지않다. 따라서쥐담즙울체간의세포질, 미토콘드리아및마이크로솜분획에서이효소의활성도변동기전을파악해봄으로써담즙울체간에서생체이물의생체변환에대한새로운지견을얻을수있을것으로생각되며아울러담즙울체로간손상이야기되는간담도질환에대한간의해독기능의일단이밝혀질것으로생각된다. Mun (1996) 에의하면쥐의총담관을결찰한후의담즙울 찰 체간에서세포질, 미토콘드리아및마이크로솜의 COMT 활성도는총담관결찰후 1, 2, 3, 7, 14, 28 및 42일에통계학적으로유의한감소를나타내었다고하였고, 혈청 COMT는정상쥐에서는나타나지않았으나총담관을결찰한쥐에서는실험전기간동안높은활성도를나타내었다고하였다. 그러나이번실험에서총담관대정맥문합만시행한간에서는 COMT의활성도는변동이없었다. 이현상은간에담즙울체의정도가미약하여서나타난결과로생각된다. Do and Kwak (2005) 은쥐를사용한실험에서총담관결찰후혈중에 TCA를주입한결과간의세포질액, 매토콘드리아, 마이크로솜에서 COMT의활성도가감소되었고혈청에서는 COMT가출현함과아울러그활성도가증가하였다고하였으며이결과는 TCA가간에서이효소의합성을억제하고또한간에서이효소의혈중유출을증가시켜나타난결과라고추론하였다. 이번실험은이사실을더욱분명하게알아내기위하여시행한것이며또한이실험에서는주입하는담즙산의종류가다르면 COMT 활성도에미치는효과도달라지는가를알아내기위하여담즙울체간에서효소들의합성에영향을주지않으며 (Ogawa et al., 1990; Kim and Kim, 1997; Rhee and Kwak, 2000) 담즙산의간독성에대해보호효과를가진다는 TUDCA (Poupon et al., 1987; Ogawa et al., 1990; Heuman et al., 1991) 를쥐에총담관대정맥문합을시킨직후상대정맥내에다량주입시켜 TUDCA의효과도관찰하여보았다. 이실험에서쥐에게총담관대정맥문합을시킨직후 TCA 를주입시켜 1일및 2일경과시켰을때간세포질액과미토콘드리아및마이크로솜분획의 COMT 활성도는대조군인총담관대정맥문합만시킨군보다유의한감소를나타내었다. 한편쥐에게총담관대정맥문합직후 TCA를주입하고 2일경과시켰을때간세포질액과미토콘드리아및마이크로솜분획의 COMT의 V max 값은대조군인총담관대정맥문합만시킨군보다유의한감소를나타내었다. 그러나이효소의 K m 값은모든실험군의간세포분획에서유의한변동을나타내지않았다. 이상의결과로보아 TCA는간의 COMT 합성을억제한다고확신할수있으며특히 TCA가 COMT 저해제 (inhibitor) 가아니라는사실 (Zollner, 1993) 과 TCA를주입한실험군에서이효소의 K m 값은변동이없으면서즉촉매효율의변동이없으면서이효소의활성도와 V max 값이감소된것은위의추론을더욱분명하게해주는결과라고생각한다. 쥐에게총담관대정맥문합을시킨직후 TUDCA를주입하고 1일및 2일경과시켰을때간세포분획과혈청에서 COMT 활성도는모두대조군의그것과별차이가없었다. 이결과를볼때 TUDCA는간의 COMT 합성에는관여하지않는다고추정할수가있었다

6 쥐에게총담관대정맥문합을시킨직후 TCA를주입하고 1일및 2일경과시켰을때혈청 COMT 활성도는대조군인총담관대정맥문합만시킨군보다약간증가되었다. 이결과로보아담즙울체시간내에 TCA의농도가증가되면혈청의 COMT 활성도도증가된다는것을알수있으며그증가의원인은 TCA에의한간의괴사 (Palmer, 1972; Drew and Priestly, 1979; Kitani et al., 1986) 로간세포막이손상되어간에서혈중으로이들효소의유출이증가되어나타난결과로생각된다. 이상이실험결과와문헌상의지견을종합해볼때담즙울체간에서 COMT의활성도감소는담즙산중 TCA에의해이효소의합성이억제되어나타난결과이며아울러담즙울체시이효소의혈중활성도증가는간의괴사로간세포막의투과성이항진되어이효소가혈중으로유출되어나타난결과로생각된다. REFERENCES Borchardt RT. N- and O-methylation in Enzymatic basis of detoxication (Jakoby WB. Ed) Vol II, pp Academic Press. NY, USA. Borchardt RT. Catechol-O-methyltransferase in Method in Enzymology (Jakoby WB. Ed) Vol 77, pp Academic Press. NY, USA. Choi HJ, Kim YH, Kwak CS. Effects of high taurocholic acid load on liver lysosomal cathepsin B and D, and acid phosphatase activities in rats with choledocho-caval shunt. J Exp Biomed Sci : Do JY, Kwak CS. Effects of intravenous administration of taurocholate on hepatic catechol-o-methyltransferase activity in common bile duct ligated rats. J Exp Biomed Sci : Drew R, Priestly BG. Choleretic and cholestatic effects of infused bile salts in the rat. Experientia : Gornall AG, Bardawill CJ, David MM. Determination of serum protein by means of biuret reaction. J Biol Chem : Greenberg DM, Rothstein M. Method for isolation and degradation of labelled compounds. in Method in enzymology (Colowick SP, Kaplan NO. Eds) Vol 4, pp Academic Press. NY, USA. Han BH, Kim YH. Effect of high taurocholate load on activity of rat liver arylesterase. Korean J Hepatol : Heuman DM, Mills AS, McCall J, Hylemon PB, Pandak WM, Vlahcevic ZR. Conjugates of ursodeoxycholate protect against cholestasis and hepatocellular necrosis caused by more hydrophobic bile salts: In vivo studies in the rat. Gastroenterology : Kim BK. Enzyme Nomenclature, IUB pp Academic Press. NY, USA. Kim SK, Kim YH. Induction of rat liver γ-glutamyl transpeptidase by bile acid load. Korean J Hepatol : Kim YH, Shin MJ. Effects of high taurocholated load on activities of hepatic alcohol metabolizing enzymes. Exp Mol Med : Kitani K, Kanai S, Ohta M, Sato Y. Differing transport maxima values for taurine-conjugated bile salts in rats and hamsters. Am J Physiol : G852-G858. Kwak CS, Kwak JS. Cell fractionation method of the rat liver. 1. Isolations of mitochondria and microsome. The Keimyung Univ Med J : Mun KC. Catechol-O-methyltransferase activity in cholestatic rat's liver induced by bile duct ligation. J Biochem Mol Biol. 1996; 29: Ogawa H, Mink J, Hardison WGM, Miyai K. Alkaline phosphatase activity in hepatic tissue and serum correlates with amount and type of bile acid load. Lab Invest : Palmer RH. Bile acids, liver injury, and liver disease. Arch Intern Med : Park SK, Kwak CS. Repression of rat hepatic cholinesterase by bile acid load. Keimyung Med J : Poupon R, Poupon RE, Calmus Y, Chritien Y, Ballet F, Darnis F. Is ursodeoxycholic acid an effective treatment for primary biliary cirrhosis? Lancet : Raxworthy MJ, Gulliver PA, Hughes PJ. The cellular location of catechol-o-methyltransferase in rat liver. Naunyn -Schmiedeberg's Arch Pharmacol. 1982; 320: Rhee BW, Kwak CS. Induction of hepatic arylamine N-methyltransferase by a tautocholate load in rats. J Korean Surg Soc : Rhee BW, Kwak CS. Effects of intravenous administration of taurocholate on hepatic thiol methyltransferase activity in cholestatic rat. J Korean Surg Soc : Rhee BW. Kwak CS. Effects of intravenous administration of taurocholate on liver and serum thiosulfate sulfurtransferase activities in cholestatic rat. J Korean Surg Soc : Zollner H. Handbook of enzyme inhibitors. 2nd ed Part B, pp 971. VCH verlagsgesellschaft mbh. Weinheim, Germany

Microsoft Word - 07-곽춘식.doc

Microsoft Word - 07-곽춘식.doc J. Exp. Biomed. Sci. 11 (2005) 45 49 Catechol-O-Methyltransferase Activity from Regenerating Liver after Partial Hepatectomy in Rats You-Hee Kim, Hye-Jung Choi and Chun-Sik Kwak Department of Biochemistry,