Significance of Blood Ketone Body Ratio as an Indicator of Hepatic Cellular Energy Status in Jaundiced Rabbits

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1 GASTROENTEROLOGY 76: , ]979 Significance f Bld Ketne Bdy Rati as an ndicatr f Hepatic Cellular Energy Status in Jaundiced Rabbits JUNJ TANAKA, M.D., KAZUE OZAWA, M.D., and TAKA YOSH TOBE, M.D. Department f Surgery, Kyt University Medical Schl, Kyt, Japan Changes in the free NAD+ /NADH rati and the energy charge (A TP + 1/ 2 ADP / ATP + ADP + AMP) f the liver were cmpared with the rati f acetacetate t f3-hydrxybutyrate f arterial bld in rabbits subjected t ligatin f the cmmn bile duct. Bth the acetacetate/ f3-hydrxybutyrate rati f the liver, which reflects the mitchndrial free NAD+ /NADH rati, and the hepatic energy charge decreased in accrdance with the decrease f mitchndrial phsphrylative activity after the ligatin. The decrease in the acetacetate/ f3-hydrxybutyrate rati f the liver was attributed t a restricted mitchndrial rexidatin f NADH due t an inhibitin f xidative phsphrylatin. Mrever, changes in the rati f acetacetate t f3-hydrxybutyrate in arterial bld were psitively crrelated with thse f the liver (r = 0.695, P < 0.01) and the hepatic energy charge (r = 0.844, P < 0.01). t is suggested that the rati f acetacetate t f3-hydrxybutyrate in bld can reflect the energy charge f the liver in jaundiced rabbits. Ketne bdy cncentratins (acetacetate + f3-hydrxybutyrate) f the liver are maintained by calric and hrmnal cntrl.1.2 These substrates are readily intercnverted by f3-hydrxybutyrate dehydrgenase in liver mitchndria. The rati f ketne bdies in the liver is in equilibrium with the rati between xidized and reduced frms f free nictina- Received Octber 20, Accepted December 2, Address requests fr reprints t: Junji Tanaka, M.D., Department f Surgery, Kyt University Medical Schl, Saky-ku, Kyt, Japan. These data are taken frm a dissertatin that was submitted by Junji Tanaka t Kyt University. This wrk was supprted in part by grants frm the Scientific Fund f the Ministry f Educatin and the Japanese Assciatin fr the Study f Metablism and Disease by the American Gastrenterlgical Assciatin /79/ $02.00 mide-adenine dinucletides (free NAD+ /NADH rati) in the mitchndria. 3 When ne assumes that acetacetate and f3-hydrxybutyrate freely penetrate e lmembranes, l the rati f acetacetate t f3- hydrxy butyrate in the bld may reflect the mitchndrial free NAD+ /NADH rati. Als, it appears pssible that the acetacetate/ f3-hydrxybutyrate rati f the bld will reflect the energy status f the hepatcytes, because mitchndrial free NAD+ /NADH ratis are clsely related t xidative phsphrylatin.' Particular attentin was given t the questin f whether knwledge f changes in the rati f acetacetate t f3-hydrxybutyrate in the bld allws a highly sensitive descriptin f intracellular energy status f the liver, i.e., the hepatic energy charge (ATP + 1/2ADP / ATP + ADP + AMP).5 n this study. evidence is presented that the rati f acetacetate t f3-hydrxybutyrate in the bld is psitively crrelated with the hepatic energy charge in rabbits subjected t ligatin f the cmmn bile duct. Materials and Methds Healthy yung male rabbits weighing kg were maintained n CR-2 (Nippn Haigshiry C., Ltd., Japan) and water ad libitum under cnstant temperature f 20 C fr 2 wk befre surgery. The animals were nt fed fr 15 hr befre peratin. Operative ligatin f the cmmn bile duct was perfrmed between 8 AM and 10 AM under intravenus anesthesia f thipental sdium (25 mg/ kg f bdy weight). Fd was given just after the peratin. Sham peratins were perfrmed by lapartmy and incisin f the hepatdudenal ligament. Bld and liver samples were taken at 12, 24, and 48 hr after surgery. n the case f the 12-hr samplings, the ligatin was perfrmed between 8 PM and 9 PM, and the rabbits were nt fed after the peratin. n the ther cases, the rabbits were nt fed fr 15 hr befre the samplings that were made between 8 AM and 10 AM. Biliary decmpressin, accmplished by re-

2 692 TANAKA ET AL. GASTROENTEROLOGY Vl. 76, N.4 mving the ligature, was perfrmed 48 hr after the initial cmmn bile duct ligatin. Fr the assay f ketne bdies and the energy charge f the liver, the left-anterir lbe f liver was clamped and frzen in situ with stainless steel tngs precled with liquid nitrgen. The prcedure was cmpleted in 10 sec. The frzen tissue was then crushed t a pwder with mrtar and pestle in a liquid nitrgen bath. Fr the assay f ketne bdies, 2 g f the pwdered tissue were hmgenized in 6 ml f ice-cld 6% (wt/vl) perchlric acid by glass hmgenizer. The hmgenate was centrifuged at 10,000 g fr 15 min at 0 _4 C. The precipitate was washed with 2 ml f ice-cld 3% (wt/vl) perchlric acid and rel:entrifuged at 10,000 g fr 15 min at 0 _4 C. Bth supernatants were cllected and adjusted t ph 6.0 with cld 69% (wt/vl) ptassium carbnate and recentrifuged at 10,000 g fr 5 min. The supernatant was used t determine the cncentratins f ketne bdies. Fr the assay f adenine nucletides, 1 g f the pwdered tissue was hmgenized in a glass hmgenizer with 3 ml f 5% (wt/vl) perchlric acid including 1 mm EDT A. Fr the assay f ketne bdies in arterial bld, 5 ml f bld were remved with a heparinized glass syringe via the abdminal arta, withut suctin, simultaneusly with remval f liver samples. n sme cases, bld samples nly were taken, withut liver samples. The 5 ml f arterial bld were added t 5 ml f ice-cld 6% (wt/vl) perchlric acid and mixed immediately. The suspensin was centrifuged at 10,000 g fr 15 min at 0 _4 C. The supernatant was adjusted t ph 6.0 and recentrifuged at 10,000 g fr 5 min at 0 _4 C. The supernatant was used t determine the ketne bdy cncentratins as sn as pssible. Beta-hydrxybutyrate and acetacetate were measured by the methds f Williamsn and Mellanby6 and Mellanby and Williamsn,' respectively. Pyruvate and lactate were measured by the methds f Czk and Lamprecht" and Gutmann and Wahlfeld,9 respectively. Adenine nucletides were measured enzymatically by the standard methd f spectrphtmetric reading at 340 nm.1o, Days 1" Ttal Bilirubin / \ "1 J \ "" \. '!-----! \ After Biliary Decmpressiri-' ' \, \ 2 3 after Bile 4 Duct ndirect Bilirubin..jt Figure 1. Time-curse f serum ttal and indirect (uncnjugated) bilirubin levels after bile duct ligatin and biliary decmpressin. BDL = bile duct ligatin. Each pint represents the mean and standard errr f values fr six r mre animals. Preparatin f liver mitchndria and measurement r xidative phsphrylatin were perfrmed by methds described elsewhere." Mitchndrial prtein was determined by the methd f Lwry et a.,'2 with bvine serum albumin as a standard. Serum ttal and indirect bilirubin were determined by the methd f Michaelssn.'3 All results are expressed as means ± standard errrs. The significance between means was determined by Student's t-test. Results Figure 1 shws the changes in serum ttal and uncnjugated (indirect) bilirubin levels after bile duct ligatin and biliary decmpressin. The ttal and uncnjugated bilirubin levels increased t a maximum 48 hr after the ligatin, and then declined slwly, pssibly due t lsening f the ligature r develpment f biliary bypass. Table 1 shws the changes in the cncentratins f ketne bdies, pyruvate, and lactate in arterial bld after the ligatin. The cncentratins f acetacetate remained unchanged, but thse f fj-hydrxybutyrate increased significantly after the ligatin (P < at 48 hr). As a result, the rati f acetacetate t fj-hydrxybutyrate decreased markedly (P < 0.001). The cncentratins f pyruvate and lactate and the ratis f pyruvate t lactate remained unchanged after the ligatin. Table 2 shws the changes in these substrates in liver tissue after the ligatin. The cncentratins f acetacetate remained unchanged, althugh thse f fj-hydrxybutyrate increased significantly 48 hr after the ligatin (P < 0.001). The ratis f acetacetate t fj-hydrxybutyrate decreased 48 hr after the ligatin (P < 0.001). The ratis f ketne bdies in the liver were relatively higher than thse in the arterial bld at each stage (Tables 1 and 2). Als, the cncentratins f ttal ketne bdies in jaundiced liver were greater than thse f sham-perated liver (P < 0.001). Pyruvate, lactate, and the rati f pyruvate t lactate remained unchanged during the perid studied. On the ther hand, the energy charge f the liver decreased rapidly after the ligatin and fell frm the nrmal level f t hr later (Table 3). Table 4 shws the changes in xidative phsphrylatin f the liver mitchrndria after the ligatin. The respiratry cntrl rati, P /0 rati, state 3 respiratin, and phsphrylative activity decreased significantly after the ligatin (P < 0.01). The mrtality rate reached a maximum at 48 hr after the ligatin. Figure 2 shws the clse relatinship between the ketne bdy ratis in liver and in arterial bld after the ligatin. The ratis f ketne bdies in liver were psitively crrelated with thse in arterial bld (r = 0.695, P < 0.01). Figure 3 shws the clse relatinship

3 April 1979 BLOOD KETONE BODY RATO AND JAUNDCE 693 Table 1. Changes in the Cncentratins f Ketne Bdies, Pyruvate, and Lactate in Arterial Bld After Bile Duct Ligatin f Rabbits (pml/ml f whle bld) Ac-Ac (p.ml/ml f whle bld) Pyruvate x 10-2 Ac-Ac B-OHB B-OHB Pyruvate Lactate Lactate Nrmal (12 hr-fast) (7) ± ± ± ± ± ± After the ligatin 12 hr (7) ± ± ± ± ± ± Sham peratin (4) ± ± ± ± ± ± hr (7) ± ± ± ± ± ± Sham peratin (4) ± ± ± ± M57± ± hr (7) ± ± ± ± ± ± Sham peratin (4) ± ± ± ± ± ± Results shwn are mean values ± S.E. with n values in parentheses. Ac-Ac = acetacetate, B-OHB =,B-hydrxybutyrate. P < 0.001, cmpared with the values in the sham-perated rabbits. between the ratis f ketne bdies in arterial bld and the hepatic energy charge after the ligatin. The ratis f ketne bdies in arterial bld were psitively crrelated with the hepatic energy charge (r = 0.844, P < 0.01). Als, when the serum bilirubin decreased int the nrmal range 2 days after biliary decmpressin (Figure 1), bth the ketne bdy ratis and the energy charge f the liver were restred t nrmal levels with a cncmitant nrmalizatin f the ketne bdy ratis in arterial bld (Table 5). Discussin As prpsed by Atkinsn,s the adenylate energy charge is an expressin f equilibrium between the energy-generating and energy-cnsuming reactins in a cell. Althugh there are many limitatins in applying a cncept f adenylate energy charge t the whle tissue,14 adenylate energy charge is a numerical expressin f the energy status f the whle tissue, which shws a balance amng the energygenerating and energy-cnsuming reactins. n this study, the adenyl ate energy charge f the liver with jaundice after bile duct ligatin decreased, accmpanying the increase f the mrtality rate f the rabbit with jaundice. Thus, the decrease in the adenylate energy charge f the liver \Yith jaundice implies a critical alteratin in hepatic cellular viability, leading t death. The decrease in the hepatic energy charge after bile duct ligatin was the result f decreased ATP cncentratin and simultaneus increase in ADP and AMP cncentratins. Under nrmal bienergic cnditins, the xidreductin energy f electrn transprt is cnverted int the phsphate-bnd energy f ATP frm ADP and Pi. 15 n the liver with jaundice, it is likely that ADP is nt efficiently phsphrylated in the mitchndria. Thus, the decrease in the energy charge f the liver results frm a relative decrease in ATP supply as cmpared with ATP utilizatin. An available explanatin fr the decreases in bth ATP cncentratin and the hepatic energy charge is cnsidered t be an impairment f ATP synthesis in the liver with jaundice. n additin, the decrease in the hepatic energy charge level (decreased ATP cncentratins) was accmpanied by a decrease in ttal adenine nucletides. Pssible explanatins fr these decreases in the ttal adenine nucletides include the fllw- Table 2. Changes in the Cncentratins f Ketne Bdies, Pyruvate, and Lactate in Rabbit Livers After Bile Duct Ligatin (pml/g wet weight) Ac-Ac (p.ml/g wet weight) Pyruvate x 10-2 Ac-Ac B-OHB B-OHB Pyruvate Lactate Lactate Nrmal (12-hr fast) (7) ± ± ± ± ± ± 0.22 After the ligatin 12 hr (7) ± ± ± b ± ± ± 0.43 Sham peratin (4) ± ± ± ± ± ± hr (7) ± ± ± ± ± ± 0.31 Sham peratin (4) ± ± ± ± ± ± hr (4) ± ± ± ± ± ± 0.42 Sham peratin (4) ± ± ± ± ± ± 0.58 Results shwn are mean values ± S.E. with n values in parentheses. At-Ac = acetacetate, B-OHB =,B-hydrxybutyrate. P < 0.01, b < P < 0.01, cmpared with the values in sham-perated rabbits.

4 694 TANAKA ET AL. GASTROENTEROLOGY Vl. 76. N. 4 Table 3. Changes in the Cncentratins f Adenine Nucetides and the Energy Charge f Rabbit Livers After Bile Duct Ligatin (f. m1!g wet liver) ATP ADP AMP Ttal Energy charge Nrmal (12 hr-fast) (7) ± ± ± ± ± After the ligatin 12 hr (6) ± 0.141" ± ± ± b ± Sham peratin (4) ± ± ± ± ± hr (7) ± 0.010" ± ± 0.011" ± ± 0.010" Sham peratin (4) ± ± ± ± ± hr (9) ± 0.201" ± ± ± 0.192" ± 0.021" Sham peratin (4) ± ± ± ± ± Results shwn are mean values ± S.E. with n values in parentheses. Ttal = ATP+ADP+AMP; energy charge = ATP+1/2ADP/ATP+ ADP+AMP. p < b < P < cmpared with the values in the sham-perated rabbits. ing. The first is a result f decreased amunts f parenchymal tissue per wet weight due t chlestasis, because we referred the cncentratins f adenine nucletides t wet weight f liver tissue in jaundiced animals. The secnd is a result f the decrease in ATP cncentratin r the hepatic energy charge which may activate adensine deaminase activity!6 The frmer is less acceptable than the latter as an explanatin f the decreased ttal adenine nucletides, because ADP and AMP cncentratins were increased in the liver tissue with jaundice. Thus, the decreases in bth the hepatic energy charge and the ttal adenine nucletides may be due t an impairment f ATP synthesis f the liver mitchndria with jaundice. n regard t the vulnerability f liver mitchndria in severe jaundice, txic effects f bilirubin n mitchndria have been reprted by many investigatrs. Bilirubin has been cnsidered t be an uncupler,17 an inhibitr f mitchndrial respiratin by inhibiting NADH xidase,'" and a factr causing an impairment f the mitchndrial membrane. 19 n this study, a mderate elevatin f uncnjugated bilirubin and a marked increase in ttal bilirubin were bserved after the bile duct ligatin f rabbits. Cncmitantly, the decrease in adenylate energy charge was bserved. The elevated level f serum bilirubin may playa rle in causing the decrease in the energy charge f the liver with jaundice. Prepared mitchndria shwed a decrease in state 3 respiratin and phsphrylative activity accmpanied by a decreased P /0 rati in vitr, althugh there remains a questin as t whether the prepared mitchndria reflect the in situ state r nt. Mitchndrial phsphrylative activity decreased cncmitantly with hepatic energy charge and mitchndrial xidreductin state, which indicate the actual state f the tissue in situ at a given time after ligatin f the cmmn bile duct. These bservatins seem t shw that a crrectin f the impaired mitchrndria was nt btained during the preparatin f mitchndria frm the liver f jaundiced animals. The impairment in A TP synthesis inducing the decrease in the hepatic energy charge is suspected t be due t the txic effects f bilirubin n mitchndria. n liver mitchndria, acetacetate is prduced in the matrix cmpartment' and underges reductin t p-hydrxybutyrate by p-hydrxybutyrate dehy- Table 4. Changes in Oxidative Phsphrylatin f the Mitchndria frm the Rabbit Livers After the Bile Duct Ligatin Hurs after BDL RC State 3 P/O PR Mrtality ('fa) 12 (6) 3.2 ± ± ± ±4.2 5 Sham peratin (4) 4.1 ± ± ± ± (5) 2.9 ± ± ± ± (6) 2.8 ± ± ± ± Sham peratin (4) 4.2 ± ± ± ± HC = respiratry cntrl rati; state 3 = state 3 respiratin (millimicrmles f xygen cnsumed per milligram f prtein per minute); PR = phsphrylatin rate (millimicrmles f ATP synthesis per milligram f prtein per minute); and P/O = ATP synthesis per xygen atm; and BDL = bile duct ligatin. p < cmpared with the values in the sham-perated rabbits. in the case f 24-hr. cmpared with thse sham perated at 48 hr. Results shwn are the mean values ± S.E. with n values in parentheses. Oxygen cnsumptin and phsphrylatin were measured at 22 C at ph 7.4 in a medium cntaining 0.3 M mannitl, 0.01 M MgC M Tris-MC buffer M ptassium phsphate buffer. 0.2 mm EDTA. and 230 'M ADP. Glutamate was added at a cncentratin f 4 mm.

5 April 1979 BLOOD KETONE BODY RATO AND JAUNDCE " m ::r m :J.0.5 '0 Ul 8 0 \ y = O.54X O Energy Charge Ratis f Ac-Ac/B-OHB (Liver) Figure 2. Relatinship between the ratis f acetacetate t {1-hydrxybutyrate in the liver and the bld in jaundiced rabbits. Crrelatin cefficient: r = 0.695, P < 0.01; regressin equatin: Y = 0.54X "C ii 1.0.: ::: ell <l Ul _Q Figure 3. Relatinship between the energy charge f the liver and the ratis f acetacetate t {1-hydrxybutyrate in arterial bld in jaundiced rabbits. Crrelatin cefficient: r = 0.844, P < 0.01; regressin equatin: Y = 2.83X t 0.9 drgenase in the mitchndrial inner membrane. 21 The equilibrium between the cncentratins f acetacetate and thse f f3-hydrxybutyrate reflects the free NAD+ /NADH rati in the mitchndria as shwn by fllwing principle 3 : acetacetate + NADH + H+ {1-hydrxybutyrate + NAD+ ' Thus, the free NAD+ /NADH rati in the mitchndria can be calculated frm the rati f the cncentratins f the substrates accrding t the equatin: Free NAD+ Free NADH acetacetate 1 -;:-;---;-----;--,--,- X - {1-hydrxybutyrate K where K is the equilibrium cnstant fr f3-hydrxybutyrate dehydrgenase. Recent studies have shwn that the mitchndrial NAD xidreductin state is f majr significance in energy prductin and in the cntrl f cell metablism n the liver f jaundiced rabbits, the equilibrium changed tward f3-hydrxybutyrate with an increased mitchndrial frmatin f reducing equivalents. There are tw pssibilities accunting fr this result. The first is increased f3-xidatin metablism with a mderate elevatin f ketne bdy cncentratins. The secnd is a result f an impaired electrn transprt system by the txic effects f elevated bilirubin level, such as an inhibitin f NADH xidase activity,'8 assciated with the decreased activity f the tricarbxylic acid cycle n the frmer case, the marked decrease in the hepatic energy charge may nt be accmpanied by inverse f3-xidatin as shwn by ther experiments. 27 Thus, it is ratinal t cnsider that the change f the NAD system in the liver mitchndria with jaundice tward a mre reduced state is the result f restricted mitchndrial rexidatin f NADH, in accrdance with a lwer rate f xidative phsphrylatin. The impairment f ATP synthesis inducing the decrease in the hepatic energy Table 5. Changes in the Ratis f Ketne Bdies and Pyruvate t Lactate in Liver and Bld, and Hepatic Energy Charge After Biliary Decmpressin Ac-Ac/ B-OHB Pyruvate/lactate X 10-2 Adenine nucletide n liver n bld n liver n bld Ttal Energy (Jl-ml/g) (Jl-ml/ml) (Jl-ml/g) (Jl-ml/ml) (Jl-ml/g) charge 48 hr after bile duct ligatin (7) ± ± ± ± ± ± hr after biliary decmpressin (5) ± ± ± ± ± ± Results shwn are mean values ± S.E. with n number in parentheses. Ac-Ac = acetacetate, B-OHB = {1-hydrxybutyrate, ttal ATP+ADP+AMP (p.ml/g f wet liver).

6 696 TANAKA ET AL. GASTROENTEROLOGY Vl. 76, N. 4 charge and the resultant decrease in the ttal adenine nucletides may be due t the suppressin f the mitchndrial electrn transprt system. On the ther hand, the rati f pyruvate t lactate, which perfrms a similar functin as the cntrlling link f phsphate ptential f glyclysis,28 remained unchanged in the rabbit liver with jaundice. t is difficult t explain this difference between the mitchndrial and cytslic NAD systems by a single mechanism. t seems likely that liver damage due t jaundice induces the decrease in the rati f xidized t reduced NAD in the mitchndria rather than that in the cytsl. Althugh many factrs are cnsidered t cntribute t changes in the rati f ketne bdies in the bld, the liver is the nly rgan that makes a net cntributin f ketne bdies t the bld stream. t has been fund that,8-hydrxybutyrate dehydrgenase is lcated in the cristae in such a way as t cmbine with the NAD f the matrix and that,8- hydrxybutyrate dehydrgenase activity is exceptinally high in liver.29 n this study, the changes in the ketne bdy ratis f arterial bld paralleled thse in liver mitchndria with jaundice. These results shw that changes in the ketne bdy ratis f arterial bld reflect the directin f the changes in the liver mitchndrial free NAD+ /NADH ratis. Thus, the changes in the ketne bdy ratis f arterial bld are cnsidered t depend upn nt nly the,8-hydrxybutyrate dehydrgenase activity, but als upn the free NAD+ /NADH ratis f liver mitchndria in the rabbits with jaundice. These results seem t be cnsistent with the fact that the mitchndrial xidreductin state crrespnds well with the rati f ketne bdies in the perfusate r the incubatin medium. 3 The mitchndrial xid reductin states f NAD were psitively crrelated with the hepatic energy charge levels after the ligatin f the cmmn bile duct. Thus, it seems reasnable t cnclude that the changes in the ratis f acetacetate t,8-hydrxybutyrate in the bld reflect the changes in the hepatic energy charge levels f rabbit livers with jaundice. References 1. Garber A, Owen OE, et al: Hepatic ketgenesis and glucnegenesis in humans. Clin invest 54: , McGarry 10, Wright PH, Fster OW: Hrmnal cntrl f ketgenesis. J Clin invest 55: , Williamsn DH, Lund p, Krebs HA: The redx state f free nictinamide-adenine dinucletide in the cytplasm and mitchndria f rat liver. Bichem 103: , Wjtczak AB: Cntrl f acetacetate and,b-hydrxybutyrate prductin in rat liver mitchndria. Bichem Biphys Res Cmmun 31: , Atkinsn DE: The energy charge f the adenylate pl as a regulatry parameter. Bichemistry 7: , Williamsn DH, Mellanby J:,B-Hydrxybutyrate. n: Methds f Enzymatic Analysis. Edited by HV Bergmeyer. New Yrk, Academic Press, nc., 1974, p Mellanby J, Williamsn DH: Acetacetate. n: Methds f Enzymatic Analysis. Edited by HV Bergmeyer. New Yrk, Academic Press, nc., 1974, p Czk R, Lamprecht AW: Pyruvate. n: Methds f Enzymatic Analysis. Edited by HV Bergmeyer. New Yrk, Academic Press, nc., New Yrk, p Gutmann, Wahlefeld AW: Lactate. n: Methds f Enzymatic Analysis. Edited by HV Bergmeyer. New Yrk, Academic Press. inc., 1974, p Bergmeyer HV (editr): n: Methds f Enzymatic Analysis, New Yrk, Academic Press, 1965: ATP, p 543; ADP and AMP, p Ozawa K, Kitamura 0, et al: Human liver mitchndria. Clin Chim Acta 38: , Lwry OH, et al: Prtein measurement with Flin phenl reagent. Bii Chern 193:265, Michaelssn M: Studies n diaz methds and a new cpperaz pigment methd. Scand Clin Lab nvest Suppl:56, Takeda H, McLaughlin A, Chance B: 113lp NMR f perfused muse liver. Science, in press 15. Lehninger AL: n: Bichemistry. New Yrk, Wrth Publishers, nc., 1970, p Chapman AG, et al: Rle f the adenylate deaminase reactin in regulatin f adenine nucletide metablism in Ehrlich ascites tumr cells. Cancer Res 36: , Zetterstrm Rand Ernster L: Bilirubin, an uncupler f xidative phsphrylatin in islated mitchndria. Nature 174: , Cuger ML: Mechanism f bilirubin txicity n tissue culture cells. Bichem Med 5:1-16, Mustafa MG, Cuger ML, King TE: Effect f bilirubin n mitchndrial reactins. Bii Chern 244: , Chapman MJ, Miller LR, Ontr JA: Lcalizatin f the enzymes f ketgnesis in rat liver mitchndria. Cell Bii 58: , Ernster L, Kaylenstierna B: n: Membrane f Mitchndria and Chlrplasts. Edited by E Racker. New Yrk, Van Nstrand Reinhld C., 1970 p Andersn KB, et al: Charges f Nictinamide-adenine dinucletides and adenylate energy charge as regulatry parameters f the metablism in Escherichia cl: Bii Chern 252: , Batenburg H, Olsn MS: Regulatin f pyruvate dehydrgenase by fatty acid in islated rat liver mitchndria. Bii Chern 251: , Veech RL, Rijiman L, Krebs HA: Equilibrium relatins between the cytplasmic adenine nucletide system and nictinamide-adenine dinucletide system in rat liver. Bichem 117: , Ogasawara N, et al: Bilirubin: A ptent inhibitr f NAD linked iscitrate dehydrgenase. Bichim Biphys Acta 327: , Harfrd S, Wetzman PDf: Evidence fr issteric and allsteric nucletide inhibitin f citrate synthase frm multiple-inhibitin studies. Bichem J 151: , Yamada T, Ozawa K. et a1: nsulin requirements fr hepatic regeneratin fllwing hepatectmy. Ann Surg 185:35-42, Stubbs M, Veech RL, Krebs HA: Cntrl f the redx state f the nictinamide-adenine dinucletide cuple in rat liver cytplasm. Bichem J 126:59-65, Lehninger AL, et al: D-,B-hydrxybutyrate dehydrgenase f mitchndria. Bi Chern 235: , Krebs HA, Patricia G, et al: Rates f ketne bdy frmatin in the perfused rat liver. Bichem J 112: , 1969