Reticuloendothelial system and hepatocyte function in

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1 Gut, 1982, 23, Reticuloendothelil system nd heptocyte function in fulminnt heptic filure J CANALESE, C D GOVE, A E S GMSON, S P WLKNSON, E N WARDLE, AND ROGER WLLAMS From the Liver Unit, King's College Hospitl nd Medicl School, London SUMMARY Kupffer cell nd heptocyte function were studied in 36 ptients with fulminnt heptic filure by mesurement of clernce of 1251 microggregted lbumin (125MAA) nd glctose, respectively. Both were impired but there ws no correltion with finl outcome, lthough on sequentil testing 48 hours lter, those ptients who survived hd significntly greter improvement in Kupffer cell nd heptocyte function. n six other ptients with similr heptocyte dysfunction but no encephlopthy, Kupffer cell function ws not significntly different from tht of controls. This is dditionl support for possible reltion between encephlopthy nd dmge to Kupffer cell function. The ltter ws lso shown to correlte with renl filure; this is consistent with the suggestion tht endotoxemi is importnt in the pthogenesis of this compliction. The mssive dysfunction of heptocytes in fulminnt Methods heptic filure hs been documented both histologiclly nd by functionl mesurement such s the PATENTS glctose clernce cpcity.' Less is known bout The 36 ptients studied (19 women, 17 men; ged Kupffer cell function.2 The ltter is importnt not yers) ll fulfilled the criteri for fulminnt only in reltion to specific functions of the reticuloendothelil system but lso becuse the degree of hep- considerbly rised serum sprtte trnsminse heptic filure.6 All hd severe heptic necrosis, tocellulr dmge my be profoundly modified by levels (36±SEM 713 U/l), nd prothrombin time ltering reticuloendothelil function.3 Blockde or prolonged by 97±8.6 s (men±sem). Liver filure stimultion of the reticuloendothelil system in experimentl glctosmine heptitis results in ptients, virl heptitis in nine (HAV in five, HBV in ws ttributed to prcetmol overdose in 24 ggrvtion or prevention respectively of the liver three, nd NANB in one), nd hlothne ssocited cell lesion. Other studies in ischemiclly induced heptitis in three. The ptients received intensive heptic necrosis hve shown tht survivl is influenced by chnges in reticuloendothelil function4 hemodilysis using the polycrylonitrile membrne cre s described elsewhere7 nd dily period of which my be relted to the filure of the dmged (RP6) nd /or hemoperfusion through Hemocoll Kupffer cells to cler gut-derived endotoxins from 1 column (Smith nd Nephew Reserch Ltd), with portl venous blood. Systemic endotoxemi hs prostcyclin s protective gent.8 All investigtions been implicted in the pthogenesis of renl filure, were mde before these procedures, before the intrvsculr cogultion, nd gstric mucosl dministrtion of blood products, nd s soon s the hemorrhges in ptients with fulminnt heptic ptient hd shown signs of developing grde V filure.5 We hve ssessed reticuloendothelil function in 36 ptients with fulminnt heptic filure by lredy estblished. nvestigtions were repeted 48 encephlopthy or on rrivl in the Unit if this were mesuring the blood clernce of microggregted hours lter. iodinted humn lbumin. At the sme time the Mesurements were mde in six other ptients heptocyte functionl cell mss ws determined by (three men, three women; ged yers) with glctose clernce test. severe heptic dmge from prcetmol overdose in whom encephlopthy hd not developed. Serum sprtte trnsminse levels were considerbly Received for publiction 27 July 1981 rised (men±sem=3±22 U/1) nd the pro- 265 Gut: first published s /gut on 1 April Downloded from on 3 My 218 by guest. Protected by copyright.

2 266 thrombin time prolonged (men±sem=49-3± 9*2 s). These ptients were studied hours fter the prcetmol overdose, time comprble with tht for ptients who did develop encephlopthy. The control group consisted of volunteer lbortory stff nd ptients with uncomplicted myocrdil infrction with n ge rnge similr to tht in the ptient group. nformed consent ws obtined from ll ptients or their reltives nd from controls before testing. Microggregted iodinted humn serum lbumin ('25MAA) ws prepred by the method of lio nd Wgner.9 After blockde of the thyroid by intrvenous injection of sodium iodide, 125MAA (5 mg/kg body weight) ws injected nd 2 ml blood smples were tken into EDTA t regulr intervls for 2 minutes;.5 ml plsm ws counted directly using Hewlett-Pckrd gmm counter. The hlf life of '25MAA in the circultion ws clculted by plotting the log,( CPM versus time. For the control subjects the men hlf life ws 137+± 1.1 min which is similr to tht found in other series using the sme system."' n six ptients low dose (. 5 mg/kg body weight) of '251MAA ws injected. At this dose removl depends more on heptic blood flow thn on Kupffer cell function;9 removl ws similr to tht found in controls given the sme dose (hlf life 3-8± 3 min nd 3 5±-1 min respectively). The intrvenous glctose clernce test ws performed by the method of Tengstrom": 35 mg/kg body weight of 2% (W/V) solution of glctose ws given intrvenously over period of up to three minutes. Zero time ws tken s the time when hlf the volume hd been given. Venous blood ws tken t regulr intervls over 4 minutes, nd mixed immeditely with 5 vol 6% perchloric cid. Glctose concentrtions in the protein-free superntnt were estimted enzymticlly with glctose dehydrogense. 12 The hlf life of glctose ws determined by plotting log,(, glctose concentrtion ginst time. Renl excretion of glctose during the 4 minute test ws less thn 1% of the totl dose (ll ptients hd indwelling ctheters). Vlues for the controls were 11-5±1-4 minutes, which re similr to those reported by other workers." Sttisticl nlysis ws by Student's t test or Wilcoxson's rnk test. Results re expressed s men + stndrd error of the men. Results 125MAA CLEARANCE CAPACTY The initil hlf life (T½/2) of 125MAA estimted in 34 ptients ws significntly longer thn tht in the control subjects (26.±1. nd 13*7±1*1 min, respectively, P<.1). n the six ptients with severe liver Cnlese, Gove, Gimson, Wilkinson, Wrdle, nd Willims dysfunction but no encephlopthy, however, the T½/2 of 14*1±*9 min ws not significntly different from tht of the control vlues (Fig. 1). There ws no significnt difference in initil T½/2 vlues between those with fulminnt heptic filure who survived nd those who died (26.6±1. nd min, respectively). Similrly, T½/2 vlues were not relted to the cuse of the liver filure. However, the second T½/2 estimtion ws significntly improved in the 11 ptients who survived nd ws unchnged in the five ptients who died (-6.4±1.9 nd -±2-1 min respectively, P<-5) (Tble). n reltion to the development of renl filure (plsm cretinine >35 L.n J LL -z 45 r 4 L *oa 3 2 k 15 1 L 5L o * DED * * 6 SEVERE HEPATC NECROSS s NORMAL CONTROL Fig. 1 '25MAA hlflife in ptients with fulminntheptic filure nd severe heptic necrosis (*prcetmol overdose, O virl heptitis, A hlothne heptitis), nd in norml controls (M). Gut: first published s /gut on 1 April Downloded from on 3 My 218 by guest. Protected by copyright.

3 Reticuloendothelil system nd heptocyte function infulminnt hepticfilure Tble Chnge in 125MAA nd glctose hlflives in ptients withfulminnt heptic filure over two dyperiod (men ±SEM) Fulminnt heptic filure Chnge in hlf life over two dys '25MAA Glctose Survived -6-4± ±5-9 N 11 6 Died ± ±7-1 N 6 5 P<-5. Survivors vs non-survivors. mmol/l nd urine output <3 ml/24 h), the initil T1/2 vlues were significntly more prolonged, reflecting the greter Kupffer cell impirment in ptients who eventully developed renl filure thn in those who did not (28.9±1*4 nd 23-9±-8 min respectively, P<.5) F 5 LX U- L 4.A i GALACTOSE ELMNATON CAPACTY The T½/2 of glctose mesured in 27 ptients with fulminnt heptic filure ws similr to tht in five ptients with severe liver dysfunction but no encephlopthy, but both were significntly prolonged compred with control vlues (36.6±3.1, 42-5±9.3, nd 11*5±1*4 min respectively, P<*1 (Fig. 2). Ptients who survived nd those who died hd the sme degree of impirment of glctose clernce (initil T½/2 vlues 37-3±6-3 nd 36.3±3*5 min respectively). n the 1 ptients in whom second mesurement ws mde t the sme time s the second T½/2 mesurement of 1251MAA (five survivors nd five non-survivors) the elimintion cpcity hd incresed in those who survived but hd decresed in those who eventully died (chnge in T½/ nd +12*±7.1 min respectively, P<.5) (Tble). There ws no correltion between the initil degree of impirment of 125MAA clernce nd the degree 9r S L Gut: first published s /gut on 1 April Downloded from :c c -Jz 3 2 L 1 L o L A S A DED 8 SURVVED SEVERE HEPAT C. NECROSS NOT NORMAL CONTROL 1 v,, DAYS POST NGESTON OF PARACETAMOL Fig. 3 Seril mesurements ofprothrombin time (), glctose hlflife (), nd '25MAA hlflife (A) in ptients survivingfulminnt heptic filure. of reduction in glctose elimintion cpcity (r=-38). Other tests performed over subsequent dys showed tht the 125MAA clernce nd glctose elimintion cpcity continued to improve in ptients who survived, shown by progressive fll in T½/2, nd returned to norml within 1 dys (Fig. 3). Fig. 2 Glctose hlflife in ptients with fulminnt heptic filure nd severe heptic necrosis (*prcetmol overdose, O virl heptitis, A hlothne heptitis), nd in norml controls (R). on 3 My 218 by guest. Protected by copyright.

4 268 Cnlese, Gove, Gimson, Wilkinson, Wrdle, nd Willims n contrst with the findings for 125MAA, the T½/2 of glctose in ptients who developed renl filure ws not significntly different from the T½/2 in those who did not develop renl filure (41.4±5-5 min nd 33-±5- min respectively). Discussion Although impired function of the reticuloendothelil system hs been reported in cses of cute lcoholic heptitis nd chronic liver disese'3 the considerble impirment shown in fulminnt heptic filure hs not been reported before. Kupffer cells usully comprise bout 8% of the fixed cells of the reticuloendothelil system.2 n the bsence of ny other known mechnism for clernce of microggregted lbumin, the prolonged hlf life of '25MAA in these ptients is most likely due to Kupffer cell dysfunction. Reduced heptic blood flow nd portosystemic shunting of blood round the Kupffer cells cn be excluded s possible cuses, s liver blood flow, s ssessed from clernce of lower dose of 125MAA ws norml. Furthermore, in ischemiclly induced heptic necrosis, vlues for liver blood flow return to norml within 24 hours but reticuloendothelil function remins bnorml.4 t is more likely tht the impirment of reticuloendothelil function is relted to the relese of cell debris ssocited with mssive heptocyte destruction. ncresed concentrtions of circulting immune complexes hve been found in fulminnt virl heptitis nd these could lso cuse reticuloendothelil blockde (personl communiction). The possible role of endotoxemi which hs lredy been referred to might lso depress reticuloendothelil function.'4 Rmsoe et l. found tht the reduction in glctose clernce from the circultion ws significntly less in ptients with fulminnt heptic filure who survived thn in those who eventully died,' lthough there ws some overlp between the two groups.'5 On the bsis of this nd the finding tht the rte of liver regenertion ssessed by the mitotic index nd the frequency of liver cells with interploid DNA is similr in both fulminnt nd uncomplicted heptitis,'6 it hs been suggested tht liver cell destruction is the mjor determinnt of prognosis in cute liver filure, heptic regenertion being less importnt. Our findings of similr prolongtion of the initil T/2 of glctose in survivors nd non-survivors would not support this suggestion. One reson for this difference my be tht in our ptients glctose ws given ccording to body weight, wheres in other studies fixed dose ws given. Although the proportion of ptients with virl heptitis in our series ws smller thn tht reported by Rmsoe et l. this is unlikely to be the explntion, becuse the T1/2 of glctose ws similr in ll groups. Our results re mpre consistent with the hypothesis tht heptic regenertion is importnt in determining the finl outcome nd is reflected in the greter improvement in T/2 for glctose nd '25MAA over 48 hours in ptients who ultimtely survive. This improvement in heptocyte function my not necessrily be ttributed to newly regenerted cells, but my reflect the return to norml function of vible heptocytes or Kupffer cells which previously hd impired metbolic function. The norml reticuloendothelil function found in six ptients with severe heptic necrosis not progressing to encephlopthy is interesting, s experimentlly induced heptic necrosis due to either ischemi or glctosmine cn be influenced by both reticuloendothelil blockde nd stimultion.4 17 An intct reticuloendothelil system my therefore be preventing the entry into, or promoting the removl from the circultion of potentil cerebrl toxins. The identity of these substnces is uncler, lthough endotoxins hve been shown to cuse cerebrl dmge in neontl cts,'9 nd levels were found to correlte with the level of encephlopthy in ptient with Reye's syndrome.2 The importnce of Kupffer cell function during the course of fulminnt heptic filure is lso reflected in the significntly worse reticuloendothelil function found in ptients who developed renl filure. This is in ccord with the current views on the mechnism of this compliction: renl filure in fulminnt heptic filure hs been ttributed to systemic endotoxemi5 which results in reduced renl perfusion.2' This is likely to be present only when reticuloendothelil function is no longer dequte to prevent the spred of endotoxins from the portl to the systemic circultion. This project represents prt of continuing progrmme, grnt supported by the MRC, into the development of liver support systems. The help of the doctors nd nurses in the liver filure unit is cknowledged. The editoril ssistnce of Miss Srh Underhill is lso grtefully cknowledged. References 1 Rmsoe K, Buch Andresen P, Rnek L. Functioning liver mss in uncomplicted nd fulminnt cute heptitis. Scnd J Gstroenterol 198; 15: Biozzi G, Stiffel C. n: Popper H, Schffner E, eds. Progress in liver diseses, vol 2, New York: Grune nd Strtton, 1965: Liehr H. Biologicl significnce of RES function in glctosmin heptitis. n: Liehr H, Grun M, eds. The reticuloendothelil system nd the pthogenesis of liver disese. North Hollnd: Elsevier, 198: Gut: first published s /gut on 1 April Downloded from on 3 My 218 by guest. Protected by copyright.

5 Reticuloendothelil system tnd heptocyte function in fultninnt hepticfilure Holper K, Olcy, Kithm A, et l. Effect of ischemi on heptic prenchyml nd reticuloendothelil function in the bboon. Surgery 1974; 76: Wilkinson SP, Gzzrd BG, Arroyo V, et l. Reltion of renl impirment nd hemorrhgic dithesis to endotoxemi in fulminnt heptic filure, Lncet 1974; 1: Trey C, Dvidson CS. The mngement of fulminnt heptic filure. n: Popper H, Schffner E, eds. Progress in liver diseses, vol 3. New York: Grune nd Strtton, 197: Trewby PN, Willims R. n: Hnson PG, Wright PL, eds. Medicl mngement of the criticlly ill. New York: Acdemic Press, 1978; Gimson AES, Lngley PG, Hughes RD. Prostcyclin to prevent pltelet ctivtion during chrcol hemoperfusion in fulminnt heptic filure. Lncet 198; 1: io M, Wgner HN. Studies on the reticuloendothelil system (RES) 1. Mesurement of the phgocytic cpcity of the RES in mn nd dog. J Clin nvest 1963; 42: Drivs G, Wrdle N. Reticuloendothelil cell dysfunction in dibetes nd hyperlipidemi. Metbolism 1978; 27: Tengstrom B. An intrvenous glctose tolernce test nd its use in heptobiliry diseses. Act Med Scnd 1968; 183: Rommel K, Bernt E, Schmitz, et l. Enzymtische glktosebestimung im blut und orlen glctose tolernce test. Klin Wochenschr 1968: 46: Wrdle EN, Anderson A, Jmes. Kupffer cell phgocytosis in reltion to BSP clernce in liver nd inflmmtory bowel disese. Dig Dis Sci 198; 25: Bencerrf B, Sebestyen MM. Effects of bcteril endotoxins on the reticuloendothelil system. Fed Proc 1957; 16: Rnek L, Andresen B, Tygstrup N. Glctosmine elimintion cpcity s prognostic index in ptients with fulminnt heptic filure. Gut 1976; 17: Milndri M, Gub J, Rnek L. Evidence of liver cell prolifertion during ftl cute liver filure. Gut 198; 21: Grun M, Liehr H, Grun W, Rosenck U, Brunsnig D. nfluence of liver RES on toxic liver cell dmge due to glctosmine. Act Heptogstroenterl (Stuttg) 1974; 21: Grun M, Liehr H, Rsenck L. Significnce of endotoxemi in experimentl glctosmine heptitis in rts. Act Heptogstroenterol (Stuttg) 1977; 24: Gillies FH, Averill DR, Kerr L. Endotoxin encephlopthy. Trns Am Neur Ass 1976; 11: Copperstock MS, Tucker RP. Possible pthogenic role of endotoxins in Reye's syndrome. Lnecet 1975; 1: Cvngh D, Ro PS, Sutton DMC, Blumgrt BD, Buchnn F. Pthophysiology of endotoxic shock in the primte. Am J Obst Gyn 197; 18: Gut: first published s /gut on 1 April Downloded from on 3 My 218 by guest. Protected by copyright.