.-

Chapter 7

Hepatotrophic Substances

By THOMAS E. STARZL. M.D .• Ph.D. otld

JOHN TERBLANCHE, Ch.l\L. F.R.C.S. (Eng). F.C.S. (S.A.)

BLOOD returning from the nonhepatic splanchnic organs via the portal venous system can spe,cifically influence the morphologic features, regenerative ca­pacity, and function of the liver. The portal blood constituents responsible for these effects have collectively been termed portal hepatotrophic factors. Much of-the 3n vivo evidence about portal hepatotrophk factors has been obtained by seeing what h~ppens to the liver when it is deprived of all or part of the portal venous return, by surgically removing nonho:!patic ~planchnic viscaa, or by infusing hormones or other substances systemically or directly into the liver circulation.

In this review, the effects of hepatotrophic substances upon hepatocytic structur~ and function are treated separately from their influence upon the regeno:!ration that follows partial hepatectomy. The failure to make this dis­tinction has pwbably been responsible for many of the controversies about no:!w developments in portal hepatotrophic physiology. This was clear in the discussions of a symposium on this subject held in May 1977. 1

HEPATOTROPHIC EFFECTS EXCLUDI~G REGE:\ERA nON

Tho:! most easily achieved portaprival state occurs \, hen ell the splanchnic venous rt:turn is diverted around the liver via an anastomosis to tho:! vena cava, leaving the liver with only an arterial supply. This procedure of portacaval shunt is also callc::d Eck's fistula, after the Russian military surgo:!on who de­scribed it in dogs more than 100 years ago.2 Based on the short-term survival of one of his eight dogs. Eck thought that a completely diverting portacaval shunt in dogs was compatible with prolonged good health. In 1893, however, Hahn, Massen. Nencki, and Pavlov3 showed that dogs v,:ith Eck's fistula de­veloped anore xia. weight loss, hepatic atrophy, and encephalopathy,

The atrophy of hepatocytes caused by Eck's fistula. as well as other

From th.: D~Pdrtments of Surgery, Denver Veterans Administration H,)spital and University of COI(lrado Medical Center, IXnvcr. Colorado; University of Cape Town. Svulh-\frica.

Th.: \\olk was \upporl~d hy rc~carch grants MRIS 8118-01 and 7227-01 frclm the Veterans Admini,uatiun; b\· rr;lnt numbers AM-I7260 and AM·077n frum th.: 0:ationdl In>lilut.:S of Health; <lnd by gr:lnt numb as RR·OO051 and RR-00069 from the General Clinic:)l R<!sc::.rch Centers Pro· ;:rdm of the Di ... i,i,)n of Re,.:arch Re~ourccs. Nation:ll In'tilUtes llf Health: and Ih.: M<!dic:l1 Re· 'c:arch CUllncil. Sputh Africa.

135

136 PROGRESS IN LIVER DISEASES

structural changes. occurs with great rapidity. being 909(- complete v .... ithin 4 days. ~-6 Ultrastructurally. the most striking and specific changes are depletion and disruption of the rough endoplasmic reticulum and reduction in the mem­brane-bound ribosomes. The same general light- and electron-microscopic changes occur after portal diversion in the livers of rats. dogs. swine. baboons. and man, with some variations in degree.; Thus the hepatic injury of Eck's fistula is common to all species studied.

What is the explanation of the changes caused by portacaval shunt? When Bollmans summarized the situation of Eck's fistula in 1961. the flow hypothesis was widely accepted. It stated that Eck's fistula syndrome was caused by a

-_ . __ . --- ___ - , _ -. - suboptimal volume as-opposed to qU:llity of hepatic blood flow. This conclusion was apparently incontrovertibly supported byexpenments in which the porta} f10\\- lost after portacaval shunt was replaced with vena caval and arterial blood. respe-ctively.!o·IO With this portal blood replacement;most ofth~advcrse-­effects of Eck's fistula in dogs wer~ avoided. Thus. portal blood seemed to

:: ___ -=-_::·,_possess. nO.physiolugically importallLspecial qualities_ - The fallacy of the flow hypothesisi'ecame 'evident during efforts to define

the necessary conditions fur successful auxiliary liver transplantation. 11 With· t\\O livers pre"cnt. fhe organ given blvod returning' from the nonhepatk' spLtnchnic organs remained healthy, \\ hercas the liver deprivl:d of such nOllr­ishment atrophied in spite of adequate:! portal flow from nonsplanchnic -sources. 12 Apparently. the liver with first access to the splanchnic venous blood was extracting something efficiently enough so that the second organ suffered from its absence.

The transplant preparations that had made the foregoing physiologic effect app~lrent had a flaw that prevented complete acceptance of what haJ b~come known as the hepatotrophic concept. There was a potential inequality of the two organs in that the homograft was under immunologic attack despite host immunosuppression, \vhereas the animal's own liver was not. Consequently, other experiments were designt!d.

:\t first, a split or partial transposition was developed that, in effect. divided the dog's o\\.'n liver into two fragmentsY'u With this operation, splanchnic venou~ blood was proviJed fvr one portal branch of the liver, whereas the other portal branch was detached and supplied with blood from the inferior vena cava. The quantity of flow was measured in many of these experi­ments n .14 and found to be generally grt!ater on the side perfused by vena caval blood. The lobes supplied with systemic venOllS blood atrophied grossly and histopathologically, whereas the lobes given normal portal blooJ hypertro­phied.

The two sides had other easily quantifiable differences. The splanchnk-fcd lobes had mN': glycogen and glucokina~e activity anJ lo\',.:r cuncentrJti,)ns of cyclic AMP and active phosphorylase. The biochemical di~~o;:iati,m was shown in many other waysl-> that are beyond the scope of this rcview. but the rt!asonable inference was that these two liver sides were living in different metaholic worlds in which hormone control played a dominant rolt!. The nature of the biochemical diffcrences suggested that cndogenous insulin. \, hich was

_ ~" ___ ", ___ ,~ __ ,,, _____ , ____________ O"'''I ..... nb. __ 01

HEPATOTROPHIC SU3STA;\CES 137

being efficiently extracted by tht! first liver tissue to which it was exposed. played an important role. The significance of endogenous insulin was further highlighted when the advantages enjoyed by the h)bes perfused by splanchnic venous blood were greatly reduced. although not eliminated, by either total pancreatectomy l)r alloxan diabetes. Hi •l; While emphasizing the role of insulin, these investigations showed equally clearly that non pancreatic hormones or other substances also contributed to the total hepatotrophic effect of splanch­nic venous blood. Although the il'!fluence of these extrapancreatic factors re­mains unchallenged. they have not been identified.

E\'entually. another kind of double liver fragment model provided much more decisive information.':i·17·ls In these experiments. one portion of the liver was fed by the effluent of hormone~rich blood returning from the pancreas. duodenum. stomach. and spleen. \\hile the opposite lobes were perfU'ied via a venou~ graft with nutrition-rich blood returning from the intestine (Fig. I A).

The histopathologic results in 60-day experiments or even as early as 4 days were dramatic. The lobules in liver lobes receiving pancreaticoduodenal V\!­

nous effluent be-came bigger and crammed with glycogen in contrast to the <;hrunken deglyco~enated lobules in lobes r.:ceiving intestinal lit:nOliS return.

PANCREATECTOMY

Splanchnic di.ision

FIG, l-Spbnchnic dil'i,il>n e"p<rim~nts. In ,he~e d,lgS. the right liver lohe:s rec~ived wnous r ... turn fwm the p~ncr,';.:ic"~,,,\tnlduodenl"pknic rt'~i,'n, ilnd the left liva It'lles receiv.:d "en,'us 1l1,),,,1 fl0rn the: inte,r1T1c">, In other c'perimenh. the inte~tinal bl'hld \\a~ direch:d into the n~ht I"bes II i,h pan.:re;lIic n,HY tel the left ,ide, IA) !':,)ndi ... beti,: dog" (S) Alloxan-induced diaht:tic J,,~,. 1\) Dn!!"> II jlh t"lJI pJndealecl"I11Y, (g,' rami.."j(ln of Surgery. GYl'lccult'f!y. and Oh'letri.:~ I~O~~9--~b2. I'J7S.)

138 PROGRESS 1:-: LIVER DISEASES

An accurate way to quantitate hepatocytic size was developed for such ex­perimentsY With light-microscopic tracing. hepatocytes were drawn on a standard thickness paper and weighed. The \ .... eights \ .... ere called size units. In Figure 2. the right lobar hepall)cytes. which had pancreatic input. had an ob­vious advantage as compared tv those on the left. which were fed with intes­tinal venous return. The cell size data coult! then be summarized in graphs or tables.

In splanchnic division experiments (Fig. l). the previously mentioned pos­sibility that insulin was the major cause for the kind of cell size difference seen

__ ._ . . ', ___ .,_ .. in IJgure 2 was strengthened by additional60-day experiments in which alloxan ~,.'" . ~.., , diabetes (Fig. I B) and pancreai~ctomy (Fig. I C)'were' superimpo~·ed;H~." The

._ .. , .. - --'-··-~.~,·:-.·~~' .. -ani'"m;ls\~:eretre6.iedd·~ilr \dfh subcutaneous' insulin, which pre~urriabty \Vas

. ; .. ; ... ;;:~_~ --:......_ ... deliye,re.d Jq bl)th sides of the liver without preference: The"size_a<.lvarii:iges .. ' .. . .... ....., '. ~_: _ .. -::J(){the right-side-d 'heopat'ocytes '\~'ere ca'neelled about 'equally in ·lhe::-IIrilrruil:;.'· .. ~~:,~-_-=.~"._.~_-- .. ":~~ubj~ct~d' to 'allQ~an'aiao~'tes' C'['pancreatectomy. In all such ·e:<perim~nls. t~ ...

~':" ... :..,'.:-·--:--:7 .... -- . - "Qeat:.!y-eqi,al~effects of allo-~an-puis()iiing -ana pancreatec"tomY'have 1:encrectto-'-

rumimizi.!.:;J.!'\.Y nFl.i.L)~. rok ,of g!u;;;)gonas a hepatotrophic factor: at least as far '-as cell si7e was conc~rned.:: ...... .. .... ..'

At the S.1me rime. these e\perirnents emphasized that insulin was not the only factor. When endogc:nou~ in~1I1in \\ as r~movcd from thl! splanchnic di­vision experiments in which subcutaneous exogenoU'-. insulin was ~iven. the dominant hep3ti..: tissue became that supplied by intestinal venous return. Translating these findings inti) more practical terms, the most fa .... orable con­dition for portal perfusion \\as with splanchnic venous blood that cont3ined normal amount:- ~')f endogenou:- ir,<;ulin. The least favorable condition W~lS per­fusion with systemic venous blood. Intermediate in quality was splanchnic

LEFT RIGHT

FIG. 2-H\!patll:lte ~haJu\\ s tra.:~J during hiq,'pathoklgi.:: e:xaminati,>n. Th~se wo::re lata cut

out ,In ~tanJard p.lj:'er and weighed .1, ;,n inJe.\ of h.:pat".:} Ie: siz.::, The: I i;ht ",b~, with th..: Llr~o::

hO::P:lti..: ,'.:11, r",<'j\,<1 \'enoU, ~10,'J frl':n Ihe p.1n.:rea,. ,wmach. dU0d.:num. :In,1 sp!~<:!n. Th~

rdati"dy ,hrun\...;-n kflloho::, \\ ilh the ,m:.:t! hcP:'I,lCYr.:S r,·,;.:ivcd into::stin'll bhlU<.1. ([ly p(rmi"i,'n

of Surgo::ry. Glncc"l.lg,.;tIl.! Oh,!~:ri,~ 1~7:17Y-l'N. 1'173.)

TOTROPHIC SUBSTANCES 139

E '.-S,mlher of Lal"'It'd Hepatocyte,· pel' 1,000 lhl'tll"l"ylt's ill L.it ,'n "f .\'(lm/(/I n. 'I!<

lIlId Dog.\ "'it" Spill/ie/mi!' Di,·i.lifJlI

Number l)f Right L,'be~ Left Ll'h:s T~ pe Dog Experiments ~f.:an Sf) ~kal1 SO

11 1.6 ~ 0.5 1.5::: 0";

hnic division (nondiabetic) 6 17.3:,: 3.8 4.0:': 1.0

hnic division (alloK3n) 4 4.9:': 0.4 17.8::: 3.6

hnic division (pancreatectomy) 5 5.1 ~ 1.0 17.5::: 3.9

:~ blood that was deficient in endogenous insulin but rich in other as yet

'~n elements. insulin effect on cell proliferation was also convincingly unmasked by :ided liver experiments l6 •17 (Table 1). The liver lobes receiving pancreatic (the right lobes in the experiments shown, Table 1) of nondiabetic dogs tled to splanchnic division had autoradiographic evidence of hepatocyte ,1:1sia relative to the lobes receiving intestinal blood, although both siJes ~ater cell renewal than normal after 60 days. This right lobar dominance iminated, heing transferred to the left side by either cdloxan or pan-:rcu­ly diahetes in those animals being treated with subcutaneous regular .. The emergence of dominant left lobes (Table I) after the elimination l)genous insulin indicated, as previously emphasized frL1m other lines l)f ce, the presence of potent but unknown additional intestinal portal fac-

full implications of portal blood deprivation on liver function arc not I, since whatever changes occur in the portaprival state are undoubt~dly

Liver function after Eck's fistula, or after the better tolerated portacaval Jsition of Child, was long thought to be essentially normal, the main ncy being inefficient clearance of ammoniaY'~o With the striking erga­hanges described earlier after portal blood deprivation. however. the are apt to be wide ranging. An example is the striking antilipidemic

of portacaval shunt in dogs,16.21-:2~ rats,~1.~6 haboons.;·13 pigs,!;':' and lJ The consequent falls in cholesterol phospholipids and possibly tri­

des may be due in part to reduced hepatic lipid synthesis.16.2~.2;.! .•. JI.3Z

effect of ponal factors upon hepatic lipid synthesis has been demon-in the same splanchnic division models shown in Figure I, after 60 Lipid synthesis in normal unaltered dogs measured either with in vitro ivo techniques was the same on both sides of the liver (Fig. 3). After nie division in nondiabetic animals, the liver perfu~ed \\ ith blood from . ..:reas and upper splanchnic organs synthesized more cholesterol th:m cr liver portion perfused with venous return from the intestine. This ,ge in cholesterol synthesis was reversed with alloxan diabetes and total ~t~ctomy. As before, these results (Fig. 3) indicated the dependence of cholesterol synthesis upon the pancreas. but the reversal effect dem­~d a major contribution by non pancreatic venous blood as well. The ,nelusions were reached in other experiments in v..hich hepatic chl1ks-

140

2 0 100 .... 90 <x: a::

80 0 0..

70 a:: 0

60 u 2

50

::E 40 ::>

30 ::E X 20 <x: ::E 10 ~ 0 0

PROGRESS 17' LIVER DISEASES

in vivo CHOLESTEROL SYNTHESIS

NORMAL

-,f+ -

-----

-R L

N = II P NS

SPLANCHNIC DIVISION

r-:r-

R L

~ondicbetic

6

<0.05

-

R L

Alloxan Diabetic

4 NS

r--

..

R L

Pancrea t ec tomy

5

<0.05

FIG. 3-ln .... i .... o chulesterol synthesi;. i;l [he ri::ht and Idt liver lobes in nurmal dugs and in dog~ sut-mitted to splan.:hnic divi~illn. In :.!l the ,planchnic divi~ion e'periments. the right lobe~ reo ceived p.ln.:reaticogastroduodeno'pJ.;mic t-k,,'.!. "hii:: the left lobes were nllurished with i;1!<!";linal "enou, t-k)l,d. The animab \~i[h splan.:hnic .!i~i'il'n "c:r<!" nl'ndiabetic. allo,\an-diat-etic. l'r <fiat-etic as the r~sult of total pan..: re;ttec tom:- . Th;: p \;!lues evmp;\re the synthe~i; r.ltcs fN the 1\,0 ~ide.,.

the greater rate of synth<!si~ being ::I~~i:;::1~J a \'alue of IOO'7~. For the other side, a proportionately lower perc.:ntage was calcu13h:d. (B: p~~mi;,ion of Surgc:ry. Gyne":Cllolgy. and mhletric, 140:~81-3':16. 11:175.)

tewl synthesis was measured aftl!r stepwise portacaval shunt in which intes­tinal 00\ .... was diverted at a fir:.t stage fl)l!owed by secondary diversion of the pancreaticogastroduodenosplenic biL)od. 1~

\Vc now return from the doubk li .... er fragment models full cycle to Eck's fistula. If insulin was a vital ponal hepatotrophic factor, the reason for its unmasking by the double liver fragment cweriments beo.me umlerstanJable. The well-known eff.ciency of insulin's removal during ~ fir~t pass thr,)ugh hepatic tissue:!:! made the insulin relatively unavail'lble for a second liver or liver fragment. At the same time the pr,1tection afforded after portal diver<;ion by flow augment3.til)[1 proceJure'> <,u..:h a~ Child's portacaval tran"rl..)sition~ (lr Fisher's portal arterializ:Hion 1" was expbined. If insulin and other hepatl1-

trophic substances were bypassed arounu a single liver. they would be rt:tumeJ to it in diluted form in direct relatil)n tel the total hepatic bk10d flow th:it these procedures increased.

If the secrets of Eck's fi"tlIla \\cre e\plained mainl}' b) depriving the liver of direct access to endogenl11l:. insulin. the experiment o;hown in Figure ..:. should be a direct test orthat h~ f'L),hesi'). :-':lmhypoglyccmic: infusilln'i l)f in"ulin

HEI'.-HOTROPHIC SUBSTANCES

Body we!1

141

FIG. 4-E.\p.:rimenls in which Eck's fistula is performed and posloperali,e infu~ions are made into the left ponal vein. (By p.:rmission of the Lanc.:t 1:821-825. 1976.)

and other substances were made for 4 days into the ligated left portal vein after Eck's fistula.:·· 6 The experiment was designed to evaluate any direct pro­tt'ctiw effect on the left lobar hepatic tissue. as well as to assess a spillover dfe\.:t un the right lobes after recirculation. The results were unequivocal. Insulin greatly reduced the acute atrophy that otherwise halved the size of the cells, and it preserved hepatocytic ultrastructure. In small doses. glucagon did not potentiate the action of insulin, and in large doses, it may have reduced the insulin benefit. Glucagon alone in either small or large doses had no ef­fect. ;'.6

The effect of insulin on hepatocytic proliferation was al.;o striking. After Eck's fistula, the mitotic rate was already increased to about three times nor­mal (from 1.6 to 4.8 per 1000 cells). Insulin more than tripled this cell renewal. with no spillover to the contralateral lobes. Glucagon alone had no effect. nor did it potentiate the action of insulin. ' ·s

Thus, relative "hepatic insulinopenia" was established as the most impor­tant element in the liver injury of E~k's fistula. It would be regrettable if the very ~l3rity with which insulin has emerged as a principal porral hepatotrophic \ubstanc(! \\ere to obscure the search for contributory factors. The observation lhat the insulin prokction in our infusion experiments \\as not complete was interpreted as a reflection of missing ancillary substances. The <;ame multifac-

-:...: ---=..:.~~:*::.;...:....:=.~-. . -- -------

PROGRESS [N LIVER DISEASES

torial theme has been consistent in all work from our laboratory on the he­patotrophic subject. However. the fact that the multifactorial control of he­patocytic integrity has not deemphasized the ccntral role of in'\ulin in maintaining li'-er cells was recl!ntly redemonstrated after removal of all the nonhepatic spbnchnic viscera including the pancreas . .1~ The intraportal infu­sion of insulin alone prevented most of t.he atrophy and other structural de­terioration of hepatocytes. and it preserved the rate of spontaneous liver celt renewal which was otherwise depressed. The hepatic protection in eviscerated animals was almost identical to that observed with intraportal insulin therapy after portacaval shunt described above and was indistinguishable from the hepatotrophic effect of ins ulin in diabetic rats . .1~ In hepatocyte tissue culture- -systems, many investigators ha .... e described analogous insulin effects.3~-~~ The role of insulin in maintaining hepatocytic mitochondrial metabolism has also been emphasized. 4 ('-41 No potentiating effect of glucagon has been demon-strated in any of these nonregeneration models. -"- .. -. --.--.-.

PORTAL BLOOD FACTORS AND REGENERATIO:"l

From the information in the fl)r;.!going section. portal hlood fa~tors are in­disputably impL1r£ant in maintaining healthy liver cl.!lIs. The assumption was a natural one that portal blood might have a specific effect on the hepatic regen­eration that folklws partial hepatectomy. This possibility was purely specula­tive. however. since hepatectomi.::s were not performed in any of our early studies. However. a portal bluod effect on regeneration after liver resection in rats was soon demonstrat.::d. 4;-H

The nature of (he regeneration-promoting substances and their origin remain in dispute. An additional question is whether they initiate regeneration or merely p~rmit the process to prL1ceed and, in either case, by what means. The conflicting conclusions re2.ched in variolls laboratories on these issue) re<;ult in part from the use of different experimental models and in part from the way in which data have been interpreted or the time after hepatectomy when the data have been acquired.

Much information about the origin of regeneration-promoting (or permitting) factors has come from evisceration procedures introduced in dogs~-' in con­junction with partial hepatectomy and adapted for rats.~~ An artifact existed in this early work in that e\og.::nous insulin was incidentally administered as part of the postoperative p~lrenteral fluid therapy. Later studies sh(m ed :J. stri:';'­ing depression and delay of regeneration after complete eviscerati,ln that could be restorl.!d tllward or even tLl normal by treatment with a combinati0n of insulin and glucagon in high tio ~e~. 47-1'

The crllci~d ~planchnic fa..:tors did not seem to be from the intestill~. Al­though an obtllnded regeneration response was found after intestinal rest:c­tion .. ;" this could not be confirmed.:>!.:;: By contrast. an almost cl1mpkte ab­sence of liver r("£,cneration after total pancreatcctl)my in rats anJ du.;:~ \\'a, rcponed.-d-,'_'I arid thi ... could be r..:stured to normal by treatment with in5u:in and glucagon.:>! The: nucial ~rbnchnil.: organ for hep:1tic rCf!enc:rati\.ln \\a~ C'Jn-

)uHSTA~CES 143

pancrl!as. and insulin and glucagon wcrl! the most critical 1creatic wk. while the other nonhepatic splanchnic organs r0l1ance.:>~

this was an excessively simplified vie\v \\as available from ~ntly confirmeJ .. ·'" that liver resection in diabetic rats i-; fol-~ regeneration. Our own investigations with split liver prep­:us hepatectomy in diabetic and nondiabetic dogs emphasized 'pancreatic blood in supponing regeneration. but they also t similar qualities in nonpancreatic splanchnic blood. ~ AI­,'ere oobo interpreted by them. Broelsch et a!. demonstrated nsplantation t!.xpt!riments that venous effluent from the je-.·: : duodenu l11 supported hepatic regeneration. albeit less well he .pancreas. 'i, I rece-ilt studyha\lc! again demonstrated the complexity of ation bY-'portal hepatotrophic factors and have-strengthened·o.c­:actori~ll hypothesis by c/early differentiating pancreatic in-;e 'origInating in the req of the intra-abdominal gastrointes-5e investigations. the removal of all the nonhepatic splanch-::d in severe inhibitil)l1 of DNA synthesis and essentially on of the histopathologic expression of liver regeneration. colon in place diJ not significantly improvc the evisceratt:d to hepatic resection. as measured with autoradiography.

lat plasma pancreaticlike glucagon was thereby kept at a lcentration. Nor did the infusion of exogenous glucagon. )11 and insulin in combination into the portal vt!in havt! a . effect upon regeneration. prior removal of the pancreas alone reduced but did not

ise to 44c;f hepatectomy. The response to nc;c hepatic rc­\e dampened by pancreatectomy. ~lost importantly. extir­of the non hepatic splanchnic viscera. while preserving the the response to hepatic rt!section even more than did pan­. Thus, removal of the pancreas and other viscera had a 1O regeneration. [:.9 and more recently Leffert and Koch60 have similarly ion as a complex series of events under multifactorial con­:ay an important regulatory role. precise ddineation of their "e difficult with any of the presently availdble experimental mone-free environment is hard to achieve in intact animals. Small amounts of hormones could have major physiologic

c'generating hepatocytes may have changing sensitivity to Igon.',1-6] The same probably applies to other hormones.

tl Portal Factors Initiate Regeneration'?

!ions conceivably could be responsible for gro\\·th initiation I Aftcr partial hepatectomy in rats or dl)gS. \\'ell-ordered

-:cur In liver cyclic r\\lP and adenyl cyclase prior to and

144 PROGRESS 1:-'; LIVER DISEASE

during r~g~neration,~l'';;.h; The various nonhepatic splanchnic evisceratior. (pancreatectomy. extirpation of all organs exc~pt the pancreas. total evisce: ation) which resulted in retarded regeneration caused severe pertubations i these hormonally controlkd "messenger" components,fl:1 Wheth~r these d~ viations have a cause-and-effect relation to the defective regeneration that wa observed or are merely coincidental remains speculative.

The potential link be{\\ een mUltiple hormone changes and regeneration i strengthened by the intriguing studies of Mac~lanus et al..~; who had prev:

, ousty shO\~nwithcultured thymlls c~Js that, increa~es in. c::clic A:\IP [cyel ,. , ... ' induce~_\yitlL,~pjl)e.phrin~,- parathvrmone,- prostaglandin ... alld calcium imme

_ "c.:,~~~~·.:c~:~':::-::,-.·:'--·dr~itely pre~\!ded,the"initiation of DN A synthesrs and active celt proliferation , The same earl):biphasic rise .. in cyclic AMP o,;:cur in rat livers l~'2and 12h

':,~c:~:c:,,;"::':~:':::;'::'-::;~~::.;:~~·-;;:"'·:-afier partial lj~p.~tectQmy ,vJth a return- toward normal as D;-';A ~ynthesis.. be , . ,- ", "',;':-":.~;, '"<'-' ,.~ •• -, ·"gan. 6;"T~~~~J!!1d,ing~ hase_be.eri'~o_nftrmedjILr:ats:~:'~; and simiJ.u:. bulJe'j~~~J ,J ~"_=-_ -"-;.,:,!=.,::,.=:..",~.:.:.--:::::-=.:.:,~~ -::=:\je-fl~~J ch::mges- rlave., b.l!en h0te(rrn-regene-r.~ti'n£ dog livers.li:! In addition. in

- :-:'-,, __ ~ •. ~ ~'_:-_ .. -:- .• crea~ed cydlc.AMP-d(pi!mlent protein kinases correlated perfecrly in r~gc!1 ',: .. _ e'rating rat ~tvers.\\'ith(h~ induction of ornithine decarboxylase,r. ,

Ornithine decarboxylase ha5 l:reerr-jmpliC'ated""-~" as thl! rate-limi~ing enzym' in the polyamine biosynthetk pathways active in regeneration. Intra"enOIl solutions containing triiodothyronine. amino acids. glucagun. and heparin in duced nuclear DN A formation and mitosis in the whole livers of unoperate, nondiabetic rats.;1 and enh,:nced ornithine decarboxylase activity followel treatment with this solution.;~ Glucagon in this stimulatory solution could b, completely replaced with a butYf} I derivative of cyclic A~I P. Ic:",ding to thi conclusion that cyclic nucleotide plays a critical role in the induction uf hepatic ON A synthesis and cell mito~is, 73

Do Nonportal Factors Initiate Regeneration? '

While portal blood factors clearly influence regeneration. they may nut ini, tiate this process but merely playa permissive role, The actual genesis ol regeneration may have a quite different explanation and could even st3.rt in the liver itself. This possibilit~· has not been fully explored. even thuugh the lit­erature is replete with reports compatible with such a hypothe~is.

Publications between 1931 and 1953 suggested that liver mitosi~ could b~ stimulated in int~ct experiml!nral animals by homologous liver mash injected intraperitoneally'~-~~ or by intravenous injections of liver fractiL)ns, 7~ ~lcJlInkin and Breuhaus were the first to demonstrate increased mitllsis in a ml1del using the already r~generating partially hepatectomized liver of the rat,~; Hl)\\e\'Cr. the first truly convincing evidence of a liver-specifk mitoti..: ~ti::1l:btl'r wa~

that a single administration of liver mash pr~pared frum weanling r~t livers tlnd given intraperitoneally to adult rats caused hepatocyte proliferation that \\ as maximum at 48 hr. 7-,'9 Although adult liver mash W:1S nl)t stimulatory_ striking stimulatory activity was found \\hen the regenerating remnant of an :tuult rat liver, 48 hr aftl!r partial hep:~tectomy, was used [0 prerar~ the Ii\er ma'h.:~' Even after a year of twicc-\\ ed..ly injections, r~g~neratir.g adul [ li\ er m,hh sti!l had a hcpati..: mitlltic stimubrory dfect. Furtht:rml)rc:. in the;,e chr,1ni..:ally

-"""""------ "-"~---"" """" "---_._----

HEP.'TOTROI'IIIC SuBSTANCES 1.t5

treat~d rats. intra-abdominal ;umors de\"elop~d at a 67'1 rate. prc~umably be­cause of the specitk stimulus to proliferation. Only one of these tumors was a Ji\"er tumor, however, while the majority were intraperitoneal retkular sar­comas. R~tts chronic.dly treated with nonregenerating adult liver mash diJ not de\elop intra-abdominal tumors.;~

The concc:pt of a stimulatory substance originating in the regenerating liver it~c1f lay dormant until J971.~o.SJ Then in 1975. a regenerative stimulator sub­stance ",us demonstrated in the supernatant after high-speed centrifugation of an extract of rat liver mash. This regenerative stimulator substance was present in very young rat livers but only appeared after partial hepatectomy in adult

- li\'ers. The extract from intact adult rat livers actually inhibited regeneration in the a~say system used (34% hepatectomized rats).'~ " Mean\\ hile, evidence was accumulating that there was a circulating plasma or serum stimulatory factor in animals with re~oenerating \i\'ers. The n:Ievant experiments were diverse and ingenious. Regen(;rative activity was increased in the intact liver of the unresected partner of a pair of parabiotic rats after partial hepatectomy in the parabiotic twin."l .-\Ithough confirmed by soml!.'l.~.,> the concept remained in dispute until clarified by the morl! efficient cross­circulatilm experiments."~-~M As total hepatectomy in one rat stimulated sig­nificant DN A synthesis in the cross-circulated paT1ner with an intact liver. the source of the humoral factor was postulated not to be in the resected liver remnant," but the rationale of this contention has subsequently been chal­lenged."O

Although suggested earlier,H9 the stimula:l)ry dfect of serum from animals with a regenerating liver was first convincingly demonstrated in a cell culture system in 1952.~') This finding has been confirmed and extended.~1-9J Serum or plasma also increased mitotic activity in vivo. ~~-~, while hepatocytes prolif­erated in normal rats subjected to multiple exchange transfusions with blood from partially hepatectomized rats."~ Finally. mitotic activity \vas increased in small liver autografts in partially hepatectomized animals. II 1 ,)-J 03 The stimulat­ing substance in the serum of rats with regenerating livers was characterized as a heat-stable protein of low molecular weight (approximating 26,OOO).1'1~

The first convincing suggestion that such humoral factMs came from the liver itself was made by Blomqvist.;~ Fisher. howev.:r. based on the experi­ments already discussed, did not favor this concept." Tht.'n Levi and Zeppa,o.~1 appeared to establish the link bet\\ een the ~erum-qimubting factors and the liver by direct investigation with an isolated perfu<;ed rat liver system. They dcmunstrated increased DNA synthesi:. in norm:!! li\'ers pafused for 1 hI" (after :1 20-min ..,t~lbilization period), using th.: cf~uent L,f:.l regenerJting r:.lt li\ cr th:.lt had been subjected to a 70% partial hepa:ecwmy 1 R or ~4 hr previollsly and testing this by either direct cross-circulation or perfuc;ion of the normal liver with reconstituted effluent. Nonregenerating intact rat li\'ers caused no in­crease in DNA synthesis in this sy-;tem.'" They subsequently showed that the cd!.., ~ynthesizing new DNA were ml):,tly hepatic parenchymal cells situated predL1min:.lntly in the peripheral region: 1 l'nfortunately. this work could not he confirmed in carefully conducted stlldie~. i"U'''; The major objection was the

•

-... -.- -

\46 PROGRESS IN LIVER DISEASES

short time (I hr) of exposure of the normal liver to the partially hepatectomized· liver effluent. Attention has on..::e again been directed to a liver source for the humoral factors. however.'~ and. if confirmed. would strongly :,upport a liver­plasma physiologic axis that is important in liver regeneratiun.

By contrast. an inhibitor of liver regeneration remains an intriguing and controversial question despite investigation over the past half century. The controversy is highlighted in a number of excellent reviews. \0,-11" Both serum and liver extract from intact adult rats have been shown to inhibit regeneration in the already regenerating liver .. ~~~.\l1J wpile this inhibitor disappears within 2 hr of partial hepatectomy and is. in fact, replaced by a stimulatory sub­stance.K2

At this time. the true role of portal blood or liver factors in initiating or potentiating or in stimulating orinhibiting liver regeneration remains to be fully elucidated:- - . - . - - ... - . ... --'.

-.-- -------- - -. ----- .. -.'" :.·~CLI~IC.-\-b-·B-lPl:.I€t\-T-lfh~S--- .. ---.. -.-

_ _. Decisions in patients for or againsfportacaval shunt, as well as the type of ~ . . ~.~ -- --'~~,~~~~. '-." ----~~---=--.=: ~~~:~-=-s-huiit~' should take into consi"d-\!r"iition -ihe-'h-~patotrophlc concept~. ~jthepatop~t.ar~~

- ..... - ... -fJ()\v is still preo;ent ill the pc'rta! vein._ th.: Warren-Zcppa shunt~" preseryes -" _. this flow while at th~ same time d~compressing esophageal variCes. The long­term results of controlled tri::J.l~ of this ingeniolls procedure are a\vaited \vith ., .. interest. If portacaval shunting does nul prove to be of benefit in cirrhotic _ patients with bleeding esophlgeal varice5."~ the evaluation of nonshunt pro­cedures will assume increasing importance. ILI

We believ~ that preservation of portal fl0W is a vital concern in patients with liver disease. However. the fact that man is resistant to the:: more s(!riolls metabolic consequences of Eel';"" fi stu 13 7 has made it feasible:: to perform the procedure with b~nefit in patients suffering from glycogen storage disease. These patients have had correction of a number of preexisting metabolic ab­normalities. as well as amazing gro\, •• th spurts.~!I.II~.II:' Continuous feeding may be an even better way of tr~ating these children or at least is an ancillary measure that can be used with "hunting"l~

Lately. our greatest interest in portal diversion has been in homozygous type II hyperlipidemia.~v.lO a disl1rd:!r that leads to I~thal cardiovascular complica­tions by adolescence. More than 20 patients throughout the world (3 in our personal experience) have had their serum lipids lowered by portacaval shunt. Only two outright failures of response have been recorded. and in both (one from Europe and one from South Africa) the shunts had clotted. The serum cholesterol concentration in our original case fell from 800 mg.'dl to nearly normal, probably as a result. at least in pitrt, of reduced hepatic chole:;terol synthesis. as mentioned earlier. Th~ bIb in serum chulesterol in our patients 2 and 3 were also dramati;;. th~ range of reduction being 40~( w 60,{. The unsightly xanthomas in the skin and tendon'S melted away \vith time. Rdief of angina in some of these patients ~nd diminution of aortic stenosis in others have suggested that rt!sorption of the same material is occurring from the damaged vascular system .

CBSTANCES 147

hic concept has suggeqed new lines of inquiry in a more the pathogenesis and/or treatment of several human disease

19 a variety of li\ er disorders and even diahetes mellitus. for liS insulin therapy may be the right drug by an inappropriate

v-workers. ll : in Volume IV of this series. pointed out that iver to regenerate in the setting of fulminant hepatic failure 1hasized in the past. In their view the available methods of ot influence mortality unless sufficient regeneration occurred could be stimulated therapeutically. As no major break­made in the management of fulminant hepatic failure. the with a better understanding of the controlling mechanisms itiators and potentiators}, methods of stimulating regenera­nts will become available. Possible therapeutic modalities herapy, as ~uggested in the past. l: Whether the answer will mixtures:I.::l L)r in pharmacologic doses of insulin ami glu­d by the study in mice with murine hepatitis,llS still remains natively. future therapy may \\ ell be with as yet unidentified .:ration. \vhich might e\'en originate fmm the damaged or itself.

REFERE~CES

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K.\. Watanabe K. PUI·

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1,,"11 in d(lg', Lan,'':l

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I ~ \brchi,'r,) T1.. P,)rter KA. Oickin~on Te. F,IfI'S TO, Slarzl TF: Physiologic requir.:­ments f,'r au\iliary Ii".:r homotran,planla­li,)n. Surf: G~neC'l'1 Ob,tet 121;16-31. 1965

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~~-.-----......- .--------

148

spbnchni.: \'c:n,lU, bloou flow ,'n th.: liH~r. Surg F,'rum 16:~80-:!8:!. 1965

14. Marchi,'r,' TL. Porur KA. Brown BI. OU.: J·B. Slarzl TE: Th.: eff.:.:! of partial porta­caval tran'p",iti<.ln on th.: canine: li .. .:r. Sur­gery 61 :723-7.~:!. 1967

15. Slarzl TE. Francavilla A. Halgrinhon CG. Fr<tncavilla FR. P<.lrt.:r KA. Brown TH. Putnam CW: The origin. hormonal nature. and action l'f h.:patotrophk substances in portal \'en~lU' I>ll.od. Surg Gyne';:lll Ohst.:t 137:179-199.1'173

PROGRE'5S 1:-'; LI\'ER DISEASES

by porta.:a\ al ;:,n:l.;;tomo,i, in rat .. fed on a

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::7. Chas.: HP. :'.k'm, T: Chole~terol metabo­li,m follo\\ing pvrta;a\,al \hunt in the rig. .-\th.:ro5~lero~i' :.!:141-1~. 1976

~8. Clrew TE. Sail. RP. J,'han~n KH. Denni" 16.' Starzl TE. L.:e ·l¥r Porter KA. Putnam CA. Stcinberi- 0: Lo\\ d.:n~~t>. and high

-CW: The int'lu.:n~~ of portal blo.,d upon d~n~ity lip,'prckin lUm()ver fllllll\\ ing p<.lr-lipid m.:taboli~m in normal and diabetic ta·;aval ;hUru In-,;u·ine. -J Lipid Re:i r;;---dogs antJ .t>l!P,)ons. SUfI! Gynecol Obste! ~1-450. 1976 ..

. '140:381-396. 1975····· ..•. _-.- ... ···.,.,..·····~9:--Sti1rZlTF:. Cha~ HP. PuttT:tlTl'€-w..-Ponet"-

-, 17. Starzi'TE. Pl'rter KA. K3Shh\"agi N. Lee KA; Porta.:a\;,! :>hunt in· hypcrlip<.lpro:.:i­naemia. Lan.:.:! ~:940-94". 1973 ,:,:,,=,~c;=..c.~:_::,.:.::.:,,=·'::":"-o:,;·-:'· .. ::::':::.:..:;..~:::-.'lY. Russe.lt Wjl.: Putl1amCW:.. The diect:

.' . of diabetes m.:i1itu> on portal blood hepa •. ·::·totrophic f:.L.:tol'~ in.dogs .. Surg _Gynecol

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\.:1 :'.IL. D,"J>qu.:t G. VauLc!le JL: Deri· vation' pOr[,'hepatiques selective:; origirre­p;lncreatiqu.: du factc:ur hepatotmphique purt,d. t\nn Chir :!3:393-40:!. 1969

19. Silen W. i\b\\d_ley DL. Weirich WI.. Har· p~r H.'\: StuJi.:s of hepatic funct;')n in dog~ \lith Ed .. Ii-tula or portacaval tran ... posi· ticln. AM.·\ :~r.:h Sur!,! 74:96-1-973. 1957

20. Owsley JQ. (i"in n.1. Clark.: JC. Harper HA. i\IcC,lll..k HJ: A ,omp;lri~l'n (If ga-1.lctll~': ckar;.lnc.: by the !iva f,lilo\\ing por· tacaval tr:tn,position or Fck fistula. SlIrg Fllnlm 9:~15-5IR. 1958

21. Winter Ie. \'.In Oolah HE. Crandell LA: Lowered ~.:rum lipid levels in the Fck th· tula dog. :\m J Physiol 133:566-571. 19~1 CllY!.: JJ. S.:hw;trtz MZ. i\laruhhio AT. Var..:o RL. Buchwald H: The eff.:c! of por· tacali.1I shunt on plasma lipids and tissu.: chlll.:slerol synthesis in the dog. Surg.:r.y 80:54-60. 1976

23. Bur;11.. W. G;,ngl A. Funllvic'i J. Grabn~r G: Effect l.lf purt~lca\'al ~hlln{ and 'Irt.:ri~tl­

iz .. ti"n of tn.: liver ()n bile acid ml!laboli~m. G.l.;uVen:"r,llog\' 6'J:3J8-3~ I. 1975

~4. CillLm~1I1 11. Co~11! J1. SchneiJa PD. \':tr~cl RL. Bllch\\ald H: The dft:i:t of,t'I.:.:!i\t' vi~ceral c;l\al shunt on pla~ma lipid~ and chllk~t..:r<ll dynamics. $lIrg.:ry S~:4:!-;ill.

1977 ~5. Edw'lld, KDG. H.:rz R. S.:aky JE. Br"J·

ky Sf':: 1.<1\'erin): (If blood pr':,,"re. rl~hl1l.1 renin sub,tr~lt.:. ,h,'J.:Sh;wl and tlil!I)'':!'id.:

-... - -----------_._--------

30 .. Stafz1 TE. 'Putri,lmCW;Kuc:p U~ P0'r1a-~ ci\.val ,hunt ,!n<!. h~ pe:rlipidc:mia. Arch Surg lif71-~~: Trr.~ .. '.

:; I. hml!~ J. S0et:~' PR. Fi,;her J f: In \'ivo u~mon':r::tiui'! ,,:' impaired chul<::;t~rora;1d f:ltty a,::id,,~ n:h~;i" fuIl,m ing rvrta.:a\.11 ~hunt (PCS) (..:t--:racted,. Ga~troent<!rolc'gy -~:I075. 1977

3:!. Bilheimer OW. G<.lI~lst.:in J L. Grundy S~I. Bro\\n ~IS: R.:~uction in chok,terol :lnd km den;ity Ii~,'?rotein syn!h.:~is afl.:r p<lr­til": 3\' 31 shunt ,:Jrg.:ry in a p~lti<!nt "ith h,.ml'l~ l!l'U\ L:nilial h~ rc:n;h,'I~,tefl'!e­mi;!. J Clin In\!-. 56:I·CO-I4.l0. 1975

:H. Fi~lu JB: E,:r .. .;ti.," of insulin by li~.:r.

.'nnu R:v ~b:! ~-+:3()9...~ 14. 19i3 34. Srarzl TE. Fr:::'!..:~\'iII:. :\. P~'rter KA. B~n­

kh,lu J: The: e::·~.:! up,'n the li,.:r of e\is­.:uation \\ ith l'~ \\ ith~lul hllrm<.ln.: rc:pla.;e· men!. Surg G~ r.~..:ol Ot-st.:t 1-16:524-531. 19711

35. R.:av.:n EP. Pe:<!rson DT. Reaven G~I: The effect of e\penm.:ntal di:\t>.:te~ mellitus :lnd insulin repla..:ement on hepatic ultra,true­lUre and pro:!;:! synthesis. J Clin Inve~! 52::!4~~fi~. I'r;

36. Gc!r,ch;:n~"r. LE. Ol.ig:\!..i T. r\nJ~r'\son ~1. :'.I,'''on J. [)a ... id,\,)fl :'.IB: Fine ~tru':lur:d and ~ro\\ th c~.!~\ct.:ri-ti..:s ()f cultured rill li\er celk I-.-'.l!in <!f(~':h. F\;:> Celi R.:s 71 :·19-:'::l. 19;~

37. Wagl~ SR. b;~t-rc:I ..... " WR. Jr. Samr'U" l.: StuJi.:s "i'! !~.: efr~ .... r> of in'\utin "n rly· c • .l~t'n ,~nth~':", ~lnJ u!~r~."trl!.;[ur~ in i ... o­bkd r.ll Ii\'~r !J':P:tto..:~ tes. Ih'.:h.:m Hi,,· phy, R.:s (\'~rr:un 53:9.n-9.p. 197}

3S. Jun!!;: L·. :-;.I,::":"1"ri 5: rrre..:t ,,[ m,ub an,!

HEPATOTROPHIC SlJBSTANCES 149

flu':;!g,'n on the DNA ~ynthesi~ of hepa- Grambort DE. Orloff MJ: Site of origin l'f l{lcyte cultules. Verh DIsch Go!, Inn ;\ted the hepatotrophic portal l,k,od f:lCIOr in-S::!! I ):;I~5-31S6. 1976 volved in liver ref!<=n.:ration. Surg Forum

39. Bc:rnaert D. Wan~on J-C, Drochmans P. ::!4:377-3i9. 1973 P(\po\\~ki .. \: Effect of in~ulin on ultrastruc- 52. Poirier RA. Clhll" CE: Role of the small ture ;tn,1 gIY':l'g.:nesis in primary cultures intestine in liver regeneration. Am Surg of aJult rat hepatocytes. J Cell BioI .. 0:555-557.1974 i4:S"'g.-900. 1977 53. Duguay l.R. Orlllff MJ: Regulation of livc:r

4ll. Old'\;! K. Yamada T. Honjo I: Role "r in- regenemtion by the pancreas in dogs. Surg ,ulin as a portal factor in maintaining the Forum 27:3~5-357. 1976 \'ia1:>i1ity'"r TiVe·r. Aiiif"'siirgJ80:716=-it!t:- C·j4;. Duguay 'L~';Orffiif MJ: 'R,']e '\:~ ·the pall-'

1974. . ___ . ····;-c·.:.~~-~~:··· . .::::.< ..... ::c.=·-,"'_c[~as in re&U'<ltion of liver rcgen.:rati,'n in .. 41. OZd\,:n'r<'- Yamaoka' y~ I'flnbtl- H.- Hon,itl -. 7~-: d~~gs.SurgForum ::!lf387-1Yli. 1977 C ." •

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-B. fi,her B. Szuch P. Fisher ER: EValuati,'n Chandlcr JG. Gr'llllboll DE. Oll"ff :-'11: pr a hunwral factor in Hver regeneration utilizing liver transplants. Canccr Res 31:322-331. 1971

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~<.J. \\'hl:lcmlll': :\0. Voorhec:s AB. Jr. Prke JB. Jr: H~pClti~ hlood Row (Illd pancrc:atic l1<'rnhl;J~, :1' m,),:ifier .. of hepatic re~ener· ~ti,Jn. Surg Furum 27:363-365. 1976

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58. Starzl TE. Fran.:a\jlla A. Pl'rta K .. \. B.:n­ichou J. Jl'ne, AF: The effect of .. pl:tnchnic viscera n:moval upon .:anine liver regen­eralilln. Surg G~ nccul Ob,.tet 14i: 1l)3-~0i. 1'178

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fl~. Fr'llh:~I,ilL1 .\. P,'r1er KA. Ikni.:h,lu J. Jc.ne, AF. St:,1/1 TE: Li\a r~~c:n..:rati,'n in J,'g"': ~1tllT'htlk'gic <lntl ch~mic'll chang~~. J Surg Res (in rrc,s)

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'I. Sglll J·C. Ch.lrtas ."'C. Chandl.:r JG. A'.IP c,lIKentra!i,ljl' priM III !h.: inilj~lti,'n

-. : .. ~ - ..

150

of D:-J A s~ nthesis f,)llowing partial hepa­tc=.;:tl)my I.H hl)rmelne infusilln. Bi,lchem Bi0phys Rc:s C0mmun 4'.1(5): 1:!01-L!07. 1972

66. Thm\\c:r S. Onl ~IG: Hormonal control of li"c=r regc:neration. Bio,;;hem J 144:361-369. 1974

67. ByU5 C\". Hc:d~e GA. Russell DH: The in­H)lvement of cycli.: I~MP-depc:ndent pro­tdn kinase!s) in the induction of ornithine:: decarboxylase:: in thc= rc=gene::rating rat liver and in the adrc=n,ti gland after unilateral ad­renale.;:tom~.' Biochim Biophy~ Acta 49S4l ):39-45.,1977

68. Cohen 55: Introduction to the Polyaminc=s. Engle-...x"lti Clift's. NJ. Prentice-Hall. 1971.

pp r-l79 69. Janne J. Raina A: Stimulation (\f spermi­

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70. Russell D. Snyda SH: Amine synthesi\ in rapidly grc'\\ i ng tissues: Ornithine decar­bo\~ bsc ;.:~ri\ it} in re;;c:nerating rat liver. chick embryo anu various tumors. Proc Natl Acad S.:i CSA 6O:1420-14~7. 1968

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