alkaline phosphatase in auxiliary transplantation

Gut, 1974, 15, 636-643

Alkaline phosphatase in auxiliary livertransplantationJ. JERSKY

From the Department ofSurgery, University of the Witwatersrand, Johannesburg, South Africa

SUMMARY Auxiliary liver allografts were inserted into baboons and allowed to undergo rejection.The incorporation of 14C leucine into alkaline phosphatase was measured in both host and donorlivers with the aid of an anti-alkaline phosphatase antibody. The results indicate that the increasedserum alkaline phosphatase levels found under these circumstances are due to increased synthesisof the enzyme by both livers. Furthermore, it was shown that in the absence of biliary obstruction,this enhanced production of alkaline phosphatase is accompanied by an increase in the excretionof the enzyme into bile. It is suggested that the stimulus for the enhanced production of alkalinephosphatase is a substance which is normally produced in the liver and excreted into the bile.

In recent years it has become clear that the increasedserum alkaline phosphatase activity associated withcertain hepatobiliary disorders is accompanied by asimilar rise in the liver itself. Although this findingis suggestive of hepatic overproduction of theenzyme, other interpretations are possible and havenot been formally excluded. For example, theincreased activity could be due to an enhancedefficiency of the enzyme as the result of a conforma-tional change in the molecule or of a change in theconcentration of various modifier substances. Suchobjections to the conclusion that the increasedactivity is due to enhanced synthesis can be overcomeby the use of a method of identification which doesnot depend on the catalytic properties of the enzyme.

In the experiments described in this paper, aspecific anti-baboon hepatic alkaline phosphataseantibody was used to isolate the enzyme from serum,bile, and liver extracts obtained from baboons whichhad previously been injected with 14C leucine. Asthis amino acid is incorporated into the alkalinephosphatase molecule, the radioactivity in theprecipitates provided an index of the rate of forma-tion of the enzyme.The experimental model chosen was that of auxi-

liary liver transplantation, as very high levels ofserum alkaline phosphatase activity occur in hepaticallograft rejection in baboons (Myburgh, Abrahams,Mendelsohn, Mieny, and Bersohn, 1971). An

Received for publication 30 April 1974.

additional advantage of this model, as will be shown,is that increased formation of the enzyme in thesecircumstances is not confined to the graft, but alsooccurs in the normal, unobstructed host liver, thusallowing a study to be made of biliary excretion ofthe enzyme. This has not been possible in previouslypublished experiments, where partial or total ob-struction to bile flow has been utilized as thestimulus to produce high serum levels of alkalinephosphatase activity.

Material and Methods

All the experiments were carried out on healthyChacma baboons (papio ursinus), each weighingabout 12 kg.

INFUSION EXPERIMENTSThe common bile duct was ligated in a baboon.Subsequently, jaundiced serum from this animaltogether with 5 tCi of 1251-labelled albumin wasinfused into three healthy baboons. The clearanceof alkaline phosphatase, bilirubin, and 1251 albuminfrom the serum was then measured over a 72-hourperiod. In addition, alkaline phosphatase activity inthe liver and bile was measured at the beginning ofthe experiment and after 24, 48, and 72 hours.

AUXILIARY LIVER-TRANSPLANT EXPERIMENTSThe net rate of formation of alkaline phosphatasewas assessed in normal baboons and in animals into

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Alkaline phosphatase in auxiliary liver transplantation6

which auxiliary livers had been inserted, by measuringthe incorporation of 14C leucine into the enzyme inthe blood and the liver tissue and gallbladder bilefrom both host and donor livers 24 hours after an

intravenous injection of the isotope (20,u Ci/kg bodyweight). In addition, alkaline phosphatase andglutamic-oxaloacetic and pyruvic transaminaseactivity was measured in these tissues and electro-phoretic analysis of the alkaline phosphatase was

carried out in starch gel.

Grouip 1Three control animals were subjected to repeatedlaparotomies under general anaesthesia in order toassess the effect of these procedures on the baselinevalues of the measured parameters. The firstlaparotomy was followed by a second four dayslater, directly after which a dose of 14C leucine was

administered. Twenty-four hours later a thirdlaparotomy was performed in order to obtainmaterial for examination.

In a further two control animals the 14C leucinewas given and the specimens were taken withoutany prior anaesthetics or laparotomies.

Group 2In five baboons, auxiliary livers were inserted (Welch,1955) and specimens were collected for baselinestudies. The animals were not immunosuppressed.After four days, 14C leucine was given and 24 hourslater further specimens were obtained. Bile couldnot be obtained from the transplanted livers, how-ever, as the gallbladder was always found to beempty, presumably because of the cholecyst-enteros-tomy which had been performed and becauserejection had sharply reduced the output of bile.

Group 3 (five baboons)The auxiliary livers were removed four days afterinsertion. 14C-leucine was then administered to the

animals and the various parameters were measuredin the remaining host livers 24 hours later.

PREPARATION AND CHARACTERIZATION OF

THE ANTI-ALKALINE PHOSPHATASE ANTIBODY

Highly purified alkaline phosphatase was preparedfrom rejecting baboon-liver allografts, using themethod of Engstrom (1964). The final product,which contained 1 milligram of protein per millilitre,was mixed with an equal volume of Freund'scomplete adjuvant and each of five rabbits wasimmunized with 500 micrograms of protein, injectedinto multiple sites. The sera from four of theseanimals contained anti-alkaline phosphatase activitysix weeks later, but were found also to react withmultiple antigens on immunoelectrophoresis againstbaboon serum and extracts of baboon liver. Afterabsorption with serum from a baboon with very lowlevels of alkaline phosphatase, the specificity of therabbit antiserum was examined in the followingways: (a) By immunodiffusion and immunoelectro-phoresis. (b) Increasing quantities of antiserumwere added to liver extracts containing knownamounts of alkaline phosphatase, glutamic-oxalacetictransaminase and lactic dehydrogenase and tosolutions of 1251-labelled human albumin. Controlsconsisted of antiserum unabsorbed with baboonserum and 'normal' rabbit serum. After standingovernight at room temperature, the tubes were

centrifuged and the residual activity in the super-natants was measured (table I). These tests were allperformed in triplicate.

In the transplant experiments, liver extracts wereprepared for testing by homogenizing 10 g of liverin a mixture of 30 ml of 001 M sodium acetate- 0.01 M magnesium acetate and 17 ml of n-butanoland collecting the aqueous phase after centrifugation.Bile was diluted 1: 20 with sodium acetate-magnesiumacetate and serum was used neat. The pH of all thetest solutions was adjusted to 7.4 before adding

Antigen (activity) Rabbit Anti-alkaline Phosphatase Serum

Absorbed with Baboon Untreated (ml) Normal Rabbit Serum (ml)Serum (ml)

0 0-2 04 08 0 0-2 04 08 0 0-2 04 0-8

Alkaline phosphatase (KAU%) mean 119 42 1 3 120 37 0 0 116 118 109 124± SE 2 2 1 1 2 3 0 0 5 2 5 4

Glutamic oxalacetic transaminase mean 2435 2645 2994 2997 2350 1218 900 1112 2306 2600 2579 3041(Wroblewskiunits %) ± SE 208 160 52 112 114 38 32 54 108 159 186 113Lacticdehydrogenase mean 133 112 119 143 117 135 121 135 122 133 124 114

± SE 7 5 7 4 2 4 4 5 6 5 8 8Albumin(C/min x 10- ) mean 342 375 370 406 358 246 216 227 386 349 349 402

± SE 7 22 28 5 18 23 13 4 19 26 10 15

Table I Reaction of rabbit antialkaline phosphatase serum with multiple antigens

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Fig 1 Disappearance-curves of bilirubin, alkaline phosphatase, and 1251-labelled albumin from the blood ofa normalbaboon after the intravenous injection of5 ,uCi of 12II albumin and 100 ml ofjaundiced serum containing 21 mgbilirubin and 4J02King-Armstrong units of alkaline phosphatase. 0-0 = bilirubin; 0- = alkaline phosphatase;A A = 125_ labelled albumin

antiserum in optimal proportions. The mixtureswere allowed to stand at room temperature for 24hours and the precipitates were then collected on022,u millipore filters for counting in a Beckmanscintillation counter.

ENZYME ASSAYS AND HISTOLOGYGlutamic-oxaloacetic acid and pyruvic transaminaseswere measured by the method of Wrobleski, andalkaline phosphatase according to King andArmstrong. Histological sections were stained withhaematoxylin and eosin.

Results

INFUSION EXPERIMENTSBilirubin was rapidly cleared from the serum, whilethe 1251-albumin levels slowly declined after equili-brating in the intra- and extravascular fluid com-partments (fig 1). The pattern of disappearance ofalkaline phosphatase resembled that of albumin,suggesting that it had entered the same 'pool' and

was not accumulating in the liver or being excretedinto the bile (or elsewhere). In support of thisconclusion, no change occurred in the concentrationof alkaline phosphatase in the liver or the bile(table II).

Hours Alkaline Phosphatase LevelsafterInfusion Liver Bile

(K-A units/g) (K-A units %)

I II III I I! III

0 4-8 3-7 3-1 168 116 688 4-8 35 2-9 142 98 71

24 4-3 3-8 3-7 111 134 8348 4-6 3-4 3-6 124 102 5972 4.9 3-6 3-2 133 108 66

Table II Sequential levels ofalkaline phosphatase inthe liver and bile of three baboons after an infusion ofjaundiced serum''A sample of each bile specimen was weighed before and afterevaporation and the alkaline phosphatase levels were adjusted toallow for variations in the concentration of the bile. The numeralsI, II, and III refer to the baboons; the serum levels obtained in Iare depicted in figure 1.

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Alkaline phosphatase in auxiliary liver transplantation

AUXILIARY LIVER TRANSPLANT EXPERIMENTS

Group 1

Initial liver alkaline phosphatase activity in thisgroup, as well as in groups 2 and 3 and in the infusionexperiments, varied between 1.4 and 4.8 King-Armstrong units per gram liver.The rate of incorporation of '4C-leucine into

alkaline phosphatase was similar in all animals.None of the parameters was affected by the repeatedanaesthetics and laparotomies. No radioactivitycould be demonstrated in the alkaline phosphataseprecipitated from the blood or bile.The liver glutamic-oxaloacetic and pyruvic trans-

aminases likewise remained fairly constant, varyingbetween 84 and 110 and 47 and 78 units per gramliver respectively.

Group 2The elevated serum transaminase levels whichfollowed transplantation were accompanied bydecreased concentration of the enzymes in theauxiliary graft, confirming the view that theseenzymes 'leak out' of damaged cells. In contrast, theraised serum alkaline phosphatase was associatedwith enhanced activity in both host and transplantedlivers as well as in the bile from the host livers(table III). This alkaline phosphatase was charac-teristic of the liver isoenzyme on kinetic studies(Hammond, Balinsky, Bersohn, and Jersky, 1973)and on starch-gel electrophoresis (fig 2). A four-foldincrease in the incorporation of '4C-leucine intoalkaline phosphatase was noted in both livers of eachanimal and significant counts appeared in thealkaline phosphatase in blood and in the bile from

Fig 2 Starch-gel electrophoresis ofserum alkalinephosphatase in a baboon before (left) andfive days afterauxiliary liver transplantation. The main band is inthe ,8-globulin region and the fainter ones near theorigin

Experiment Serum AuxiliaryLiver Host Livers Bilefrom Host LiversNumber Transplants

Transplantation

Before After Before After Before After Before After

Alkaline phosphatasel 1 17-6 29-8 3.5 14-1 3-2 42-7 66 1 763(King-Armstrong units) 2 16-2 46.0 3-1 26-0 4-7 37-4 120 1 293

3 10.0 23-1 4-6 18-3 2-8 20-9 77 9874 11-6 400 3-8 26-6 4-3 13-7 152 1 216S 28-4 56 8 2-9 21-4 3-4 31-3 91 1 032Mean 16-8 39-1 3-6 21-3 3.7 29-2 101 1258

Glutamicpyruvate 1 12 53 51-3 22 5 502 61.2transaminasel 2 25 33 46-8 25-5 78-4 76-5(Wroblewski units) 3 27 880 47.0 2-8 49.7 41-6 Not done

4 27 76 67-9 19-2 60-0 51-35 21 320 71-2 42-1 58-3 57-1Mean 22 272 569 22-4 59.5 57.9

Table III Alkaline phosphatase and glutamic-pyruvic transaminase values bejore and five days following insertionofan auxiliary liver

'In serum and bile, the figures represent units % and in the livers, units per gram of liver.

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the host livers (fig 3a). Histologically, evidence ofrejection was found in the grafts, while the hostlivers appeared normal (fig 4).

Group 3The findings in the host livers after prior removal ofthe grafts were the same as those seen in the hostlivers in group 2 (fig 3b).

SPECIFICITY OF THE ANTI-ALKALINE PHOS-

PHATASE ANTIBODYA single sharp precipitin line was obtained on im-munodiffusion (fig 5). On immunoelectrophoresis,two precipitin lines were seen (fig 6).

Table I summarizes the results of the experimentsin which the ability of the antisera to react withvarious antigens was tested. Both the absorbed and

Fig 3 Incorporation of 14C-leucine into alkaline phosphatasein various tissues before and fivedays after auxiliary liver trans-plantation. In figure 3a is depictedthe results obtained when the 14C-leucine was injected with theauxiliary livers in situ; figure 3bshows the findings in the host whenthe grafts had been removed 24hours previously. The results areexpressed as counts per minute perg liver tissue, per ml bile, and per100 ml serum. In each case theleft of the two columns representsthe pretransplant figures T.L. =

ifl auxiliary liver graft; H.L. = host[j liver; H.B. = gallbladder bile

from the host liver; S = serum

Fig 4 The histological appearances of the host liver(left) and the auxiliary liver in the same animal, fivedays after insertion of the graft

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Fig 5 On immunodiffusion against a baboon-liverextract (A) the unabsorbed rabbit anti-baboon-alkalinephosphatase serum (C) yielded a broad band ofprecipitation. After absorption with baboon serum

low in alkaline phosphatase, this antiserum (B) nowproduced a single, sharp precipitin line. The line betweenC and B is the result of the reaction of the unabsorbedantiserum against the baboon serum in B

Fig 6 Immunoelectrophoresis. The wells contained anextract of baboon liver (see text) and the trough was

filled with absorbed rabbit anti-baboon-alkaline-phos-phatase serum. In addition to the dense precipitin linein the P-globulin region, a faint second precipitin linecan just be made out

unabsorbed antisera were able to precipitate all ofthe alkaline phosphatase out of solution. However,while the unabsorbed antiserum also reacted with1251-labelled albumin and with glutamic oxalacetictransaminase, the absorbed antiserum did not.Neither antiserum could be shown to react withlactic dehydrogenase. It is concluded from theseexperiments that the absorbed antiserum is largely,but not entirely, monospecific.

Discussion

It is now generally accepted that the alkalinephosphatase activity found in the liver probablyarises in that organ and does not accumulate therefrom extrahepatic sites (Combes and Schenker,1969). The results of kinetic studies on alkalinephosphatase extracted from normal and trans-planted baboon livers (Hammond, Balinsky, Bersohn,and Jersky, 1973) and of electrophoresis of serumalkaline phosphatase in patients with liver disease(Newton, 1967) and in liver transplantation inbaboons (fig 2) support this view. More direct proofis provided by the many experiments in which infusedhomologous alkaline phosphatase has failed toaccumulate in the liver or be excreted in the bile(Cantarow and Miller, 1948; Wang and Grossman,1949; Clubb, Neale, and Posen, 1965); similarresults were obtained in the present experiments(table I, fig 1). Furthermore, it would be expectedthat if the alkaline phosphatase in hepatobiliarydisease did originate outside the liver, insertion ofan auxiliary liver should have no effect on serumlevels of the enzyme, as excretion should proceednormally in the host's own liver. The fact that serumalkaline phosphatase rises after this procedure, eventhough biliary excretion not only does not diminishbut actually increases (table II), strongly suggeststhat the increased enzyme activity arises in the hostliver. As will be shown later, the increased alkalinephosphatase activity in these experiments is notconfined to the host's liver but also occurs in theauxiliary liver.

Possible explanations for the increased hepaticalkaline phosphatase activity found in hepato-biliary diseases include an enhanced efficiency of theenzyme, release of enzyme from preformed inactiveprecursors, an increased rate of synthesis (or adiminished degradation in the face of a constantrate of production), and accumulation in the liveras a result of failure of biliary excretion.Numerous workers have evaluated the possibility

that the increase in alkaline phosphatase activityin hepatic biliary disease is due, not to any increasein the number of enzyme molecules, but to a changein the efficiency of action of the enzyme (Gutman,1959). This could result from the release of activatorsor the removal of inhibitors, or from a conforma-tional change in the enzyme molecule. The formerpossibility has been repeatedly excluded by com-paring the enzyme activity in sera obtained beforeand after bile duct ligation with that found aftermixing two samples. The enzyme activity of themixture has always proved to be the mean of theactivities of the unmixed samples, indicating thatthere has been no change in the concentration of

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modifying substances. However, increased efficiencycould also result from conformational changes inthe enzyme molecule, as has been shown to be thecase in the steroid-mediated increase in alkalinephosphatase activity seen in cultured HeLa cells(Griffin and Cox, 1966). Mixing experiments wouldnot differentiate between increased activity due tothis mechanism and that resulting from an actualnet increase in the amount of enzyme produced.Similarly, the experiments of Kaplan and Righetti(1970), in which it was shown that cyclohexamidecould eliminate the increase in alkaline phosphataseactivity which normally follows bile duct ligationin rats, do not exclude the possibility that proteinsynthesis is necessary for a conformational changein the enzyme molecule or for the release of enzymefrom precursors. The fact that the steroid-mediatedconformational change in HeLa cell alkalinephosphatase can be prevented by cyclohexamideprovides a precedent for such an interpretation oftheir data.

In the present series of experiments, it has beenshown that there is an increased rate of incorpora-tion of '4C-leucine into alkaline phosphatase at thesame time that alkaline phosphatase activity rises.This finding argues very strongly in favour of thehypothesis that the increased enzyme activity is dueeither to increased synthesis or diminished degrada-tion of alkaline phosphatase, although two possibleobjections to this conclusion can be raised and shouldbe considered. The first is that the accumulation ofenzyme within the liver merely results from failure ofbiliary excretion. If this were so, an increase in theradioactive alkaline phosphatase per gram of liverwould result even though there had been no trueincrease in the rate of production of the enzyme.That this is not the explanation is shown by the fact,discussed below, that increased intrahepatic in-corporation of 14C-leucine into alkaline phosphatasecan be demonstrated in the host livers, where normalquantities of enzyme-rich bile are being produced.The second and more serious objection concerns thespecificity of the antigen-antibody reaction used toisolate the radioactive enzyme. It is likely thatmaterial other than antibody and alkaline phos-phatase was present in the precipitate, even thoughthis could not be demonstrated. However, althoughthe imperfections of this method of isolatingalkaline phosphatase are acknowledged, it isbelieved that valid conclusions can be drawn fromthese experiments for the reasons discussed pre-viously. It is not possible, by the techniques used inthese experiments, to distinguish between increasedsynthesis of alkaline phosphatase and diminisheddegradation.The exact relationship between obstruction to bile

flow and raised levels of hepatic alkaline phosphataseactivity has been particularly controversial. Theclassical 'retention' theory, according to whichalkaline phosphatase is normally cleared from theserum into the bile and accumulates when bile flowis obstructed, has been disproved by the findingsthat the isoenzyme in these conditions is of hepaticorigin and that infused alkaline phosphatase is notexcreted via the biliary tract. Nevertheless, theenzyme is normally present in bile in small quantitiesand large increases in activity occur in certain cir-cumstances. Thus, ligation of one hepatic ductresults in an increased amount of alkaline phos-phatase in the bile of the unobstructed lobe (Polin,Spellberg, Teitelman, and Okumura, 1962). Similarlyin the baboons which received auxiliary hepatictransplants, markedly enhanced alkaline phosphataseactivity has been demonstrated in the bile from theirown livers. Like the enzyme of non-hepatic origin,liver alkaline phosphatase is not cleared from theserum into bile (fig 1, table II), so that the highbiliary levels in each of these experiments must haveoriginated in the unobstructed lobe and the host'sown liver respectively. This implies that in thesesituations the unobstructed liver tissue is alsoproducing increased quantities of the enzyme whichis then being excreted into the bile. Evidence thatthis is so is provided by the finding of an increasedrate of incorporation of 14C-leucine into alkalinephosphatase in the host's liver even after removalof the auxiliary transplant, thus excluding thepossibility that the labelled enzyme originated in thegraft when both livers were inplace. At the same time,there is a parallel increase in the quantity of radio-active alkaline phosphatase in the bile from thehost's liver (fig 3b). It would therefore seem that inthe absence of biliary obstruction, augmentedhepatic production of alkaline phosphatase isaccompanied by an increased excretion of theenzyme into bile. Presumably when obstruction ispresent, the alkaline phosphatase which wouldotherwise be lost in the bile, is retained and in thislimited sense there appears to be some validity to ahighly modified 'retention' theory.The primary event, however, is an increase in the

production of alkaline phosphatase by the liver. Asa result of this, enzyme passes out of the cells intothe blood and, if there is no biliary obstruction, in-to the bile. The passage of alkaline phosphatase intothe blood is not dependent on biliary obstructionand can occur in its absence. Thus, after removal ofan auxiliary transplant, injection of '4C-leucine intothe baboon results in an increased production of theenzyme by the unobstructed host liver and this isaccompanied by the appearance of radioactivealkaline phosphatase in the serum (fig 3b) even

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Alkaline phosphatase in auxiliary liver transplantation 643

though there is no interference with biliary outflow.The basic cause of the hepatic overproduction ofalkaline phosphatase is unknown. Biliary obstruc-tion is clearly not essential; in the experimentalsituation described in the present paper the pheno-menon was induced in apparently normal liver tissue.As there was no connexion between this liver andthe graft other than via the bloodstream, it wouldappear that a humoral agent originating in the graftwas responsible for 'switching on' the host liver. Itis tempting to speculate that this hypothetical agentis a chemical which is normally produced in the liverand excreted into the bile at a rate near to themaximum possible. If that were the case, biliaryobstruction would result in the retention of thissubstance and overproduction of the enzyme wouldensue in both obstructed and unobstructed livertissue. The fact that no overproduction of alkalinephosphatase occurred after infusion of jaundicedserum into normal baboons could be explained bypostulating either that too small a dose was adminis-tered or that the substance is relatively unstable.

This work was carried out in the Michael and JanieMiller Laboratory. The biochemical tests wereperformed by Dr T. Ipp of the South AfricanInstitute for Medical Research. The financialassistance of the Medical Research Council ofSouth Africa is gratefully acknowledged.

References

Cantarow, A., and Miller, L. L. (1948). Nonexcretion of jaundice-serum alkaline phosphatase in bile of normal dogs. Amer. J.Physiol., 153, 444-446.

Clubb, J. S., Neale, F. C., and Posen, S. (1965). The behavior ofinfused placental alkaline phosphatase in human subjects.J. Lab. clin. Med., 66, 493-507.

Combes, B., and Schenker, S. (1969). In Diseases of the Liver, 3rded., edited by L. Schiff. Lippincott, Philadelphia and Toronto.

Engstrom, L. (1964). Studies on bovine liver alkaline phosphatase,purification, phosphate incorporation. Biochem. biophys. Acta(Amst.), 92, 71-78.

Griffin, M. J., and Cox, R. P. (1966). Studies on the mechanism ofhormone induction of alkaline phosphatase in human cellcultures. II. Rate of enzyme synthesis and properties of baselevel and induced enzymes. Proc. nat. Acad. Sci. (Wash.), 56,946-953.

Gutman, A. B. (1959). Serum alkaline phosphatase activity in diseasesof the skeletal and hepatobiliary systems: a consideration ofthe current status. Amer. J. Med., 27, 875-901.

Hammond, K. D., Balinsky, D., Bersohn, I., and Jersky, J. (1973).Kinetic studies on alkaline phosphatase from baboon liver.Int. J. Biochem., 4, 511-520.

Kaplan, M. M., and Righetti, A. (1970). Induction of rat liver alkalinephosphatase: the mechanism of the serum elevation in bileduct obstruction. J. clin. Invest., 49, 508-516.

Myburgh, J. A., Abrahams, C., Mendelsohn, D., Mieny, C. J., andBersohn, I. (1971). Cholestatic phenomenon in hepatic allo-graft rejection in the primate. Transplant Proc., 3, 501-504.

Polin, S. G., Spellberg, M. A., Teitelman, L., and Okumura, M.(1962). Origin of elevation of serum alkaline phosphatase inhepatic disease. Gastroenterology, 42, 431-438.

Wang, C. C., and Grossman, M. I. (1948). Nonexcretion of serumalkaline phosphatase by the liver and the pancreas of normaldogs. Amer. J. Physiol., 156, 256-260.

Welch, C. S. (1955). A note on transplantation of the whole liver indogs. Transplant. Bull., 2, 54-55.



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