Brettschneider, Lawrence

Groth, Carl G

Guptill, Joanne

Kashiwagi, Noboru

Starzl, Thomas E

English

PubMed

D-Scholarship@Pitt

I EFFECT OF IIOMOGRAFT ISCHEMIA UPON SERUM PROTEINS AFTER ORTHOTOPIC LIVER TRAXSPLANTATION NOBORLT KASHIWAGI, 1I.D. CARL G. GKOTH, 5i.D. LAIVRBNCE BRET'I'SCHNEIDER, M.D. JOANNE GUPTILL, B.S., M.T. THOMAS E. STARZL, KD. ,  PH.D. Denver, Colo. Frolrl the Unlvers~ty  of Colorado School of Medicine and Denver Veterans Admlnisti atlon Hospital Reprinted from SURGERY St. Louis Vol. 63, Yo. 2, Pages 247-251, February, 1968 (Copyright 0 1 9 6 8  by The C V Mosb) Company) (Prlnted in the U. S A ) Effect of homograft ischemia upon serum proteins after orthotopic liver transplantation T here have been several reports con- cerning the protein metabolism of orthotopic liver homografts after their transplantation to nontreated recipients. Serial changes in plasma  protein^^-^, or turnover rates of amino acid  isotope^^-^ were measured. In  dogs which received livers that had not been badly damaged by ischemia, it was found that plasma concentrations of protein frac- tions were initially well maintained6 with the possible exception of albumin,%nd that the rate of protein synthesis was generally increased until shortly before death.3-5 These short-term observations did not sug- gest that grave defects of protein synthesis would constitute a major problem after this kind of operation. However, subsequent studies by Brettschneider and co-workers1 have shown that drastic falls in serum pro- teins often occur after orthotopic homotrans- plantation of livers damaged after preserva- tion for a number of hours, and that the presence or absence of these changes pro- vided an important clew to the long-term prognosis of recipient animals treated with immunosuppressive agents. Thr uork is supported by Grants FOj-TW-1154, AM 06283, AM 06344 HE 07735, AM 07772, A1 04152, FR 00051, FR 00069 from the United States Public Health Se, vice. Received for publication June 23, 1967. "Fellow in Su~gery, University of Colorado Srhool 01 Medicine, Denver, Colo. **Fellow in Surgery, University of Colorado School of Medicine, Denver, Colo. NOBORU KASHIWAGI, M.D." CARL G. GROTH, M.D."" LAWRENCE BRETTSCHNEIDER, M.D. JOANNE GUPTILL, B.S., M.T. THOMAS E. STARZL, M.D., PH.D. DENVER, COLO. From the University of Colorado School of Medicine and Denver Veterans Administration Hospital I n  the present study this question has, therefore, been re-examined by measuring the plasma proteins in dogs which were un- der immunosuppressive therapy and which received both badly and minimally injured homografts. The use of immunoelectropho- retic techniques permitted qualitative study of the way in which specific protein fractions were affected, first by ischemic injury and then by subsequent modified rejection. METHODS Mongrel dogs weighing 10 to 23 kilograms were used. Immunosuppressive therapy for the nonoperated control animals as well as for those subjected to transplantation was with daily azathioprine, a progressively di- minishing seven-day postoperative course with prednisone beginning at 5 mg. per kilo- gram per day, and daily subcutaneous in- jections of 1.0 to 2.5 ml. horse antilyrnpho- cyte globulin (ALG) . Orthotopic liver trans- plantation was carried out with standard techniques1 ; arterial reconstruction was with end-to-end hepatic arterial anastomosis. The methods of liver preservation have been described elsewhere1 and will be alluded to under the different experimental groups. Postoperatively, frequent measurements were made of serum glutamic oxalacetic trans- aminase (SGOT) , serum glutamic pyruvic transaminase (SGPT) , alkaline phosphatase, and bilirubin. Vol.  63, No. 2, f ip .  247-251 February, I968 SURGERY 2 4 7 2 4 8 Kaslziwagi et al. Surgery February 1968 Total serum proteins were determined with refrectometry."' Electrophoresis was done with a microzone electrophoresis c e l l . ~~mmunoe l ec t ropho re s i s~  was pcr- formed with Scheidegger'sR technique of us- ing rabbit anticanine sera.$ Experimental groups. Group I-Controls. Five nonoperated ani- mals were treated with the 3 immunosup- pressive agents. Group 2-Minor ischemic injury. Four dogs received orthotopic liver homografts which were cooled in the donor by intra- portal infusion with chilled electrolyte solu- tion and then transferred within an hour. Group 3-Moderate ischemic injury. Six dogs received livers which had been stored extracorporeally for 8 to 24% hours with a combination of hypothermia, portal and hepatic arterial perfusion with diluted blood, and hyperbaric oxygenation. This preserva- tion technique was the most effective one tested in a recent study.l Group 4-Severe ischemic injury. Twelve dogs received homografts after 8% to 24% hours' preservation by techniques similar to those described in Group 3. However, cer- tain details, such as optimal perfusion rate, had been changed.l By the first postopera- tive day there was evidence by standard liver function tests of grave ischemic injury. RESULTS Changes in first week. Group I .  The total serum protein and its fractions were unchanged in these five dogs during the seven-day period of study. Group 2. The average SGOT on the first postoperative day was 119 SF units (range 70 to 175) as compared to a normal range in this laboratory of 0 to 40 units, indicating that damage to these organs was not severe. '*T.S. Meter, American Optical Co., Buffalo, N. Y. +Beckman Instruments, Inc., Fullerton, Calif. :The apparatus for electrophoresis in agar was manu- factured by LKB-Produckter AB, Stockholm, Sweden. Veronal buffer was used a t  a p H  of 8.6 and ionic strength of 0.05 in agar and 0.1 in tank. OHyland Division Travenol Laboratorics, Inc., Los Ange- les, Calif. Before transplantation, both donor and recipient animals had serum proteins which were within normal. Subsequent changes were expressed as percentage deviations from the preoperative recipient values. During the first postoperative week, these levels were maintained at 85 to 110 percent (average 100 percent) whether the grafts were sub- sequently rejected at an early or late time. For example, one animal died of acute re- jection after seven days, despite which pro- tein concentrations were well maintained during the first six days (Fig. 1 ) .  During this period there was no statisti- cally significant change in the electrophoretic pattern, although the a,-globulin was slightly depressed in all four animals, to an average of 85 percent. The results of immunoelectro- phoresis of the sera were consistent with these findings, in that ceruloplasmin, a z M ,  % ELECTROPHORESIS IMMUNOELECTROPHORE61S TOTAL PROTEIN --f-- so ALBUMIN * ALBUMIN * HAPTOGLOBIN 9 CERULOPLASMIN t 42. t .(¶L t @, GLOBULIN TRANSFERRIN 1 BOA andP,c t 50 PI. * 150, PRE-OWOST-OP 9 TIME IN DAYS B 2 DEPRESSED 'OR ABSENT Fig. 1. Relentless rejection which occurred after orthotopic liver transplantation in a dog despite treatment with azathioprine, prednisone, and an- tilymphocyte globulin. Note that the protein frac- tions as measured with electrophoresis were rela- tively stable. The proteins measured with immunoelectrophoresis are listed a t  the same level as the electrophoretic classes in which these are included. Volume 63 Number 2 Egect of ischemza on proteins 2 4 9 and alpha-2 lipoprotein (a2L), which have the electrophoretic mobility of a2-globulin, all appeared to be reduced in at least three of the four dogs. These changes occurred only after four or five days. At this time the ,Bla precipitation band also became indis- tinct. The y-globulin had already become ele- vated in two dogs, which later died of re- jection after seven and 21 days. The two others, which lived for 29 and 100 days re- spectively, had early depression of the yglobulin. However, -these alterations could not be well seen by immunoelectrophoresis. Group 3. The average SGOT on the first postoperative day was 381 SF units (range 110 to 645). The increased ischemic in- jury was reflected in a depression of the total serum protein to 79 to 97 percent of the original values (average 87 percent) during the first seven days. There were PRE - OP FOST- OP DAY I DAY 2 DAY 4 DAY 5 Alb Fig. 2. Changes seen by immunoelectrophoresis in the serum of a dog which received a damaged liver homograft. Note the early postoperative dis- appearance of several precipitation bands includ- ing ceruloplasmin (Cp),  am, ~ I L ,  PIA, YY, and y ~ .  These tended to return, but never completely to control status. variable minor declines in the concentrations of all the electrophoretic classes of  rotei ins, including the y-globulins, which were de- pressed to 79 percent. With immunoelectro- phoresis the subgroups of the a,-globulins appeared depressed in the same general way as in Group 2. Group 4. l 'he  SGOT rise on the first day was to an average of 920 SF units (range 305 to 3250). Daily total proteins in eight dogs were depressed during the first week to an average of 83 percent (63 to 100 per- cent). Changes in the protein fractions were highly variable. Only one of these dogs sur- vived for more than 13 days; the exceptionai animal died after 19 days. In  these eight dogs, as well as in four others in which electrophoresis was not per- formed, immunoelectrophoretic studies often revealed a striking selective attenuation or even a total loss of discrete protein moieties. An example is shown in Fig. 2. On the first postoperative day precipitation bands had completely disappeared for the ceruloplas- min, az5[ and azL components of the a,-glob- ulin. In addition, the pla portion of PI-glob- ulin and the yA and y$I portions of the p2- globulin could not be found. Most of the precipitation bands which remained were shorter or less distinct than in the control sample. During the ensuing days there was a gradual return of some of the missing pre- cipitation bands, but the a,L as well as PIA and ylI remained absent until the death of the animal on the sixth day. Qualitatively, similar findings were evident in all 12 ex- periments, although frequently the losses were less extreme than those illustrated. Subsequent changes in dogs surviving chronically after operation. Several of the animals in Groups 2 and 3 had good hepatic function from the beginning. In others, early evidence of injury, including minor depres- sions in some of the serum proteins, had re- turned to or toward normal within a week or 10 days. In  eight such dogs it was possible to ascribe subsequent changes to rejection. The serum proteins remained relatively normal in these animals, often for weeks or 2 5 0 Kashiwagi et al. Surgery February 1968 months. With the onset of fatal late rejec- tion from three weeks to three months post- operatively there were declines in two of three dogs in total serum protein and most of its component fractions, except for the y globulins, which rose in two instances. The serum proteins in the other five animals were little changed by rejection crises which reversed spontaneously; four of these dogs are still alive from two to four months post- operatively. DISCUSSION For kidneys the degree to which tissue anoxia during preservation adversely influ- ences the long-term prognosis can be pre- cisely investigated in the absence of an im- munologic barrier, since autografts can be used for testing. With livers this approach is not feasible for obvious technical reasons, and an assessment of the long-term suit- ability of preserved hepatic homografts can be obtained only if these are transplanted to recipients which are provided with effective immunosuppression. All animals in this study were treated with immunosuppression. Cooled and immedi- ately transplanted liver homografts were used, and the results during the first week were generally consistent with previous re- ports of protein metabolism after orthotopic transplantation to untreated recipients. Serum protein levels and individual pro- tein fractions were maintained at near nor- mal levels, as would be expected with good liver function. The only detail which dif- fered from earlier observations was that increases in a,-globulin ~oncentration"~ were not seen. These animals continued to have normal or near-normal serum proteins until fatal rejection occurred. At that time an over-all decrease in serum protein con- centration developed in some dogs several days before death. There was a variability in immune globulin concentrations both early and late after operation. However, quickly developing rejection was more com- mon in animals with elevated y globulins. The most important factor in early post- operative protein metabolism seemed to be the degree of ischemic injury imposed upon the homografts. When this was moderate there were significant but reversible changes in some of the protein components, particu- larly those of a, class. With more severe damage there was often complete disap- pearance for several days of some of the immunoelectrophoretic bands. These tended to return, but never completely to normal. All of these dogs eventually died in less than 19 days. In  such experiments the after-effects of ischemia in combination with an un- analyzable further immunological injury to the homografts apparently precluded long- term survival. SUMMARY Serum proteins were studied with electro- phoresis and immunoelectrophoresis in drug- modified recipients of orthotopic hepatic homografts. The homografts that were pro- vided had been subjected to minor, moder- ate, and severe ischemic injury. In  the early postoperative period serum protein and its components were well maintained in the re- cipients of minimally traumatized livers and depressed in the others in rough proportion to the degree of prior anoxic damage. The significance of these, as well as of subsequent changes in serum proteins, are discussed in relation to evaluation of liver preservation techniques. REFERENCES 1. Brettschneider, L., Daloze, P. M., Huguet, C., Porter, K.  A., Groth, C. G., Kashiwagi, N., Hutchison, D. E., and Starzl, T. E.: The use of combined preservation techniques for extended storage of orthotopic liver homo- grafts, Surg. Gynec. & Obst. In press. 2. Kohn, J.: A new supporting medium for zone electrophoresis, Biochem. J. 65: 9, 1957. 3. Kukral, J. C., Littlejohn, M. H., Butz, G. W., Jr., and Starzl, T.  E.: Biochemical studies of the homotransplanted canine liver, S. Forum 12: 112, 1961. 4. Kukral, J. C., Littlejohn, M. H., Williams, R. K., Pancner, R. J., Butz, G. W., Jr. and Starzl, T. E.: Hepatic function after canine liver transplantation, Arch. Surg. 85: 173, 1962. 5. Kukral, J. C., Riveron, E., Weaver, J., Ney- Volume 63 Number 2 man, B., Vaitys, S., Barrett, B., and Starzl, T. E.: Metabolism of plasma protein fractions after orthotopic homografts and autografts of the dog liver, S. Forum 17: 218, 1966. 6. Moore, F. D., Wheeler, H. B., Demissianos, H. V., Smith, L. L., Balankura, O., Abel, K., Greenberg, J. B., and Dammin, G. J.: Ex- perimental whole organ transplantation of the liver and of the spleen, Ann. Surg. 152: 374, 1960. 7.  Reiss, E.: Eine neue Methode der quantita- tiven Eiweissbestirnmung, Arch. Exper. Pathol. Pharmakol. 51: 18, 1904. Eflect of ischemia o n  proteins 2 5 1 8. Scheidegger, J. J.: Une micro-methode de l'immunoClectrophortse, Internat. Arch. Al- lergy 7: 103, 1955. 9. Starzl, T. E., Kaupp, H., Brock, D. R., and Linman, J. W.: Studies on the rejection of the transplanted homologous dog liver, Surg. Gynec. & Obst. 112: 135, 1961. 10. Starzl, T .  E., Marchioro, T. L., Portor, K. A., Taylor, P. D., Faris, T. D., Herrmann, T. J., Hlad, C. J., and Waddell, W. R.: Factors determining short- and long-term survival after orthotopic liver homotransplantation in the dog, SURGERY 58: 131, 1965. 

Effect of homograft ischemia upon serum proteins after orthotopic liver transplantation.

Kashiwagi, N

Groth, CG

Brettschneider, L

Guptill, J

Starzl, TE

Name not available

Effect of homograft ischemia upon serum proteins after orthotopic liver transplantation

http://d-scholarship.pitt.edu/3537/1/31735062124478.pdf

Effect of homograft ischemia upon serum proteins after orthotopic liver transplantation.

Abstract

Similar works

Full text

Available Versions

D-Scholarship@Pitt

Name not available

Name not available