Lysosomal Enzyme \g=b\-GlucuronidaseAfter Hepatic Cryosurgery Release of

Naofumi Nagasue, MD

three portions of different segments of the liver frozen at \p=m-\60C for five minutes by contact freezing with Freon. The activities of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum lactic dehydrogenase (SLDH), and serum \g=b\-glucuronidase were determined serially at regular intervals thereafter. The concentrations of serum potassium and sodium were also measured. Several hematologic measurements were made to detect abnormalities of the coagulation system. The activities of \g=b\-glucuronidase, transaminase, and SLDH were raised substantially after freezing, reaching a peak between three and six hours postoperatively. There was a tendency toward hyperkalemia, but no substantial changes were found in serum sodium concentrations. The hematologic data showed no evidence of intravascular coagulation after hepatic cryosurgery. Accordingly, if and when cryosurgical procedures are applied to the liver as a clinical trial, acidosis or hypoxia must be carefully avoided postoperatively. In

eight rabbits,


applied clinically Cryosurgerypalliative malignant regions oropharyngeal has been




treatment of




of the or facial have been most effectively treated by this procedure.1 J More¬ over, several reports indicate that cryosurgery could be a beneficial method for palliation of tumors in the anorectal area, prostate, uterus, or breast.24 Experimentally, it has been also shown that cryosur-



for publication Nov 11, 1974. From the Second Department of Surgery,

Medicine, Fukuoka, Japan. Reprint requests to Second Department Hospital, Fukuoka, Japan (Dr. Nagasue).


Kyushu University School of Surgery, Kyushu University

gery is useful for resection of parenchymal organs, such as the pancreas, kidney, spleen, or liver.-9 In 1968, Cooper and Hirose" reported that cryosurgery could provide a way to safely resect liver parenchyma as well as to destroy well-circumscribed neoplasms within the liver. They suc¬ cessfully performed resection of a hemangioma in the left lobe of the liver by cryosurgical technique in a 51-year-old man. In 1970, Neel et al7 demonstrated that cryosurgery to the liver induces proper necrosis of the parenchyma in monkeys without any systemic adverse effect and that the degree of cryonecrosis can be potentiated by simultaneous hepatic inflow occlusion. In 1971, Healey et als also showed the safety of cryosurgery to the liver in dogs reflected by the histological changes and enzymatic responses follow¬ ing the procedure. The latter two groups measured the activities of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) and observed a transient increase in these enzymes after cryosurgery of the liver. It is known that the liver contains large amounts of ly¬ sosomal enzymes (acid hydrolases) that exert proteolytic properties in cells or in the circulating blood with an opti¬ mum activity at an acid pH.1" Deleterious actions of acid hydrolases are well demonstrated in several surgical con¬ ditions, such as in some types of profound shock11-13 or in organ preservation in connection with transplantation.14" The purpose of the present study was to find whether acid hydrolases are released into the circulation after he¬ patic cryosurgery and, if so, to ascertain whether there are any harmful effects on animals in the concept of ly¬ sosomal theory.

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Fig 1.—Three portions of different contact freezing.

segments frozen

at —60 C


eosin, x70).


Eight rabbits of both sexes weighing kg were used for experiments. All the animals were anesthesized with 30 mg/ kg of pentobarbital sodium after fasting 12 hours. Laparotomy was performed with an upper midline incision, and three portions 1.6 to 2.5


of different segments of the liver were frozen at -60 C for five minutes. Using the tonsillar probe, three frozen areas of nearly the same size were produced by contact freezing in three different segments of each animal (Fig 1). The operative procedure took about 30 minutes in all the experiments. Kanamycin sulfate (200 mg) was administered intraperitoneally, and the operative wound was closed primarily by one-layer silk suture. The animals were returned to the cage and were given water freely postopera¬ tively. Blood sampling was performed through the catheter intro¬ duced into the superior vena cava from the right external jugular vein.


Analysis The activities of serum ß-glucuronidase were determined by the method of Fishman et al" and were given in Fishman units. The SGOT and SGPT levels were assayed by the method of ReitmanFlankel. The serum lactic dehydrogenase (SLDH) level was as¬ sayed by the method of Cabaud-Wroblewski. The concentrations serum potassium and sodium photometric method.



Fig 2.—Histologie findings of frozen liver 36 hours after cryosur¬ complete necrosis of hepatic parenchyma (hematoxylin-

gery. Note



by the usual


Hemoglobin, red blood cells, and thrombocytes were calculated by the standard laboratory techniques. Prothrombin time was de¬ termined by the Quick one-step method. Plasma fibrinogen was measured by the gravimetric method. The statistical comparison for significance was carried out ac¬ cording to the Student t test and a value of less than .05 was considered to be significant. RESULTS

All the animals survived more than 24 hours after the operation when the final blood sampling was accom-

pushed. However, half the animals were very inactive, es¬ on the first postoperative day. At autopsy, per¬


formed 24 hours postoperation, focal necrotic lesions were observed, surrounded by irregular hemorrhagic zones. The histologie study of the lesions revealed complete necrosis of the frozen areas replaced by polymorphonuclear leuko¬ cytes, and the border was clearly demarcated, although leukocyte infiltration was also seen in the surrounding liver parenchyma (Fig 2). The changes in serum /3-glucuronidase activities are shown in Fig 3. In all cases, ß-glucuronidase increased sig¬ nificantly (P < .02), with a peak reaction between three and six hours postoperatively. Although the individual re¬ sponse varied within wide limits ranging from three to seven times of the initial values, the pattern of enzyme ac¬ tivities seemed to be similar in all cases. Very similar re¬ sponses were also observed in SGOT, SGPT, and SLDH, but the elevation of SGPT was more prolonged than that of the other two enzymes (Fig 4 through 6). Table 1 shows the changes in serum potassium and so¬ dium concentrations. The postoperative increases in po¬ tassium were substantial, but no remarkable changes were found in the sodium concentration. Table 2 shows the changes in hématologie factors. No substantial changes were observed after hepatic cryosur¬ gery. COMMENT The present study showed that a substantial release of lysosomal enzyme /8-glucuronidase occurs following he¬ patic cryosurgery. Several authors have reported immedi¬ ate or long-term effects of cryosurgery to the liver and suggested the feasibility and safety of this procedure.5·7-9 However, the present results might suggest some objec¬ tion to this because deleterious effects of acid hydrolases have been well documented.

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Fishman units

Karmen units



1000' 500



£rr?"° op.




12 Time in hours Fig 3.—Changes in serum ß-glucuronidase activities after hepatic cryosurgery.



12 6 Time in hours


Fig 4.—Changes in SGOT activities after hepatic cryosurgery.


Karmen units

0 Fig 5.—Changes



6 12 Time in hours

in SGPT activities after


hepatic cryosurgery.

OL Fig 6.—Changes






Time in hours hepatic cryosurgery.

in SLDH activities after

Table 2.—Hématologie Data After Hepatic Cryosurgery Table 1.—Serum Potassium and Sodium Concentrations After

Hours After Operation

Data_Initial_3_6_24 2.8 ±0.1 3.5 ± 0.2* 3.9±0.1t 4.8 ± .ß

K+, mEq/liter Na% mEq/liter *

t j

Hours After

Hepatic Cryosurgery

140 ± 1

140 ± 2

139 ± 2

140 ±2

Release of lysosomal enzyme beta-glucuronidase after hepatic cryosurgery.

Lysosomal Enzyme \g=b\-GlucuronidaseAfter Hepatic Cryosurgery Release of Naofumi Nagasue, MD three portions of different segments of the liver froze...
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