Phospholipid metabolism of dog liver under hypoxic conditions induced by ligation of the hepatic artery

527

Biochimica et Biophysics Acta, 664 (1981) Elsevier/North-Holland Biomedical Press

527--531

BBA 57816

PHOSPHOLIPID METABOLISM OF DOG LIVER UNDER HYPOXIC CONDITIONS INDUCED BY LIGATION OF THE HEPATIC ARTERY

JUN MATSUMOTO a, TOSHIRO TANAKA b, MASAMI GAMO c, KUNIHIKO SAITO ‘** and ICHIO HONJO ’ a Third Department of Internal Medicine, and Departments of b Surgery and ’ Medical Chemistry, Kansai Medical School, Moriguchi, Osaka, 570 (Japan) (Received November 17th, 1980)

Key words: Artery ligation; Hypoxemia; liver)

Phospholipid

degradation; Phospholipase;

(Canine

summary Ischemic hypoxic liver was induced in dogs by ligation of the hepatic artery. About 67% of the dogs died of liver necrosis within 1 or 2 days (severe cases), and the rest survived (mild cases). In the severe cases, the decreases in the contents of total lipids, phospholipids and proteins of the liver after 24 h were 24, 46 and 12%, respectively, of the original values. The marked decrease in phospholipids was due to decreases in the microsomal and mitochondrial fractions. In the mild cases, similar but smaller decreases occurred and decrease of phospholipids occurred only in the microsomal fraction. The main phospholipids were choline and ethanolamine glycerophospholipids, and their molecular species were analyzed. In the severe cases, ligation resulted in relative increases in mono- and diene species and a decrease in polyene species. No increase in phospholipase activity was found at various times after ligation of the hepatic artery. Penicillin-treated dogs all survived and showed little decrease in liver phospholipids.

Introduction The liver is supplied with blood by the hepatic artery and the portal vein. After ligation of the hepatic artery, dogs die within 2 or 3 days [l]. However, when they are treated with penicillin just before its ligation they survive [2]. These phenomena have been confirmed repeatedly [ 3-1. * To whom cormspondence should be addressed. Abbreviations: POPOP. 1,4-bis(2-(S-phenyloxazolyl)benzene;

OOOS-2760/81/00000-00000/$02.50

PPO. 2.5-diphenyloxazole.

@ Elsevier/North-Holland

Biomedical Press

Under hypoxic conditions, cells of various organs, such as rat liver [lo] and kidney [ll], are injured and die, possibly owing to degradation of constituent phospho~pids, which participate in cell functions as main component of biomembranes. Endogeneous phospholipase(s) regulates membrane function by altering the membrane lipid composition. This paper describes changes in phospholipid metabolism in dog liver under ischemic hypoxic conditions induced by ligating the hepatic artery. Materials and Methods

Ligation of the hepatic artery. Healthy mongrel dogs, 5-12 kg, were starved for 24 h, anesthetized by intravenous injection of isozole (10 mg/kg) and subjected to laparotomy. First, a piece of the peripheral part of normal liver lobe was removed for me~urement of control (0 time) values where liver necroses occurred preferentially. Then the common hepatic artery, gastroduodenal artery and right gastric artery were ligated [12]. After the operation, the dogs were left on the operation table for about 9 h and supplied with physiological saline solution. When necessary, further isozole was injected. After 3, 6 and 9 h, respectively, the peripheral parts of the lobe, about 3 g, were removed. Then dogs were transferred to a cage and supplied with water ad libitum, Food was given after about 24 h. Mood tests. The following items were determined at various times after ligation of the hepatic artery: glutamic oxaloacetic transaminase and glutamic pyruvic transaminase [ 131, immunoreactive insulin [ 14], immunoreactive glucagon f 151, blood sugar [ 161, ammonia [ 171 and free amino acids [ 181. Lipid extraction. According to the method of Bligh and Dyer 1191, 3 g of wet liver was homogenized with 5.9 ml of H20 in a Ultra Turrux (Ika Werk Co., F.R.G.). A sample of 0.5 ml of homogenate was used for determination of nitrogen by the Kjeldahl method or of protein by the method of Lowry et al. [ ZO] with bovine serum albumin as a standard. The rest of the homogenate was mixed with 0.5 ml of HZ0 and 20 ml of CHJOH in a vortex mixer and 10 ml of CHC& was added (one-phase system), The mixture was stirred and centrifuged at 3000 rev./min for 5 min and the supernatant was collected. The extraction was repeated four times. All the supernatants were combined and made up to 95 ml with the one-phase system and mixed with 25 ml each of CHC13and HzO. The mixture was centrifuged, and the CHC13 layer was removed, washed once with 10 ml of CH~OH/H~O (10 : 9, v/v) diluted with benzene and evaporated in a rotary evaporator. The total lipids were dissolved in CHCi&H,OH (1 : 1, v/v) and stored at -20°C under nitrogen. Separation of simple and complex lipids. According to the method of Hanahan et al. [21], total lipids, corresponding to 1 mg of lipid phosphorus, were dissolved in CHC13 and applied to a column (1 X 5.5 cm) packed with 1.5 g of silicic acid (~~inc~odt Chemical Works, U.S.A.)/hyflosuper ccl (Nakarai Chemicals, Japan) (1 : 0.5, w/w). Simple lipids were eluted completely with 30 ml of CHCl, and then complex lipids with 50 ml of CHJOH. No simple lipids were detected in the CHJOH fraction by micro-thin-layer chromatography, and the recovery of lipid phosphorus was over 95%. Isolation of choline g~ycerophospholi~~ds and etha~olami~e g~yce~ophospho-

529

lipids. The phospholipid fraction, 200 pg P, was dissolved in CHC13,applied to a preparative thin-layer plate (20 X 20 X 0.06 cm) with an Autoliner (Desage Co., F.R.G.) and developed with CHC13/CH30H/Hz0 (65 : 25 : 4, v/v/v). The plate was sprayed with 0.01% Rhodamine 6G in Hz0 and spots were located under ultraviolet light. Choline glycerophospholipids and ethanohunine glycerophospholipids were extracted from the plate by the method of Bligh and Dyer [ 191. The choline glycerophospholipids were further purified by column chromatography on alumina (1 g, 1 X 3 cm, 200 mesh, Nakarai Chemicals, Japan). The final preparations of both samples appeared homogeneous on thin-layer chromatography and the recoveries were 100 pg P (50%) and 46 pg P (23%), respectively. Constituent fatty acids of choline glycerophospholipids and ethanolamine glycerophospholipids and their molecular species. Fatty acid methyl esters were

prepared by refluxing with 10% CH,OH-HCl for 2 h. A Shimazu GCSBF gas chromatograph was used with a flame ionization detector. The column (2 m X 3 mm) was a glass spiral tube packed with 10% Silar 10 C coated on gas chrom Q, 100-120 mesh. The column temperature was 180°C and was raised to 200°C at a rate of lO”C/min after elution of Cu):*. The molecular species of choline glycerophospholipids and ethanolamine glycerophospholipids were analyzed as described before [ 22-241. The phospholipids were hydrolyzed with Bacillus cereus phospholipase C and the resulting ‘diacylglycerols’ were converted to trimethylsilyl or t-butyldimethylsilyl derivatives. The apparatus used was a Shimazu LKB-9000, GC-MS PAC 300, and the column was a spiral glass tube (2 m X 3 mm) packed with 1% OV-1 on Chromosorb W, AW, HMPS, 80-100 mesh. The flow rate of helium, used as carrier gas, was 30 ml per h. The mass spectrometer was operated at an electron energy of 22.5 eV, and ionizing current of 60 PA and an acceleration voltage of 3.4 kV. The temperatures of the injector, column, separator and ion source were 310, 285, 300 and 31O”C, respectively. In the selected ion revival technique, repetitive scans were obtained at 12-s intervals. The molecular species were analyzed by monitoring the following three kinds of ions: (l), a molecular ion, [M-15]’ for trimethylsilyl or [M-57]’ for t-butyldimethylsilyl derivatives; (2) a characteristic ion of the acyl residue, [RCO + 741’; and (3) base peak m/e 129 for trimethylsilyl and m/e 171 for t-butyldimethylsilyl derivatives. The relative amounts of individual species were calculated by computer from the peak areas based on the molecular ions. Cellular fractionation. A sample of 3 g of wet liver was homogenized in a Potter homogenizer with 27 ml of 0.25 M sucrose/l0 mM Tris-HCl (pH 7.4)/ 1 mM EDTA, and the homogenate was centrifuged at 800 Xg for 10 min. The supernatant was centrifuged first at 15 000 X g for 10 min and then at 105 000 X g for 60 min. The protein and phospholipid contents of each fraction (Table I) were consistent with the values reported by Hogeboom et al. [25] and Kihara [26]. Phospholipase activities. 1-[9,10-3H,]PahnitoylS-acyl-sn-glycero-3-phosphocholine and l-acyl-24 1-14C] oleoyl-sn-glycero-3-phosphocholine were prepared by the methods of Akesson et al. [27] and Pugh and Kates [28], respectively. These substrates were chromatographically pure and had specific activities of 0.76 and 2.49 Ci/mol, respectively. The assay system in a total volume of 0.4

530

TABLE I LIPID AND PROTEIN RUPTION (n = 2)

CONTENTS

OF SUBCELLULAR

FRACTIONS

BEFORE

AND AFTER

Total lipids (me/g wet tissue)

Lipid phosphorus @g/g wet tissue)

Tissue nitrogen (me/g wet tissue)

Before

Before

Before

After (24 h)

Severe cases (Groups A and B) Homogenate 30.8 800 X g pellet 13.2 15 000 X g pellet 5.8 105 000 X g pellet 11.6 Supernatant 2.3 Before

After (24 h)

INTER-

After (24 h)

23.6 16.4 2.7 3.8 4.4

1027.0 428.7 195.9 400.7 51.7

526.1 373.2 56.6 96.2 55.1

26.2 9.7 3.1 2.8 17.7

24.7 14.7 1.0 1.0 14.7

After

Before

After

Before

After

(72 h)

(72 h)

(72 h)

Mild cases (Group C) Homogenate 800 X g pellet 15 000 X g pellet 105 000 X g pellet Supernatant

32.0 12.5 5.3 12.3 3.2

29.1 13.6 5.3 7.9 3.5

998.3 387.9 154.7 398.8 56.0

853.2 410.0 158.3 218.7 44.7

24.0 8.2 2.5 2.8 16.9

23.0 9.7 2.6 1.9 14.0

ml consisted of 2.5 mM phosphatidylcholine (3H, 75 000 dpm or 14C, 16 000 dpm), 100 mM Tris-HCl buffer (pH 7.4) and 20 mg enzyme protein. The murture was incubated at 30°C for 30 min. The enzyme was prepared by homogenizing 0.2 g of liver with 0.2 ml of 100 mM Tris-HCl buffer (pH 7.4), which roughly corresponded to 30 mg of bovine serum albumin. Three other systems were also used, containing Triton X-100 at a final concentration of 0.5% and/or 10 mM CaClz. The reaction was stopped by adding 1.0 ml of CH30H followed by 1.0 ml of CHC13 and 0.5 ml of Hz0 (two-phase system of Bligh and Dyer [ 191). The reaction products in the CHCl, layer were separated by thin-layer chromatography in CHC13/CH30H/Hz0 (65 : 25 : 4, v/v/v). The radioactive spots on thin-layer plates were located with a Birchorer, Model 450A, radiochromatogram spark chamber camera, and were scraped directly into vials and counted in a Packard Model 2650 scintillation counter, using 10 ml of scintillator containing 0.9 g POPOP and 15.0 g PPO in 3 1 of toluene. Values were corrected for quenching by the external standard ratio method. The hydrolysis rate was expressed by the relative amount of fatty acids liberated from the substrate [ 291. The total recovery of radioactivity of the substrate and hydrolysis products was found to be about 95%. Results Clinical symptoms of dogs after ligation of the hepatic artery Fifteen dogs operated were’ roughly classified into three groups. Group A: the dogs (4/15) gradually became unable to move, showed weak reflexes and died after about 24 h. Group B: the dogs (6/15) were moribund about 24 h after the operation, but were still alive. Group C: the dogs (5/15) were in a

531

0-o Hours

10 after

20 Interruption

Fig. 1. Chronological changes of glutamic pyruvic transaminase (0). tiutamic oxaloacetic tramwninase (A). immunoraactive glucagon (o), immunoreactive insulin (0). ammonia (A) and blood sugar (m) in blood after intenuption.

(n = 3).

Fig. 2. Chronological changes of plasma amino acids, valine (o), leucine (b), phenylalanine (0). isoleucine (0) and tyrosine (v), after interruption, (n = 3). 0, ratio branched chain amino acids: aromatic amino acids.

relatively good condition after 24 h. They were all killed after 72 h, except one which survived for 49 days. Blood tests

Groups A and B showed similar extents of liver dysfunction: i.e., glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and immunoreactive glucagon increased remarkably with time, whereas immunoreactive insulin was unchanged. Ammonia increased gradually after a temporary decrease (Fig. 1). Free amino acids increased, whereas the ratio of branched chain amino acids to aromatic ones decreased (Fig. 2). Group C showed only slight abnormality in liver function. Accordingly, the dogs were divided into two types on the basis of liver dysfunction: severe cases (Groups A + B, 10/15) and mild cases (Group C, 5/10). Unless otherwise stated, only data on the severe cases are described in the following sections. Macroscopic and histological findings As described by Urabe [ 61, soon after interruption

of the blood supply the whole liver became reddish-purple and then purple. Dark purple spots appeared after 3 h and after 12 h they were located in the peripheral parts of liver lobes, which were the commonest sites of liver necrosis. The rest of the liver remained reddish-purple or purple. The dark purple spots or necrotic parts showed marked histological atrophy and fatty degeneration of liver cells, and dilation of venous sinusoids after 24 h,

532

Fig. 3. Histology of dog liver after interruption. (X 100, hematoxylin

eosin St~iM)

but liver cells around Glisson’s capsules appeared relatively normal (Fig. 3). Other part of the liver showed only slight changes in macroscopic and histological appearance. Regional difference in lipid composition of liver No appreciable differences in lipid composition were found in eight sites of normal liver lobes, although the compositions varied in different dogs. Chronological changes in lipid and protein contents of liver Fig. 4 shows that the contents of total lipids, phospholipids and protein per g of liver from the dark purple or necrotic parts decreased with time; in Group B the values after about 24 h were 76, 54 and 88%, respectively, of the original values. The decrease in phospholipids was especially remarkable. Other parts of the liver showed no appreciable decrease in protein and lipid contents. Liver from mild cases (Group C) showed similar but much slighter changes, the contents of phospholipids after 24 and 72 h being 92 and 79%, respectively, of the initial value. Changes in lipid and protein contents of cellular subfractions In the severe cases (Groups A and B), decrease in phospholipid content of the whole homogenate was due to decreases in the contents in the 15 000 X g and 105 000 X g pellets (Table I). But in the mild cases (Group C), phospholipids decreased only in the 105 000 X g pellet, not in the 15 000 X g pellet (Table I). A similar change was found in protein content.

501 0’

10

20

I

I

0

Hours after InterrUPtIOn

10

20

‘70 Hours

after

Interruption

Fig. 4. Chronological changes in lipid (0). phospholipid (A) and protein (0) contents of the liver after interruption. I, severe cases; II, mild cases; (n = 3). Fig. 5. Chronological changes of main molecular species of choline glycerophospholipids, ‘32’ (0). ‘34’ (o), ‘36’ (o), ‘38’ (A)* ‘40’ (0). ‘32’ means the carbon numbers of C-l and C-2 fatty acid residues, e.g. dipalmitoyl phosphatidylcholine.

Phospholipid composition The contents of choline glycerophospholipids and ethanolamine glycerophospholipids in normal liver isolated by preparative thin-layer chromatography were 50 and 23%, respectively. Other minor components were also present. Lysophosphatidylcholine was not detected. There was no difference in the contents of these components after interruption of the blood supply. Molecular species of choline glycerophospholipids and ethanolamine glycerophospholipids The main constituent fatty acids of these phospholipids were 16 : 0, 18 : 0, 18:1,18:2,20:4and22:6.After24h,theamountsof16:0,18:1and 18 : 2 fatty acids were increased, and those of 20 : 4 and 22 : 6 fatty acids were decreased (Table II). The molecular species of these phospholipids were mainly ‘34 : 1’ (mainly 16 : O/18 : l), ‘34 : 2’ (mainly 16 : O/18 : 2), ‘36 : 1’ (mainly 18 : O/18 : 1), ‘36 : 2’ (mainly 18 : O/18 : 2), ‘36 : 3’ (mainly 18 : l/ 18 : 2), ‘36 : 4’ (mainly 16 : O/20 : 4), ‘38 : 3’ (mainly 18 : O/20 : 3), ‘38 : 4’ (mainly 18 : O/20 : 4 and 16 : O/22 : 4), ‘38 : 5’ (mainly 16 : O/22 : 5 and 18 : l/20 : 4) and ‘38 : 6’ (mainly 16 : O/22 : 6). The molecular species of ethanolamine glycerophospholipids were more unsaturated and longer chain fatty acids than those of choline glycerophospholipids. After 24 h, monoene and diene species, such as ‘34 : 1 ‘, ‘34 : 2’, ‘36 : 1’ and ‘36 : 2’, were increased, whereas polyunsaturated species, such as ‘38 : 4’, ‘38 : 5’ and ‘38 : 6’, were decreased (Table III). The chronological changes in molecular species of choline glycerophospholipids are shown in Fig. 5: total ‘34’ species increased and ‘38’ species decreased with time.

534 TABLE II FATTY ACID COMPOSITIONS OF CHOLINE AND ETHANOLAMINE FROM LIVER BEFORE AND AFTER INTERRUPTION (n = 3) Fatty acid

14 : 0 14 : 1 16 : 0 16 : 1 18 : 0 18 : 1 18 : 2 20 : 1 20 : 3 20 : 4 22 : 4 22 : 5 22 : 6

GLYCEROPHOSPHOLIPIDS

Choline glycerophospholipids

Ethanolamine glycerophospholipids

Before

After

Before

After

(%)

(24 h) (%)

(%)

(24 h) (%)

0.6 0.2 16.7 1.8 29.0

0.2 0.1 26.9 1.1 25.4 9.2 17.7 0.1 0.5 16.0 0.5 1.0 1.3

0.4 0.7 14.8 1.7 30.8 5.6 7.1 0.1 0.4 24.6 2.0 3.1 8.7

8.0 14.1 0.1 0.8 22.8 0.9 2.1 2.9

0.4 17.0 1.4 31.9 6.5 11.4 0.2 0.8 24.1 1.1 1.1 4.1

TABLE III MOLECULAR SPECIES OF CHOLINE AND ETHANOLAMINE LIVER BEFORE AND AFTER INTERRUPTION (n = 2) Molecular species

32 : 0 32 : 1 34 : 0 34 : 1 34 : 2 34 : 3 36 : 0 36 : 1 36 : 2 36 : 3 36 : 4 36 : 5 38 : 2 38 : 3 38 : 4 38 : 5 38 : 6 40 : 4 40 : 5 40 : 6 40 : 7

GLYCEROPHOSPHOLIPIDS

Choline glycerophospholipids

Ethanolamine glycerophospholipids

Before

After

Before

After

(%)

(24 h) (%)

(%)

(24 h) (%b)

1.1 0.4 1.3 10.5 16.1 1.9 0.6 6.9 15.2 4.5 6.5 1.2 0.6 5.6 18.8 5.5 2.2 0.1 0.4 0.6 -

2.4 0.5 2.4 14.5 21.6 2.0 0.5 7.6 19.1 4.7 5.8 0.8 0.3 3.2 10.5 3.0 1.1

0.2 3.3 6.5 0.2 0.2 4.1 13.1 5.6 6.8 0.7 0.8 7.1 30.7 9.1 6.3 0.8 1.5 2.4 0.6

0.4 9.6 12.6 0.6 0.8 7.6 16.2 5.0 6.4 0.1 1.0 7.0 25.3 4.5 1.6

7

0.3 1.0

FROM

535 TABLE IV PHOSPHOLIPASE

ACTIVITIES

Enzyme source

OF LIVER BEFORE AND AFTER

INTERRUPTION

(n = 3)

Free fatty acids liberated (46) C-l

c-2

Before (0 time)

0.9

0.2

After (24 h) Necrotic parts Non-necrotic parts

2.2 0.3

0.1 0

Phospholipase activities The phospholipase activities of the necrotic

and non-necrotic parts of the liver of Groups A and B at various times after interruption of the blood supply were compared with the control. No significant differences were found (Table IV), even when Triton X-100 and/or CaCl, was added to the assay system. To ascertain the presence of some inhibitor or activator for phospholipase A2 activity in the liver, Crotalus adamanteus phospholipase AZ was added, and its activity was quantitatively recovered. Effect of penicillin G on phospholipid metabolism of the liver

Four penicillin-treated dogs (Group D) showed similar changes to Group C in clinical symptoms, values for blood components, macroscopical and histological appearance, and total lipid, phospholipid and protein contents. They did not die within 24 h after interruption. Discussion Since Haberer’s [l] work in 1906, all dogs in which the arterial flow to the liver was interrupted have died of liver necrosis within 2 or 3 days. Two possible mechanisms of the liver necrosis have been proposed. One is that proliferation of anaerobic bacilli, such as Clostridium welchii, under the hypoxic conditions was induced and production of phospholipase C by these bacteria resulted in phospholipid degradation and liver necrosis [ 30,311. The second is that disorder of the portal system resulted in irreversible localized stagnation and aseptic ischemic liver necrosis [ 5,6,8]. The release of liver ferritin into the blood might be related to irreversible stasis in the intrahepatic portal system [ 71. The second proposal is widely accepted. In our study, proliferation of bacilli was found in only one of 22 dogs (5%) and death of the dogs is attributable to aseptic ischemic liver necrosis. The relationship between ischemic liver necrosis and phospholipid metabolism was the decrease in phospholipid content of the liver. In the severe cases it was found in both the microsomal and the mitochondrial fractions but in the mild cases it was found only in the microsomal fraction. This suggests that under hypoxic conditions decrease in phospholipids proceeds from the microsomes to the mitochondria and that when the decrease extends to the mitochondria, liver function is irreversibly damaged, resulting in death.

536

The reasons why liver phospholipids decreased and phospholipase was activated under hypoxic conditions are unknown. Chien et al. [lo] pointed out that when the hepatic artery and portal vein of rats were ligated, the liver increased in the intracellular Ca” content, which would activate the Ca-dependent phospholipase(s). In fact, however, they did not describe the elevation of the phospholipase activity but found the depletion of the liver phospholipids. In our experiment, the increase in phospholipase activities in the liver could not be detected directly at any time after interruption, but liver phospholipids decreased with time. Besides hypoxia, intracellular Ca” of the liver was also elevated after galactosamine administration [ 321. In addition some membraneactive substances, e.g. melittin [ 33-351, direct lytic factors [ 351, chlorpromazine [36] and basic protein [37,38] facilitate the initiation of endogenous phospholipase activities. The mild cases observed in this experiment (Group C, 33%) must have had individual anomahties, such as a hepatic collateral system, and one dog survived for 49 days. Pennicillin did not inhibit phospholipase A2 activity with egg yolk phosphatidylcholine but moderately inhibited with egg yolk [36], and the difference would depend on the physicochemical conditions of the substrates. In our experiment, it is possible to think that in vivo penicillin prevented the depletion of mitochondrial phospholipids by the activation of endogenous phospholipase(s) and mitochondrial dysfunction. Acknowledgements The authors wish to express their thanks to Drs. M. Yamamoto and Y. Sameshima for their helpful discussion. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. References 1 2 3 4 5 6 7 8 9 10

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6.396402 57-63