Quantitative analysis of regenerating and degenerating areas within the lobule of the carbon tetrachloride-injured liver

ARCHIVES

OF

BIOCHEMISTRY

Quantitative within GEORGE

AND

BIOPHYSICS

Analysis

the Lobule

448460 (1965)

111,

of Regenerating of the Carbon

R. MORRISON:

and

Degenerating

Tetrachloride-lniured

FRANCES E. BROCK, ROBERT E. SHANK

IRENE

Areas Liver’

E. KARL,

AND

Department of Preventive Medicine, Washington University School of Medicine, St. Louis, Missouri Received April 26, 1965 The livers of rats were studied 48 hours after acute carbon tetrachloride injury in order to determine the degree to which four areas of the lobule participate in both regenerative and degenerative processes and to relate alterations in enzyme activity in each area to these processes. With histological stains, the intralobular distribution of mitotic activity, neutral fat and necrosis were quantitated. Microchemical techniques in conjunction with microdissection of lyophilized sections were used, and determinations were made of total protein, total hemoglobin, total lipid, and of nine enzymes in all four areas of the liver lobule. Regenerative processes were shown to dominate the portal midaone area of the injured lobule while degenerative processes were localized largely in the central area. Within the degenerated central area, activities of alkaline phosphatase, phosphoglucoisomerase, and glucose-6-phosphate dehydrogenase were elevated above control levels, while activities of lactic dehydrogenase, isocitric dehydrogenase, malic dehydrogenase, glutamic dehydrogenase, glutamic-alanine transaminase, and p-hydroxybutyryl-CoA dehydrogenase were decreased. In the portal midzone, a decreased activity of phosphoglucoisomerase and an elevated activity of lactic dehydrogenase were the only alterations in enzyme activities which were interpreted as being related to the regenerative process when correlations were made between the intralobular patterns of enzyme activities and the intralobular patterns of the manifestations of regeneration and degeneration. Present concepts of the biochemical changes which follow hepatic injury are based primarily upon alterations as determined in homogenates of liver specimens. The information obtained from such biochemical studies is necessarily limited because they do not take into account certain and pathological characteristics anatomical

of the hepatic lobule. The liver is a composite organ; several different cell types contribute to the struc1 Supported by the U.S. Army Medical Research and Development Command, Office of the Surgeon General, under Contract No. DA-49-007-MD-1024, and by grant AM-06309-2, National Institute of Health, U.S.P.H.S. 2 John and Mary Markle Scholar in Medical Science.

ture of the lobule. It has been estimated that only 65 % of the cells in a homogenate of normal liver are parenchymal cells (1). Moreover, quantitative measurements reported from this laboratory have demonstrated biochemical differences between parenchymal cells in various areas of the normal liver lobule (2). In hepatic injury, a further complication is the extraordinary capacity of the liver to regenerate, with the result that degeneration unaccompanied by regeneration is uncommon within a lobule. This is the first reported endeavor to separate regenerating from degenerating cells in the hepatic lobule by means of microdissection and to make quantitative microchemical measurements of several metabolic processes which characterize each type of

448

CARBON

TETRACHLORIDE-INJURED

LIVER

LOBULE

449

FIG. 1. Typical Ccl*-injured rat hepatic lobule. Superimposed lines indicate the approach to microdissecting the area between the portal tract on the right and the central vein on the left into four approximately equal areas; portal (P), portal mideone (PM), central midzone (CM), and central (C). Hematoxylin and eosin st,ained section; x275.

cell. To perform this separat’ion, advantage was taken of the characterist’ic zonal distribution of degeneration and regeneration in t,he lobule after various forms of hepatic injury. The microchemical procedures for analysis developed by 0. ET. Lowry and his associates (3) were used, and groups of hepatic cells from regenerating and degenerating areas of the rat liver lobule were studied 48 hours after carbon tetrachloride (CCL) injury, when mitotic activity, lipid accumulation, and necrosis in the liver are at peak levels. In four areas of the hepatic lobule the results of enzyme assays are interpreted in terms of the quantitat’ive morphological and chemical manifest’ations of regeneration and degeneration. EXPERIMENTAL Animals. Normal female Sprague-Dawley rats (Holtzman Rat Company) weighing 14&170 gm were maintained on a Purina Chow diet. Carbon

tetrachloride was diluted with 10 parts mineral oil and 0.3 ml/l00 gm body weight of this mixture was injected intraperitoneally after a la-hour fast. Control rats were injected with 0.3 ml of mineral oil/100 gm bodyweight. After continuing the fast for an additional 48 hours, four control and four Ccl,treated rats were killed by decapitation. During exsanguination, carotid blood was collected for analyses. Immediately thereafter, the liver was rapidly excised, weighed, and sampled for frozendried sections, fixed histological stains, and homogenates. Frozen-dried sections. A small biopsy from the periphery of the left lateral lobe of the liver was frozen in liquid nitrogen. Sections were cut at 22 p in a cryostat at -25” and dehydrated in VLZCUO at -40”. Lyophilized sections were stored for less than 2 months at -20” in evacuated tubes. Preliminary experiments revealed no loss in activity of the enzymes measured under these conditions of storage. Dissection of lyophilized sections and weighing of dissected segments were carried out in a dehumidified room at 18”. Representative parts of the lobule were dissected out under

450

MORRISON

a microscope at a magnification of 72x. The identity of the lobular structures was readily established on unstained sections and was confirmed on alternate stained sections. Only the lobules adjacent to the capsule were utilized. Segments dissected from various areas of t,he lobule weighed 0.05-0.10 rg on a quartz beam balance. After weighing, segments were placed in microtest tubes for immediate enzyme analyses. For various determinations, the area of the lobule between the portal tract and the central vein was divided into four approximately equal areas. The areas dissected out for analyses are depicted in Fig. 1. They are P, groups of hepatic cells in areas adjacent to the portal tract; PM, groups of cells from the half of the mideone closest to the portal tract; CM, groups of cells from the half of the midzone closest to the central vein; C, groups of hepatic cells from areas adjacent to the central vein. Each area of three lobules from the livers of every rat were assayed. Fixed stained sections. Fixed sections of the left lateral lohe of the liver were cut at 6 p and stained with either hematoxylin and eosin or with Oil Red 0. For various morphological determinations, the area of the lobule between the portal tract and the central vein was divided into four equal segments of 0.1 mm width; an eyepiece micrometer was used. These four segments correspond to the port,al, portal midzone, central midzone, and centrolobular areas dissected from lyophilized tissue for chemical assays. Morphological measurements. Counts of numbers of cells in meta-, ana-, and telophase of mitosis were made on sections of each liver stained with hematoxylin and eosin. The counts were made in 25 hepatic lobules chosen at random from the periphery of each liver so that a total of 100 lobules from control and from CCll-injured livers was counted. Counts were made in the four areas of the lobule under investigation. Counts of polymorphonuclear leukocytes and of parenehymal cells with degenerated nuclei were also made in the four areas of the lobule under investigation. These counts were made in six consecutive lobules from t,he periphery of each liver, and a total of 24 lobules from control and Ccl,injured livers was counted. In order to determine the mitotic index (mitoses/lOOO parenchymal cells) and necrotic index (degenerated nuclei/1000 parenchymal cells) in each area of the lobule, the total number of parenchymal cells was counted in the four areas of the six lobules which were utilized to count degenerated nuclei. Sections stained with Oil Red 0 were used to

ET AL. estimate the intralobular distribution of neutral fat in each liver. Estimates of the amount of neutral fat accumulated within parenchymal cells were graded on a scale having six arbitrary grades, grade six being for parenchymal cells with the cytoplasm completely filled with fat droplets and grade one for cells in which fat was barely discernible. Parenchymal cell weights were determined by counting the hepatic parenchymal cell nuclei in weighed and stained segments dissected from portal, portal midzone, central midzone, and central areas of the lobule. The lyophilized sections were 22 p in t,hickness, the diameter of the average parenchymal cell. After weighing, segments were stained with 1% cresyl violet. The original counts were corrected for the error introduced by the probability that all nuclei visible do not have their centers within the section, by multiplying by the ratio of the section thickness to the section thickness plus mean nuclear diameter (4). Nuclear diameters were det.ermined in each section by measuring diameters of 10 nuclei. Determination of chemical constituents. In each liver, triplicate determinations were made of the four areas of the lobule under investigation for protein, heme protein, and lipids. Segments of the lobule dissected from lyophilized sections of each liver were pooled into samples of 1 .O /rg dry weight for measurement of protein or heme protein. Total protein was measured by the Folin phenol method (5) in a final volume of 0.05 ml in the spectrophotometer. The total heme protein was measured by a method3 which converts the heme moiety to porphyrin for readings in a final volume of 0.05 ml in the fluorometer. Because the livers were not perfused to remove circulating red blood cells, the heme protein measurements were essentially hemoglobin determinations. Total lipid was determined gravimetrically by difference after extraction with hexane and anhydrous ethanol at room temperature, according to the procedure of Lowry (6). Advantage was taken of the fact that neutral fats are extracted primarily by n-hexane while phospholipids are removed primarily by ethyl alcohol. Segments dissected from lyophilized sections of each liver weighing approximately 0.1 pg dry were extracted with 50 ~1 n-hexane for 6 hours at 25” and reweighed prior to extraction with anhydrous alcohol. This initial extraction was termed “neutral fat,” although most of t,he cholesterol and probably small amounts of phospholipids are removed by hexane. Preparation of homogenates. Samples from the periphery of the left lateral lobe of the liver with 3 G. R. Morrison,

unpublished

procedure.

CARBON

TETRACHLORIDE-INJURED

LIVER

TABLE

ANALYTICAL

451

LOBULE

I

CONDITIONP

Incu-

Ref.

Bu5er

dhmple w wt.. Y1mtion (Irg) ~OlUilE

Additions

011)

Alkaline

phosphatase

p-OH butyryl-CoA dehydrogenase

(7)

AMPie, 10.0

0.5 M, pH

b

Sodium pyrophosphate, 0.09 M, pH 6.4

-Glucose-g-phosphate dehydrogenase

(8)

AMPic, 9.8

Glutamic acid dehydrogenase

(9)

Phosphate, pH 7.6

Glutamic-alanine transaminase

0.1 M, pH

0.05 M,

Disodium p-nitrophenyl phate, 8 mM MgC12, 2 mM

phos-

0.8

10

Acetoacetyl-CoA, 0.4 mM Nicotinamide, 1 mM Sodium amytal, 2 mM DPNH, 0.3 mM

0.2

30

Glucose-6-phosphate, 5 mM Sodium EDTA, 0.5 mM TPN+, 0.35 mM

0.5

G

Sodium a-ketoglutarate, 3.5 mM 1 Ammonium sulfate, 0.1 mM DPNH, 0.5 mM

0.3

30

Sodium a-ketoglutarate, 10 mM Alanine, 96 mM Pyridoxal phosphate, 4 mg% DPNH, 0.75 mM Lactic dehydrogenase

0.4

9

AMPzd, 0.1 M, PH 8.9

(33)

Isocitric acid dehydrogenase

(10)

Tris, 0.1 M, pH 8.1

Sodium isocitrate, 5 mM Nicotinamide, 20 mM MnClz, 0.25 mM TPN+, 1 mM

0.2

40

Lactic acid dehydrogenase

(11)

Tris, 0.1 M, pH 7.4

Sodium pyruvate, 2 mM Nicotinamide, 20 mM DPNH, 2 mM

0.1

40

Malic acid dehydrogenase

02)

AMP~C, 0.1 M, PH 10.5

Sodium malate, DPN+, 5 mM

0.1

45

Tris, 0.1 M, pH 8.1

Glucose-&phosphate,

Phosphoglucoisomerase

(8) /

-

-!-

5 mM

30 mM

0.3 -

10

-

Q Incubations were for 30 minutes at 38” except for @-OH butyryl-Coil dehydrogenase (30 minutes at. 25”) and alkaline phosphatase (60 minutes at 38”). Suitable blanks were always included for both standards and samples. All buffers contain 0.05% bovine plasma albumin. b D. Azarnoff, unpublished procedure. c AMPI indicates 2.amino-2-methyl-1-propanol. d AMP, indicates 2-amino-2.methyl-l, 3-propanediol.

fresh weights of -500 mg were homogenized by hand at 0” in ground glass homogenizers with 10 volumes of water. Aliquots were immediately stored at -20”. Within the first week of storage these aliquots were thawed and diluted with ap-

propriate buffers containing 0.05% bovine serum albumin for individual analyses. Preparation of whole blood and serum. Carotid blood was collected in both heparinized and untreated tubes. Ten pl of blood was immediately

452

MORRISON

ET AL. (b) p-GlucoisonLeruse. Absorption at 500 rnp of blanks and samples in HzSO( was found to increase at different rates at room t.emperatures after the primary development of the color at GO”. Consequently, timing was individualized for each tube and samples were read 15 minutes after incubation with the HpSO1-resorcinol mixture at 60”. After this interval, standard curves were still linear. (c) Glucose - 6’ - phosphate dehydrogenase. To achieve linearity and improved activity of this enzyme, it was necessary to add 0.5 mM EDTA to the buffered substrate mixture. The activity was not measured at its optimal pH but was measured at pH 9.8, where the Ks of 6-phospho-gluconate dehydrogenase is SO high (2.2 X 10m4M as determined in this laboratory) that it contributes less than 5y0 to the readings.

frozen in liquid nitrogen and lyophilized at -30” before storage in vucuo at -20”. The clotted blood was permitted to stand for 1 hour at 25”, and the serum which separated was stored at -20”. Within the first week of storage, whole blood and serum samples were thawed and diluted with appropriate buffer containing 0.05% bovine plasma albumin for enzyme analyses. Conditions for enzyme assays. Conditions which permit enzyme activities to be measured at nearly optimal concentrations of substrate and coenzymes, and at optimal pH are listed in Table I with References 7-12. The appropriate, complete reaction mixture at 0” was pipetted into microtest tubes containing dried sections at room temperature or homogenates at 0”. The tubes were incubated in a Dubnoff shaker. Reactions were stopped by transferring the tubes to an ice bath and rapidly adding either acid, alkali, or a large diluting volume of appropriate buffer. Fluorescence of reduced pyridine nucleotides or of the alkaline derivatives of oxidized pyridine nucleotides was determined in l-ml volumes in a Farrand model A fluorometer. Phosphoglucoisomerase and alkaline phosphatase activit,y was measured in 0.5 and 0.05 ml volumes, respectively, in a Beckman DU spectrophotometer. The following modifications of analysis are not included in the references in Table I. (a) Glutamic dehydrogenase. The activity was increased by changing from tris to a 0.05 M potassium phosphate buffer. Nicotinamide and adenosine diphosphate were not added. Because of the low temperature coefficient of this enzyme, incubation time was individualized for each tube and was begun as soon as the buffered substrate was added to each tube.

RESULTS

The four areas of the normal and Ccl,injured liver lobule selected for dissection were studied for morphological, chemical, and biochemical evidence of regeneration and degenerat’ion. Morphological Jindings. Table II shows the counts of mitotic figures, degenerated parenchymal nuclei, and polymorphonuclear leukocytes as well as an estimate of the quantity of neutral fat in each area of the lobule. In the port’al midaone of the CCld-injured lobule, mitotic figures were twice as numerous as in the portal area and 4.5 times more numerous than in Dhe central area. The central half of the segment dissected from

TABLE

II

~NTRALOBULAR DISTRIBUTION OF MORPHOLOGICAL MANIFESTATIONS OF REGENERATION 48 HOURS AFTER CC14 ADMINISTRATION DEGENERATION IN THE ROT LIVER Constituent

Group of rats

Mitoses/KKl lobules Parenchymal cells/25 lobules Mitotic index (mitoses/lOOO cells)

ccl, cc14

Neutral

Control ccl,

fat (arbitrary

units)

Necrotic index (degenerated nuclei/1000 cells) Polymorphonuclear leukocyt,es (number/1000 cells) a Portal half of segment. * Central half of segment.

Control cc14

Control cc14 ccl,

AND

Area of lobule Portal

Portal midzone

Central midzone

Central

52 1620 0 8.1

92 1390 0 16.6

69 1890 0 9.1

40 2923 0 3.4

+?d +1 0 0 0

+%

+2 0 0 3

+wa +6n oa 65”

+w* +2* 0” 89ob 40

+w +x2

o* 4-w 0 1000

95

CAR.BON d 18 !-

TETRACHLORIDE-INJURED

Mtoses

Relative levels of Neutral fat esfi-

MIDZONE

MIDZONE

FIG. 2. Intralobular distribution of major manifestations of regeneration and degeneration.

the central midzone consists of necrotic cells, and the viable half contained the majority of the mitotic figures in this segment. The finding that mitosis clearly dominated the midzone 48 hours after an injection of CC&, which results in necrosis of one third of the area of the lobule, is in agreement with t,he radioautographic studies of Grisham.4 On stained sections 36 % of the area of the average CC14-injured lobule was necrotic. Degenerated nuclei and polymorphonuclear leukocytes occupied the entire central segment and one half of the centralmidzonal segment (Fig. 2 and Table 11). In the middle of t’he central midzone, these manifestations of necrosis ended rather abruptly at a boundary of cells heavily laden with neutral fat droplets. The accumulation of neutral fat droplets within parenchymal cells decreased progressively from this boundary to the portal t.racts. The intralobular distribution of fat as observed on Oil Red O-stained sections differed from the chemical measurements of lipid in that the center of the injured lobule had fewer neutral fat droplets on stained sections than other areas of the lobule but more “neutral fat” and total lipid by chemical determinations than other areas of the lobule. A possible explanation for this apparent discrepancy is that the lipid in the central area of necrosis is not in a form, e.g. cholest,erol, readily stained by Oil Red 0. Chemical constituents. Measurements in 4 J. W. Grisham,

personal

communication.

LIVER

LOBULE

453

lyophilized sections for protein, heme protein, total lipid, and “neutral fat” are tabulated along with average parenchymal cell weights (Table III). In control livers the intralobular differences in protein, t’otal lipid, and “neutral fat,” concentrations were minimal. In t,he CCll-injured liver the total lipid was approximately bwice control levels in each area of the lobule. This increase in lipid was largely due t’o “neutral fat,” particularly t,oward the center of the lobule. The “neutral fat” was 12.8, 8.9, 4.7, and 4.7 ~g/lOO erg of dry liver above control levels in central, central midzone, portal midzone, and portal areas of the lobule, respectively. Despite these elevations in Iipid content, t.he periportal concent,ration of prot’ein was equal to that, in control livers and the centrolobular concentrat’ion was only 4.Fj% below control levels. Total hemoglobin was 50 % higher in the centrolobular segments of control liver t’han in the other three areas of the lobule. This is in keeping wit,h the observation in t.his study that t’he centrolobular sinusoids were more dilated. The hemoglobin content in centrolobular sections of CCll-injured livers was 50 % above control centrolobular levels. This elevat’ion is understandable in the light of the cent’rolobular hemorrhage which is customarily associated with Ccl, injury. Elsewhere in t’he CCL-injured lobule, segments revealed less hemoglobin than in corresponding control segments, suggesting that in these areas swollen parenchymal cells had compressed the sinusoids. The average weight of the normal fasted parenchymal cell was 0.00215 pg dry, a value in close agreement with that which can be calculated from the data of Price and Laird (13). In t,he portal and port’al midzonal segments of the CC&injured lobule, the average parenchymal cell weighed 26 % more than its control counterpart. This is consistent with the general observations that parenchymal cells sustaining sublethal CC& injury are swollen and the evidence that regenerating parenchymal cells have an increased mass (14). Enzyme activities. The nine enzymes measured are known to be located primarily in three fractions of the hepatic cell: (a) mitochondria (glutamic dehydrogenase, isocitric

454

hIORRISON

ET AL.

TABLE

III

INTRALOBULAR DISTRIBUTION OF CHEMICAL MANIFESTATIONS OF RE~ENER.~TION .YNDDEGENERATION IN THE RAT LIVER 48 HOURS AFTER CC14 ADMINISTR:\TION

Groupof rats

Constituents Total protein (pg/lOO pg dry) Total heme protein (rg/lOO pg dry) Total lipid (/*g/100 pg dry) “Neutral fat” (pg/lOO pg dry) Avg. parenchymal cell weight (rg dry/cell)

Control ccl, Control CCL Control ccl, Control cc14 Control cc14

Areaof lobule Portal 66.2 66.5 0.93 0.72 11.1 19.1 2.6 7.3 0.00219 0.00294 TABLE

65.8 65.7 1.01 0.99 13.5 26.0 3.5 12.4 0.00191 0.00157

67.0 65.9 1.04 0.66 10.7 20.2 2.7 7.4 0.00236 0.00280

Group of rats

Phosphoglucoisomerase Glucose-B-phosphate dehydrogenase Lactic acid dehydrogenase p-OH butyryl-CoA dehydrogenase Isocitric acid dehydrogenase Malic acid dehydrogenase Glutamic acid dehydrogenase Glutamic-alanine transaminase Alkaline phosphatase

Q Enzyme

activities

represent

Control CCL Control cc14 Control CCla Control CCL Control ccl, Control ccl, Control cc14 Control ccl, Control ccl, the average

Central 63.8 60.9 1.51 2.26 13.1 29.1 3.0 15.8 0.00214 0.00107

IV

ENZYMES OF CARBOHYDRATE MET~~BOLISM IN CONTROL AND CCln-INJURED Enzym.#

-.

Portal midzone Central midzone

RAT

LIVER

LOBULES

Enzyme activities (mpmoles/Mgdry wt./hour)a Portal

Portal midzone Central midzone

-

39.8 37.9 0.90 0.99 156.3 154.6 18.2 14.9 14.5 11.0 93.7 80.4 10.7 10.4 11.7 9.5 0.082 l.ooo

37.6 31.8 1.03 1.17 137.3 154.6 18.8 13.5 16.0 11.8 95.3 77.3 10.6 10.1 10.5 8.2

of triplicate

determinations

dehydrogenase, malic dehydrogenase and p-OH butyryl-CoA dehydrogenase) ; (b) t,he cell sap (lactic dehydrogenase, isocitric dehydrogenase, malic dehydrogenase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase, and glutamic-alanine transaminase) ; and (c) along the surface lining bile canaliculi (alkaline phosphatase). Mean activities of enzymes in microdissected sections of the lobule are expressed on a dry weight basis in Table IV and on a protein basis in Fig. 3.

35.6 35.6 1.01 1.83 122.3 108.3 16.3 9.6 16.7 9.3 88.1 57.2 11.8 8.6 5.4 4.8 0.065 0.580

Central 36.8 40.8 1.27 3.47 98.7 73.9 14.4 3.3 17.9 5.1 82.9 32.3 13.2 3.5 3.9 2.1 0.067 0.204

for each of four rats

In livers of control rats the activity of each enzyme showed a portal-central gradient within the lobule. Because these gradients were roughly linear, the midzone activities closely approximated the average of the activities within the lobule as a whole. In CCL-injured livers the intralobular distribution in the activity of each enzyme differed from that in the control lobules. This difference can be described for an enzyme by one of the five intralobular patterns. The general characteristics of these patterns, depicted

CARBON

TETRACHLORIDE-INJURED

LIVER

A.

LOBULE

455

C.

GDH: Glutamic dchydrogcnosc MDH: Molic dehydrogenose BOHDH:p-OH butyrl CoA dehydrogenose ICDH: lsocitric dchydrogenase

LDH: Lactic dehydrogcnose PO1 : P-giucoisomerase GPT: Glutomic-alonine tronsominase

E%,o’ LDH

P

PM CM

P

C

1 P

I I I PM CM C

C

D.

B.

0

PM CM

0

I P

t I PM CM

I C

FIG. 3. Distribution of enzyme activities within the rat liver lobule 48 hours after CC14 injury. Activities on a protein basis in the injured lobule are expressed as a percentage of control levels. Areas within the lobule are neriportal (P), portal midzonal (PM), central midzonal (CM), and central (C).

in Fig. 3, are a centrolobular depression in activity (Fig. 3A), a centrolobular elevation (Fig. 3B), a midzonal rise with a centrolobular depression (Fig. 3C, lactic dehydrogenase), a midzonal depression wit,h a cent’rolobular elevation (Fig. 3C, phosphoglucoisomerase) and a periportal elevation in act,ivity (Fig. 3D). In the control livers (Table IV), two of the mitochondrial enzymes had centrolobular activities above and two had centrolobular activities below periportal levels. The midzonal activit’ies of isocitric dehydrogerlase and glutamic dehydrogenase were, respectively, 16 and 11 mpmoles per microgram per hour, and their activities wele both 123%

centrolobular of periportal

activities. Malic dehydrogenase and /?-OH butyryl-CoA dehydrogenase had midzonal

activities of 91 and 17 mpmoles per microgram per hour, respectively, and their centrolobular activities were, respectively, 88 and 79 % of periportal activities. In the CCll-injured livers, the activities of these four mitochondrial enzymes are described by a single intralobular dist’ribution pattern (Fig. 3A). Periportal activities were 3-24 % below control levels, and the centrolobular activities were 59-76 % below control levels. The changes in midzonal activities were between t’hese two ext’remes. In the control livers only one enzyme located primarily in t)he cell sap had a centrolobular activity above the periportal level. Glucose-6-phosphate dehydrogenase had a midzonal activity of 1.0 mKmole per microgram per hour and a centrolobular activity 141% of t,he periportal activity. In the CCL-

456

MOR.RISON

ET AL.

TABLE

1’

ENZYMES OF CARBOHYDRATEMETABOLISMINBLOOD AND LIVEROF CONTROL AND CClp-INJURED Enzymea

Phosphoglucoisomerase Glucose-6-P dehydrogenase Lactic acid dehydrogenase p-OH butyryl-CoA dehydrogenase Isocitric acid dehydrogenase Malic acid dehydrogenase Glutamic acid dehydrogenase Glutamic-alanine transaminase Alkaline phosphatase

Group

of rats

Control ccl, Control cc14 Control ccl, Control cc14 Cont.rol ccl, Control cc14 ConBrol ccl, Control cc14 Cont,rol ccl,

Liver,

fresh mates

homog-

Serum,

fresh

11.0 10.7

-

37.5 36.9 5.2 3.1 6.8 4.0 34.9 21.9 4.9 3.1 4.0 2.4 41.5 218.0

0.0233 0.0192 0.0010 0.0024 0.0079 0.0125 0.0283 0.0246 0.0010 0.0054 0.0039 0.0169 0.0075 0.0112

RATS

Whole blood, lyophilized homogenate

0.643 0.643 0.266 0.250 1.617 1.636 0.004 0.010 0.002 0.030 0.208 0.297 0.031 0.036 0.039 0.042 Ob Ob

a Enzyme activities represent the average of triplicate determinations for each of four rats. lJ Determinations negative with very high blank values, which are believed to be due to constituents of the red blood cells absorbing in the region of the Soret band.

injured liver, this intralobular distribution pattern was accent,uat.ed (Fig. 3B). The periportal activity was 111% of control levels, the centrolobular activity was 286 % of control levels, and the midzone activit.y was between these two extremes. The remaining three cell sap enzymes revealed centrolobular activities below periport~al levels in the cont.rol liver. Lactic dehydrogenase, glutamic-alanine t,ransaminase, and phosphoglucoisomerase had midzonal activities of 130, 8, and 36 mkmoles per microgram per hour, respectively, and their centrolobular activities were, respectively, 37, 67, and 8 % below periportal activities. In the CC&-injured livers, the intralobular distribution patterns of these three enzymes were irregular and were characterized by different midzonal reversals in their portal central gradients (Fig. 3C). Lactic dehydrogenase had a periportal activity equal to the cont’rol level, a portal midzonal elevation t,o 114 % of the control level, and a reduced centrolobular activity, 78 % of the control value. Glut,amic-alanine transaminase had a periportal activity that was 80 % of the control level; a cent.ral mid-

zone elevation, 89 %> of the control level; and a centrolobular depression 56% of the control level. Inversely related to these two was the pattern of phosphoglucoisomerase with a periportal activity close to control levels; a reduced portal midzone activity, 86% of the control value; and a centrolobular elevation, 114 % of the control value. Except for the cent,ral midzone elevation in glutamic-alanine transaminase, the midzonal and centrolobular alterations in these three enzymes were significant. In both the control and CCll-injured livers, alkaline phosphatase had a linear portal central gradient with the periportal levels of act,ivit.y being highest. The midzonal activity of alkaline phosphatase was 0.065 mpmole per microgram per hour in the control livers, and centrolobular activity was 18 % less than periportal levels. Carbon tetrachloride injury accentuates this pattern of intralobular dist’ribution (Fig. 3D) since the periportal act.ivit,y was 1215% of the control activity, the centrolobular activity was 320% of the control, and the midzonal activity was between these two extremes. Act.ivities in fresh liver homogenat,es are

CARBON

TETRACHLORIDE-INJURED

LIVER

LOBULE

Control

Levels

457

+ R= elevation due to Regeneration - R=depression due to Regeneration +D = elevation due to Degeneration - D= depression due to Degeneration

A

16

,/+D

-16

X Corrected

for Controls

FIG. 4. Hypothetical patterns for intralobular distribution of enzyme activities. Curves are based upon the relative morphological and chemical evidence for regeneration and degeneration tabulated above.

expressed on a wet weight basis (Table V). Generally they reflected the midzonal activities on a dry weight basis in microdissected segments of the control and Ccl,injured lobules when corrections are made for an estimated 68 % moisture in fresh livers. Alterations in homogenates of the CC&injured livers revealed a reduction below control activities for all enzymes except alkaline phosphatase, lactic dehydrogenase, and phosphoglucoisomerase. Activities for the latter two enzymes were equal to controls, apparently reflecting the opposing alterations in the midzonal and centrolobular activities found for these enzymes in microdissected segments of the injured lobule. Glucose-6-phosphate dehydrogenase was not measured in homogenates, but has been found to be increased in similar homogenate studies.s 6 I. E. Karl, R. Schwartz, S. McNicol, H. Zarkowsky, and R. E. Shank, unpublished data.

Because of centrolobular hemorrhage in the CC14-injured lobule, contributions to enzyme activities by whole blood must be considered. Calculating from the intralobular hemoglobin levels in Table III and the enzyme activities in lyophilized whole blood in Table V, it is concluded that no more than 0.5 % of the centrolobular activity of any enzyme can be attributed to whole blood in dissected sections. DISCUSSION

The morphological and chemical findings indicate that all areas of the hepatic lobule of the rat participate in regenerative and degenerative processes after Ccl, injury. Because the two processes are most active in different areas of the lobule, it has been posslible to distinguish between alterations in metabolic functions which characterize regeneration and those which characterize degeneration by separating the lobular areas by microdissection prior to analyses.

458

MORRISON

The degree to which each area of the lobule is involved in regeneration and degene&ion has been estimated by selecting mitotic act’ivity and total lipid as appropriate expressions of the two processes. Intralobular mitotic activit,y and tot,al lipid are compared with controls in Fig. 2 along with morphologically determined levels of neutral fat accumulation and parenchymal cell necrosis. Mitotic activity is an index of regenerative activity. Total lipid appears to be a valid expression of degeneration since its intralobular distribution pattern parallels a composite of the patterns for degeneration without necrosis (neut’ral fat’) and degeneration with necrosis (degenerated nuclei) seen in histological stained sections. The quantitative levels of mitotic activity and tot,al lipid are tabulated under Fig. 4. It is demonstrated that the portal midzone had t,he highest mitotic index, 4% times greater than that in the center of the lobule, while the centrolobular area had the highest total lipid content, 1% times greater than that in the portal midzone. In each area of the lobule, these quantit,ative manifestations of regenerat’ion (R) and degenerat,ion (D) may be plotted either alone (+R or -R, and +D or -D) or in combination (+R+D, -R + D, +R - D, -R - D) to derive 8 intralobular patterns. Five of these patterns are depicted in Fig. 4 and are considered to be expressions of the combined influence of regeneration and degeneration upon functions in various areas of the lobule. It, is therefore anticipated that alterations in enzyme activities due to regenerat,ion, degenerat,ion, or both will have intralobular patterns resembling one of these 8 patterns. For example, the +D pattern is the type of intralobular distribution which would be anticipated for an enzyme with its activity unaltered by regenerative processes but elevated by degenerative processes. Similarly, the +R-D pattern is t,he type of intralobular distribution anticipated whenever the activit’y of an enzyme is elevated by regenerative processes and at the same time depressed by degenerative processes. In viewing the 8 possible patterns, it should be emphasized that a midzonal reversal in the portal central gradient of an enzyme’s activity indicates a regenerative patt’ern re-

ET AL.

gardless of whether degenerat)ion also alters the act’ivity. In contrast,, a degenerative pattern, uninfluenced by regeneration, has relatively linear portal central gradients. In the t,reated rats, an effort- has been made to interpret changes in enzyme activities in various areas of these lobules in the light of the quant’itative morphological and chemical evidence for regeneration and degeneration in each area of the injured lobule. Each pattIerr for the distribution of an enzyme’s act’ivity wit,hin the Ccl,-injured lobule (Fig. 3) is comparable wit,h one of the five morphological and chemical patterns of regeneration and degeneration depicted in Fig. 4. The act,ivity pattern of glucose-6phosphate dehydrogenase resembles the +D pattern. Four other enzymes had patterns suggesting that degeneration alone is responsible for t’heir altered activities; glut’amic dehydrogenase, isocitric dehydrogenase, malic dehydrogenase, and P-OH butyrylCoA dehydrogenase were reduced in a manner resembling the -D pattern. The activity pattern of phosphoglucoisomerase resembles the -R+D pattern and indicates a reduct,ion in act,ivity due to regeneration and an elevation due to degenerat,ion. The act,ivities of glutamic-alanine transaminase and especially of lactic dehydrogenase resemble the reciprocal patt,ern, +R - D. The remaining activity pattern, alkaline phosphatase, compares with none of the morphological and chemical patterns. This may be related to the evidence that this enzyme is located largely along bile canaliculi (15). These findings demonstrate that there are biochemical as well as morphological differences between regenerating and degenerating hepatic cells. Among the degenerating centrolobular cells, the specific activity of cell sap enzymes participating in glycolysis (phosphoglucoisomerase) and t’he pentose pathway (glucose-B-phosphate dehydrogenase) are elevated, while the activities of cell sap enzymes important in gluconeogenesis (lactic dehydrogenase and glutamic-alanine transaminase) are moderately reduced and the act,ivit’ies of enzymes functioning within mitochondria (glutamic dehydrogenase, isocitric dehydrogenase, malic dehydrogenase and P-OH butyryl-CoA dehydrogenase) are great,ly reduced. Among regenerating mid-

CARBON

TETRACHLORIDE-INJURED

zonal cells similar degenerative changes are less evident, and superimposed alterations attributed to regeneration include a reduction in the activity of the enzyme participating in glycoloysis plus elevated activities for the cell sap enzymes participating in gluconeogenesis. Inasmuch as enzyme activities are only interpreted after correlating them with morphologlal and chemical findings, little if any of the midzonal alterations for enzymes part’icipating in the pentose pathway or functioning in the mitochondria can be attributed t.o regeneration. Earlier invest,igations are in agreement wit’h our interpretation that alkaline phosphatase, glucose-6-phosphate dehydrogenase, and the mitochondrial enzymes are less active in the liver following CC14 injury as a result of degeneration. Using homogenates 48 hours after Ccl, injury in rats, other investigators have demonstrated alterations in enzyme activities similar to those in this report5 (16-19). While homogenate studies do not permit separation of the biochemical manifestations of regeneration from those of degeneration when both processes coexist as they do 48 hours after CC&, certain enzymic alterations due to degenerat,ion have been defined using homogenates by taking advantage of the finding that degeneration precedes regeneration by several hours. The mitotic index in the rat liver is maximal at 48 hours after Ccl, administration (20), and the increase in DNA synthesis has not begun at 12 hours aft’er injury (5). Consequently alterations in enzyme activities during the first 12 hours after CC& administration have been interpreted as degenerative changes. In one study,5 all enzymes evaluated in the present’ report were measured in liver homogenates at intervals after Ccl, injection. The specific acbivities of alkaline phosphatase and glucose-6-phosphate dehydrogenase were elevated as early as 6 hours after Ccl, and significantly elevated by 12 hours, but> t,he other 7 enzyme activities were still not significantly abnormal by 24 hours. Other studies during the first’ 12 hours after Ccl, administration also support the conclusion that, alkaline phosphatase and glucose-6-phosphate dehydrogenase are elevated in response to degeneration (16, 21). Support for our conclusion that) the activi-

LIVER

LOBULE

459

ties of the mitochondrial enzymes are depressed as part of the degenerative process comes from studies with electron microscopy and cytochemical analyses of subcellular fractions during the early hours after CCL, injury. All investigators agree that structural changes in mitochondria are present by 6 hours (22-24), and quantitative depressions in mitochondrial function are measurable by 12 hours (25-27). Regeneration after partial hepatectomy has been extensively studied by using liver homogenat,es. At 48 hours after partial hepatectomy both elevations (28) and reduct’ions (29) in alkaline phosphatase activity, reduced activities of glycolytic enzymes (29), and minimal changes in transaminase activity (29, 30) have been reported. These alterations are in agreement with our midzonal findings 48 hours aft’er Ccl, injury. After partial hepatectomy the mitochondrial population of the average rat liver cell is decreased (31) and the activities of mitochondrial enzymes are reduced (29, 30, 32). Since the latter appear to be manifestations of cellular regeneration, we may not be justified in interpreting the reduced midzonal activities of mitochondrial enzymes after Ccl, injury as manifestations entirely due to degeneration. A final evaluation of the significance of the biochemical variations observed in this study in regenerating and degenerating areas of the injured lobule must be deferred pending accumulation of basic information regarding the control and mechanisms of each process as they relate to the turnover of enzymes in parenchymal cells. However, three hypotheses are suggested that might prove useful as a guide for future investigation. It would appear that the regenerating cell has preserved many of the specialized functions which characterize normal parenchymal cells. The activities of regenerating cells are close enough to control activities to indicate that they utilize lactate for the production of circulating glucose and generat,e energy for regeneration by way of the tricarboxylic acid cycle. The second suggestion is that severely damaged parenchymal cells have preserved little of their specialized funct,ions. The constellation of enzyme activities in t,hese cells indicate that they may

460

MORRISON

be utilizing glucose primarily in met’abolism which produces lactate and generates t’he energy needed for repair through glycolysis. The third hypot’hesis deals with alkaline phosphatase. Inasmuch as this enzyme is located largely along bile canaliculi and normally has an intralobular gradient increasing toward the portal t)racts, it seems reasonable to suggest that in CCL injury the intralobular pattern of alkaline phosphatase is essentially a response to degeneration with an elevated activity in the central area of necrosis. The phosphatase elevations in other areas might be due not so much to cellular regenerative activity as t’o the flow of the enzyme via bile canaliculi toward the portal bile ductules. Whether or not there is an increased lipid synthesis and a decreased lipid catabolism in the centrolobular area, the direct intralobular correlation between lipids and glucose-6-phosphate dehydrogenase and the indirect intralobular correlation bet’ween lipids and P-hydroxybutyryl-CoA dehydrogenase are of interest. REFERENCES 1. ABERCROMBIE, M., AND HARKNESS, R. D., Proc. Roy. Sot. (London), SeT. B 138, 544 (1951). 2. SHANK, R. E., MORRISON, G., CHENG, C. H., KARL, I., AND SCHWARTZ, R., J. Histochem. Cytochem. ‘7, 237 (1959). 3. LOWRY, 0. H., Harvey Lectures Series 68, (1963). 4. ABERCROMBIE, M., And. Record 94,238 (1946). 5. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 6. LOWRY, 0. H., J. Histochem. Cytochem. 1, 420 (1953). 7. LOWRY, 0. H., ROBERTS, N. R., WV, M-L., HIXON, W. S., AND CRAWFORD, E. J., J. Biol. Chem. 207, 19 (1954). 8. BUELL, M. V., LOWRY, 0. H., ROBERTS, N. R., CHANG, M-L., AND KAPPHAHN, J. I., J. Biol. Chem. 262, 979 (1958). 9. LOWRY, O.H., ROBERTS, N. R., AND LEWIS, C., J. Biol. Chem. 220, 879 (1956). 10. ROBERTS, N. R., COEHLO, R. R., LOWRY, 0. H., AND CRAWFORD, E. J., J. Neurochem. 3, 109 (1958).

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(1952). 17. BRUNS, K., BND NEUH~US, J., Biochem. 2. 326, 242 (1955). 18. MOLANDER, D. W., AND FRIEDMAN, M. M., Clin. Res. Proc. 4, 39 (1956). 19. TSUBOI, K. K., AND STOWELL, R. E., Cancer Res. 11, 221 (1951). 20. LEEVY, C. M., HOLLISTER, R. M., SCHMID, R., MACDONALD, R. A., SND D.~VIDSON, C. S., Proc. Sot. Exptl. Biol. Med. 102, 672 (1959). 21. ISSELBACHER, K. J., AND JONES, W. A., Gastroenterology 46, 424 (1964). 22. ASHWORTH, C. T., LUIBEL, F. J., SANDERS, E., AND ARNOLD, N., Arch. Puthol. 76,212 (1963). 23. SMUCKLER, E. A., ISERI, 0. A., AND BENDITT, E. P., J. Exptl. Med. 116, 55 (1962). 24. CAMERON, G. R., AND KARUNARATNE, W. A. E., J. Pathol. Bacterial. 42, 1 (1936). 25. CHRISTIE, G. S., AND JUDAH, J. D., PTOC.Roy. Sot. (London), Ser. B 142, 241 (1954). 26. JUDdH, J. D., AND REES, K. R., Biochem. Sot. Symp. 16, 94 (1959). 27. RECKNAGEL, R. O., AND LOMB~RDI, B., J. Biol. Chem. 236, 564 (1961). 28. TSUBOI, K. K., YOKOHAMA, H. O., STOWELL, R. E., AND WILSON, M. E., Arch. Biochewc. Biophys. 48. 275 (1958). 29. SANCHEZ, E. Q., SOBER~N, G., PALACIOS, O., LEE, E., AND KURI, M., J. Biol. Chem. 236, 1607 (1961). 30. GREENBAUM, A. L., GREENWOOD, F. C., AND HARKNESS, R. D., J. Physiol. 126, 521

(1954). 31. ALLARD, C., DE LAMIRANDE, G., AND CANTERO, A., Cancer Res. 12, 580 (1952). 32. NOVIKOFF, A. B., AND POTTER, V. R., J. Biol. Chem. 173, 223 (1948). 33. ROSEN, F., ROBERTS, N. R., AND NICHOL, C. A., J. Biol. Chem. 234, 476 (1959).