Clinical Science and Molecular Medicine (1977) 52,585-590.
Enzyme activities of cells of different types isolated from
livers of normal and cholestatic rats
A. M . WOOTTON, G. NEALE A N D D. W . MOSS
Departments of Chemical Pathology and Medicine, Royal Postgraduate Medical School, London
(Received 10 September 1976; accepted 27 January 1977)
SY
1. The distribution of three membranebound enzymes, alkaline phosphatase, 5’nucleotidase and y-glutamyl transferase, has
been examined in rat liver fractionated into
parenchymal cells, a Kupffer cell fraction and a
biliary tract fraction by perfusion with collagenase solution.
2. In control rat livers the highest enzyme
concentrations were found in Kupffer cell
preparations. The fraction enriched with biliary
tract cells showed concentrations two- to fourfold those in isolated parenchymal cells.
3. At 24 h after ligation of the bile duct there
was a fourfold increase in the alkaline phosphatase activity in parenchymal cells and a
2.5-fold increase of the same enzyme in the
biliary tract fraction. The concentration of
alkaline phosphatase in Kupffer cells remained
unchanged.
4. 5’-Nucleotidaseand y-glutamyl transferase
concentrations tended to decrease after bileduct ligation for 24 h, the most marked changes
being found in Kupffer cells.
5. After bileduct ligation for 7 days the
overall activities of hepatic alkaline phosphataSe and y-glutamyl transferase increased fourfold and there was a small rise in 5‘-nucleotidase. For alkaline phosphatase and y-glutamyl
transferase the changes were found primarily
in parenchymal cells and in the biliary tract
fraction. There was a small rise in the activity
of 5‘-nucleotidase in parenchymal cells and a
fall in activity in Kupffer cells.
Correspondence: Dr D. W. Moss, Department of
Chemical Pathology, Royal Postgraduate Medical
School, Du Cane Road, London W12 OHS.
6. These data show marked differences in
the distribution and behaviour of three enzymes
which are often used as alternatives in the
assessment of patients with liver disease.
Key words: alkaline phosphatase, cholestasis,
5’-nucleotidase, y-glutamyl transferase, liver.
Iniroductioo
Changes in the activities of several enzymes in
serum are of established value in the investigation of hepatobiliary disease. Since the liver
consists of several morphologically and functionally distinct cellular components, it is to be
expected that the extent to which the activities
of individual enzymes will be affected in disease
will depend on the degree of involvement of
their respective cells of origin. For example,
empirical observations have established leakage
from parenchymal cells as the origin of the
raised serum aminotransferase activities in
infective or toxic hepatitis. Elevated serum
activities of alkaline phosphatase (EC 3.1.3.1),
5’-nucleotidase (EC 3.1.3.5) and y-glutamyl
transferase (EC 2.3.2.2) are particularly associated with cholestasis. Originally it was thought
that the elevated serum alkaline phosphatase
was due to a failure of excretion of the enzyme
by the liver but more recent studies of bileduct-ligated animals have shown that induction
of the enzyme takes place within the liver
(Polin, Spellberg, Teitelman & Okumura,
1962; Sebesta, Bradshaw & Prockop, 1964;
Kaplan & Righetti, 1970). However, the site of
the increased enzyme activity has not been
determined. Some or all of the increased alka585
586
A . M . Wootton, G . Neale and D. W. Moss
line phosphatase activity of serum may represent
leakage of this newly synthesized enzyme.
The activities of 5’-nucleotidase and yglutamyl transferase increase slowly in the liver
of rats with ligated bile ducts and more rapidly
in serum, in contrast to the rapid rise in the
activity of alkaline phosphatase in the liver
(Kryszewski, Neale, Whitfield & Moss, 1973).
However, the relatively small changes in the
activities of these enzymes in whole liver could
mask much greater changes within individual
cells, such as those of the biliary tract.
We now report the results of investigations
in which the livers of rats with occluded bile
ducts and of normal control animals have been
separated into morphologically distinct fractions by perfusion of the organ with collagenase
solution in situ. It has been possible to study
isolated parenchymal cells, a Kupffer cell
fraction and a fraction enriched with biliary
tract tissue. The activities of alkaline phosphatase, 5’-nucleotidase and y-glutamyl transferase
have been determined in fractions from the
livers of normal rats and rats in which the bile
ducts had been ligated 1 or 7 days before
perfusion.
Materials and methods
Male Spragu+Dawley rats were anaesthetized
with pentobarbitone (50 mglkg body weight)
and the livers were perfused in situ with collagenase solution [type I, Sigma London Chemical Co.; 0.033% (wlv) in Krebs-Henseleit
bicarbonate buffer solution] by the method of
Berry & Friend (1969). Silicone-treated glassware was used throughout but hyaluronidase
was not used. Some rats received no previous
treatment; four had undergone a sham operation 24 h before perfusion, and the bile ducts of
others had been sectioned between double
ligatures 24 h or 7 days before perfusion.
Completeness of obstruction was verified at
the subsequent perfusion operation. The perfusion technique was modified in the rats which
had been operated upon 7 days previously, as
adhesions formed between the liver and the
duodenum, thus preventing cannulation of the
portal vein. Reverse perfusion was therefore
used, with perfusate entering via the right atrium
and inferior vena cava, and leaving by the
severed portal vein, perfusate then being
pumped back to the reservoir.
Approximately 1 g of tissue was removed
intact from each perfused liver as a control
sample. Parenchymal cells and a residual
biliary tract fraction were prepared from the
remaining lobes as previously described (Wootton, Neale & Moss, 1975). The isolated cells
were collected in silicone-treated glassware in
order to prevent adherence of Kupffer cells to
the glass surfaces. After centrifugation at 50 g
for 1 min to sediment most of the parenchymal
cells, the supernatant was treated with protease
[type VI, Sigma London Chemical Co.; 0.25%
(w/v)] and shaken for 1 h at 37°C to digest
unsedimented parenchymal cells. The remaining
Kupffer cells were recovered by centrifugation
at 250 g for 5 min. Erythrocytes which had
sedimented with the Kupffer cells were removed
by osmotic lysis. The cells were then centrifuged
again and washed.
On average 6 g of fractionated liver lobes
yielded the following average weights of the cell
fractions: parenchymal cells, 2.5 g; Kupffer
cells, 0.1 g; residual biliary tract fraction, 0.5 g.
The recovery of cells was estimated to be about
45% for the parenchymal fraction but only
some 15-30% for Kupffer cells, assuming that
these two cell types constitute %95% and
5-10% of the weight of liver respectively
(Lentz & Di Luzio, 1971).
Alkaline phosphatase activity was extracted
from the perfused unfractionated lobe and
from each cellular fraction of the liver with
butan-1-ollwater (1 :2, v/v) and was measured
by the method of Hausamen, Helger, Rick &
Gross (1967) at 30°C. 5’-Nucleotidase and
y-glutamyl transferase were extracted with
sodium deoxycholate solution (1 %, wlv, in 1%,
w/v, NaHC03). The former was determined
at 37°C by the method of Campbell (1962) and
the latter at 25°C by the method of Szasz
(1969). These extraction procedures had been
shown previously to give maximal and reproducible extraction of enzymes (Kryszewski
et al., 1973). The protein content of each extract
was measured by the method of Lowry, Rosebrough, Farr & Randall (1951).
Activities of the enzymes in the perfusate
were low, indicating that not more than 5% of
the total enzyme was lost during perfusion. This
was confirmed by the close agreement between
all three enzyme activities of perfused but unfractionated liver and those found by Kryszewski
et al. (1973) in whole, unperfused rat liver.
Enzymes in isoluted rut liver cells
587
TABLE
1. Speclfic actiuities of alkallne phosphatase, S'-nucleotidase and y-glutamyl transferase in perfiued
unfractionated liver and in the separate fractions for control and cholestatic rats
Results are given as mean v a l u e s f s e ~with the number of experiments in parentheses. P is the probability
that the difference from the control value is due to chance (unpaired t-test).
Specific activity (i.u./g of protein)
Alkaline phosphatase
Whole liver
Parenchymal cells
Kupffer cells
Biliary tract
S'-Nucleotidase
Whole liver
Parenchymal cells
Kupffer cells
Biliary tract
y-Glutamyl transferase
Whole liver
Parenchymal cells
Kupffer cells
Biliary tract
Control
1 day after bile duct
ligation
7 days after bile duct
ligation
101+20 (11)
71*10(11)
467+ 166 (5)
198+27 (11)
394+88 (lI)P<O.Ol
298+30 (11)P<O~001
422*80 (5) P = 0.81
493+72 (11) P< 0.001
432+ 66 (10) P< 0.001
200+ 63 (10)P< 0-05
696+ 142 (5) P = 0.33
1109+ 143 (10)P< 0.001
39+ 3 (1 1)
38+6 (11)
441 f 128 (5)
119+15 (11)
36+ 5 (10) P = 0.60
34+ 8 (10)P = 0.70
2l4+ 72 (5) P = 0.16
125f21 (10)P = 080
48+ 3 (10)P< 0.05
SO+ 3 (10)P = 0.12
241 102 (5) P = 0.26
98+ 14 (10)P = 0.33
1.1+_0*2
(11)
0*4+0-1(11)
23*3+6.9 (5)
12-4f3.0 (I I )
0.9+_0-2
(10)P = 0.64
0 *5 + 01 (10)P = 0.39
3.2+ 1.2 (5) P< 0.05
7*4+1.5 (10)P = 0.17
45k0.6 (10) P< 0001
1.7+03 (10) P< 0.001
l7.6+ 5.6 ( 5 ) P = 0.54
21*4+27(10)P< 0.05
Statistical comparisons were made by unpaired t-test.
Samples of the different fractions from each
preparation were examined by light-microscopy
to check the extent of crosstontamination
with other types of cell. The cells in the parenchymal cell preparations were uniform in size,
and most cells appeared intact and were able to
exclude Trypan Blue. The cells composing the
Kupffer cell fractions were much smaller than
the parenchymal cells, which formed less than
1 % of this fraction. Approximately 40% of the
cells in this fraction were phagocytic, for they
contained carbon particles after perfusion of
the liver of a rat previously injected with India
ink. A film of Kupffer cells, stained for yglutamyl transferase activity (Rutenberg, Kim,
Fischbein, Hanker, Wasserkrug & Seligman,
1969),showed that this enzyme activity was not
uniformly distributed amongst the cells and did
not coincide completely with phagocytic activity. Stained sections from biliary tract preparations showed these fractions to be enriched with
bile ducts, and that connective tissue and some
parenchymal cells were also present. The appearance of the several fractions was not
+
markedly different whether obtained from normal or cholestatic rats.
The specific activities (i.u./g of protein) of
alkaline phosphatase, y-glutamyl transferase
and 5'-nucleotidase in perfused whole liver
from normal rats and in the different fractions
(Table l), were not significantly different in
cells from rats which had and those which had
not undergone a sham operation. For each
enzyme, the specific activities were greater in
the Kupffer cell and biliary tract fractions
than in parenchymal cells or whole liver. The
differences between the biliary tract and parenchymal cell fractions were statistically highly
significant (P< 0.001).The level of significance
was lower (P< 0.05) for the parenchymal/
Kupffer cell comparison, because of the smaller
number of Kupffer cell fractions obtained.
The specific activity of y-glutamyl transferase
in whole liver is low (Ideo, Morganti & Dioguardi, 1972) and the concentration of this
enzyme in parenchymal cells is also particularly
low.
When the different numbers and relative
sizes of the various types of cell which together
constitute the whole normal liver are taken into
account we can estimate the contribution of
each fraction to the total enzyme activity of the
organ. These calculations assume that the aver-
588
A . M . Wootton, G . Neale and D. W . Moss
age protein content of 1.11 g in the liver of a
200 g rat is distributed uniformly throughout
the constituent cells, so that 0.97 g is contributed by parenchymal cells, 0.06 by Kupffer
cells and 0.08 g by biliary tract cells. These
figures are based upon the estimated relative
weights of different cells given by Lentz &
Di Luzio (1971), and the average weight of the
biliary tract residue. The specific activities of
the enzymes in the cellular extracts are assumed
to be representative of the specific activities
in the whole cells.
These calculations show that parenchymal
cells contribute at least half of the total alkaline
phosphatase and 5’-nucleotidase activities of the
whole liver of control animals, although these
cells contain lower activities of the enzymes
than are found in non-parenchymal cells. However, the greater mass of parenchymal cells is
not sufficient to offset their extremely low
activity of y-glutamyl transferase, so that nearly
90% of the total activity of this enzyme in
whole liver is present in non-parenchymal cells.
After experimental ligation of the bile duct
there were changes in the specific activities of
the three enzymes in the perfused, unfractionated liver and in each of its fractions (Table 1).
One day after bile-duct ligation there was a
statistically significant fourfold elevation of
specific activity of alkaline phosphatase in both
whole liver (P< 0.01) and parenchymal cells
(P< 0.001). A smaller significant rise (P< 0.001)
also occurred in the biliary tract fraction, but
the contribution of this rise to that in wholeliver activity was less important, as this fraction
forms a smaller proportion of the mass of the
whole organ than do the parenchymal cells.
Seven days after bile-duct occlusion the specific
activity of alkaline phosphatase was still
greater than normal in whole liver and parenchymal cells, but with some evidence of a
decline in parenchymal cell activity. The
specific activity of the biliary tract fraction,
however, showed a further increase (P =
0.001). The slight increase in Kupffer cell
activity at 7 days was not significantly different
(P> 0.3) from that in this fraction from either
normal rats or rats with bile ducts obstructed
for 1 day.
In contrast to the changes in alkaline phosphatase activity, there was no significant increase in the specific activities of either 5’nucleotidase or y-glutamyl transferase in any
fraction 1 day after ligation of the bile duct
(P>0.15). Seven days after bile-duct ligation,
both 5’-nucleotidase (P< 0.05) and y-glutamyl
transferase (P< 0.001) showed significantly
increased specific activities in whole liver,
compared with normal liver. The magnitude of
these increases is similar to that found by
Kryszewski et al. (1973). These enhanced
activities appear to be due to increases in parenchymal cell ( P i 0.001) and biliary tract (P<
0.05) activities for y-glutamyl transferase. However, although the activity of 5’-nucleotidase in
parenchymal cells was slightly increased 7 days
after occlusion, the difference is not statistically
significant (P = 0.12).
Discussion
Histochemical studies have shown that the
enzymes alkaline phosphatase, 5’-nucleotidase
and y-glutamyl transferase are not uniformly
distributed amongst the various cellular components of the liver in the rat and other animals.
However, histochemical evidence is not unanimous in assigning these enzymes to particular locations within the liver. In normal rats,
Hagerstrand & NordCn (1972) found alkaline
phosphatase to be located in sinusoidal walls
and in connective tissue around bile ducts and
arteries, with a few preparations showing
canalicular staining. Ronchi & Desmet (1973)
observed faint staining for alkaline phosphatase only in the canaliculi of a narrow periportal
area of normal rat liver. Activity of this enzyme
has also been detected histochemically in
Kupffer cells (Van Wersch, 1963), and Kupffer
cells magnetically separated from rat liver after
iron loading also contained alkaline phosphatase (Davydov & Mayanskaya, 1974). 5‘Nucleotidase has been demonstrated histochemically in sinusoidal and canalicular microvilli (Novikoff & Essner, 1960), and its activity
was greater in a canaliculi-enriched fraction
from rat liver than in the microsomal fraction
(Song, Rubin, Rifkind & Kappas, 1969).
According to Issa, Mullock & Hinton (1976),
5’-nucleotidase is located predominantly in the
plasma membrane of hepatocytes, with some
activity also in other cell types and in periportal
connective tissue. The low activity of y-glutamyl
transferase in normal rat liver was located in
Kupffer cells and bile-duct epithelium by
Rutenburg et al. (1969). Its presence in biliary
Enzymes in isolated rat liver cells
epithelium has been confirmed by Hagerstrand
& Norden (1972) and Ronchi & Desmet (1973).
The morphology of the liver fractions obtained in this study showed a clear separation
of parenchymal and non-parenchymal cells,
and our observation that the specific activities
of all three enzymes are greater in non-parenchymal than in parenchymal cells seems
correspondinglywell founded. y-Glutamyl transferase is almost entirely non-parenchymal but
appreciable concentrations of 5’-nucleotidase
and alkaline phosphatase are also present in
parenchymal cells. The separate identities of the
two non-parenchymal fractions are less well
established, however. The absence of significant differences in specific activities of any
enzyme between the biliary tract and Kupffer
cell fractions which make up the non-parenchymal cells may be due to the fact that, whereas
the parenchymal cell fraction is composed
almost entirely of hepatocytes, both the nonparenchymal fractions are morphologically
more heterogeneous. Kupffer cells do not themselves constitute a homogeneous population.
For example, Bissell, Hammaker & Schmid
(1972) isolated a sinusoidal cell population
from rat liver. Some of the cells ingested heattreated erythrocytes but nearly all the liver
cells ingested colloidal particles. Heterogeneity
in the Kupffer cell fraction prepared in our
study was implied by the non-uniform distribution of y-glutamyl transferase activity, which
was not completely coincident with the phagocytic activity for carbon particles. Furthermore,
the method of preparing Kupffer cells is such
that biliary cells of similar size may be included
in this fraction (Grant & Billing, 1975), so that
these two non-parenchymal cell fractions
probably overlap in cellular composition.
Nevertheless, although separation into pure
non-parenchymal cell types is, at best, only
partially complete, our results suggest that both
Kupffer cells and biliary tract cells of rat liver
containhighconcentrationsof the threeenzymes.
The most striking early effect of bileduct
ligation on the distribution of enzyme activities within the rat liver is the fourfold increase
in specific activity of alkaline phosphatase in
parenchymal cells, which is observed 1 day
after obstruction. Isoenzyme characterization
shows this enzyme to be of an electrophoretically rapidly migrating and moderately heatstable form (Wootton er al., 1975) correspond-
589
ing to the form of the enzyme appearing in the
plasma of cholestatic rats (Kaplan & Righetti,
1970). The increases in both liver and plasma
alkaline phosphatase activity can be inhibited
with cycloheximide (Kaplan & Righetti, 1970;
Kryszewski et al., 1973). Thus it appears
probable that the increased parenchymal cell
activity is the origin of some or all of the immediate change in plasma activity. The increase in specific activity in the biliary tract
fraction is part of a continuing process, which,
after 7 days, amounts to a fivefold increase in
the specific activity of this fraction compared
with the corresponding fraction from normal
liver. Alkaline phosphatase deriving from
biliary tract cells may contribute to the elevated
activity in plasma especially as the duration of
obstruction increases.
The activities of both 5’-nucleotidase and
y-glutamyl transferase in plasma also increase
markedly within 24 h of ligation of the bile
duct in the rat, with a continuing rise for the
latter enzyme (Kryszewski et al., 1973). Although the changes in plasma activities of all
three enzymes are thus similar after obstruction,
there is no rapid change in specific activity of
either y-glutamyl transferase or 5’-nucleotidase
in any fraction of the liver such as occurs in
parenchymal cells for alkaline phosphatase.
These observations therefore show that similar
changes in plasma enzyme activities do not
necessarily imply correspondingly similar alterations within the tissues from which the enzymes
are presumed to originate.
The rapid increase in 5’-nucleotidase and
y-glutamyl transferase activities in plasma after
occlusion of the bile duct may be due to release
of enzyme from cell membranes, especially
from non-parenchymal cells, which show initially high concentrations of these enzymes.
A similar process may also operate for alkaline
phosphatase but, as already discussed, the
increase in parenchymal cell activity of this
enzyme probably contributes a considerable
part of the raised activity in serum in early
obstruction. The three enzymes may also be
differently located within cell membranes and
this may contribute to different patterns of
enzyme release: 5‘-nucleotidase, for example,
is thought to be present on the outer surface
of cells (Trams & Lauter, 1974).
The specific activities of alkaline phosphatase
and y-glutamyl transferase in the biliary tract
590
A . M . Wootton, G.Neale and D. W. Moss
fraction are both increased in more prolonged
biliary obstruction, although the effect is
greater for the former enzyme. The proliferation
of biliary epithelium, which begins 2 days after
obstruction of the bile duct (Vinnik, Kern &
Corley, 1963), would increase the amount of
this enriched source of enzymes that is potentially available for their release into the circulation and so contributes to the continued
elevation of enzyme activities in plasma.
Acknowledgments
We thank Mrs C. Spry and colleagues in the
Nuffield Department of Clinical Medicine,
Oxford, for valuable advice on the perfusion
technique.
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