Visualization of the transport of primary and secondary bile acidsacross liver tissue in rats: in vivo study with ¯uorescent bile acids
Piotr Milkiewicz1,*,², Charles O. Mills1,², Stefan G. Hubscher2, Rene Cardenas3, Teresa Cardenas1,Ann Williams1, Elwyn Elias1
1Liver and Hepatobiliary Unit, Liver Research Laboratories, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK2Department of Pathology, University Hospital Birmingham, UK
3Faculty of Sciences, National University of Mexico, Mexico
Background/Aims: Lysyl ¯uorescein conjugated bile acid analogues (LFCBAA) closely parallel their natural counter-parts. To assess LFCBAA as a tool for the visualization of bile acid transport within liver tissue.
Methods: Wistar rats were administered physiological concentrations of the primary bile acid analogue cholyllysyl¯uoroscein (CLF) and of the secondary bile acid analogue lithocholyllysyl ¯uorescein (LLF) and serial liver biopsieswere taken at ®xed intervals. Both compounds were also injected retrogradely into the biliary tree. Frozen sectionswere examined by ¯uorescence microscopy.
Results: Both CLF and LLF were rapidly taken up from sinusoidal blood but differed signi®cantly in their hepatichandling. CLF was rapidly transported into bile, whereas LLF transport was slower and produced signi®cantly morebile duct ¯uorescence. LLF clearance showed a lobular gradient with last remaining bile acid being con®ned largely tozone 3. Both compounds were avidly taken up by cholangiocytes after injection intravenously or retrogradely into thebiliary tree.
Conclusions: Visualization of LFCBAA by ¯uorescence microscopy may yield further information regarding hepa-tobiliary bile acid localization during studies of physiological and pathological mechanisms involved in transport of bileacids. The presence of both compounds within cholangiocytes strongly suggests that they may undergo a degree ofchole-hepatic recirculation.q 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.
Keywords: Fluorescent bile acids; Cholangiocytes; Chole-hepatic circulation
1. Introduction
Cholyl lysyl ¯uorescein (CLF) and lithocholyl lysyl ¯uor-
escein (LLF) belong to a group of lysyl ¯uorescein conju-
gated bile acid analogues (LFCBAA) which express several
physico-chemical and biological properties similar to
natural bile acids, including rapid secretion into bile [1±
4]. LFCBAA have been used in several studies investigating
the physiology and pathophysiology of bile acids transport
both in in vivo and in vitro [5±9]. The presence of ¯uores-
cein in the LFCBAA molecule should permit the micro-
scopic visualization of the passage of these compounds
within liver tissue and therefore provide new data on
patterns involved in lobular and cellular transport of bile
acids. The aim of the study was to establish a histological
method for following the movement of bile acids within rat
liver tissue using LFCBAA.
2. Materials and methods
2.1. Materials
Cholyl-lysyl-¯uorescein (CLF) and lithocholyl-lysyl-¯uorescein (LLF)
were synthesized according to the method of Mills et al. [1,3]. The synthetic
procedure gave a high yield of both compounds, which appeared as a single
spot after high-performance thin layer chromatography. Fig. 1 shows the
Journal of Hepatology 34 (2001) 4±10
0168-8278/01/$20.00 q 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.
PII: S0168-8278(00)00076-3
www.elsevier.com/locate/jhep
Received 6 October 1999; received in revised form 16 June 2000; accepted
19 June 2000
* Corresponding author. Tel.: 144-0121-472-1311 ext 3974; fax: 144-
0121-627-2497.
E-mail address: [email protected] (P. Milkiewicz).² These authors contributed equally to this work.
chemical structures of CLF and LLF. Pentobarbital sodium was obtained
from May and Baker, Dagenham, UK, Dormitor from Smith±Kline±Beec-
ham, UK and Vetalar was obtained from Upjohn±Pharmacia, UK.
2.2. Animals
All experimental protocols were approved according to the Animals
Scienti®c Procedures Act 1986. Male Wistar rats (230±240 g body weight)
were allowed free access to standard laboratory diet (41B maintenance
diet, Pilsbury, Birmingham, UK) and tap water ad libitum. Anaesthaesia
was achieved using intraperitoneal pentobarbital or Dormitor and Vetalar.
After laparotomy, the common bile duct was cannulated in the upper half
with portex polythene tubing (20 cm long ID� 0.29 mm, OD� 61 mm.
AR Horwell, London, UK) and bile ¯ow established. The body tempera-
ture of animals was monitored by rectal probe and maintained at
37.5 ^ 0.58C by constant temperature regulator. CLF and LLF in the
dose of 2 mmol were administered via the right jugular vein into 3±4
rats in each group. The bile was collected into preweighted tubes in 10
min intervals. Liver tissue samples, weighing 0.076 ^ 0.012 g were taken
before (control) and 1, 3, 5, 7, 10, 30, 45, 60 min after injection in CLF
and LLF group. In order to establish the effect of sequential tissue
sampling on bile ¯ow and total bile salt concentration in bile, liver biop-
sies were not performed in control rats.
2.3. Retrograde injection of CLF and LLF into the biliary
tree
CLF and LLF in the dose of 2 mmol in 40 ml of physiological saline were
injected into the bile duct via portex polythene tube using Hamilton syringe
and subsequently washed with physiological saline according to the method
of Iqbal et al. [10]. Liver tissue samples were obtained at 1, 3, 5, 8, 10 and
15 min after injection of LFCBAA.
2.4. Processing of biopsies
Tissue samples were transferred (within seconds) into liquid nitrogen and
then stored at 2708C. Frozen sections 6 mm thick were cut on a Leitz
cryostat (Leitz Model Kryostat 1720) at 2208C. Sections were then trans-
ferred to a glass slide and allowed to dry in the dark at room temperature for
1 h in order to allow ice crystals to melt. This procedure did not cause
redistribution of LFCBAA. Specimens were then analyzed under a ¯uor-
escence microscope (Axioscop, Zeiss, Germany). Semiquantitative analy-
sis of the distribution of CLF and LLF in specimens was performed by two
investigators (S.G.H.and P.M.). Representative pictures were documented
as color slides on Kodak Ektachrome 160T ®lm.
2.5. Processing of bile
Bile was collected in pre-weighed tubes and bile volume was determined
gravimetrically (with the assumption that 1 g is equivalent to 1 ml of bile).
Measurement of CLF and LLF in bile was performed as previously
described [1,11]. Total bile salt concentration was analyzed enzymatically
with commercially available 3 alpha dehydrogenase kit (Sigma Diagnos-
tics, UK).
3. Results
Bile ¯ow, biliary output of total bile salts and biliary
excretion of CLF and LLF are presented in Figs. 2±4.
Thin layer chromatography (TLC) showed that both
LFCBAA were excreted into the bile in an intact form as
already demonstrated by C.O.M for CLF [1] and LLF (C.O.
Mills, unpublished data). Biliary drainage and sequential
biopsies did not have any signi®cant effect on bile ¯ow
and biliary output of total bile salts during ®rst 90 min of
the experiment. However, both parameters started to dimin-
ish thereafter and the decrease reached statistical signi®-
cance at 100±110 min of the experiment. For these
reasons the study time was limited to 90 min (30 min for
establishing the bile ¯ow and 60 min after injection of CLF
and LLF).
Histological ®ndings are summarized in Table 1.
P. Milkiewicz et al. / Journal of Hepatology 34 (2001) 4±10 5
Fig. 1. Chemical structures of cholyl lysyl¯uorescein (CLF) and litho-
cholyl lysyl¯uorescein (LLF).
Fig. 2. Bile ¯ow in CLF group, LLF group and controls. Biliary drai-
nage and biopsies did not in¯uence the bile ¯ow during ®rst 90 min of
the experiment, but this parameter started to decrease thereafter
reaching statistical signi®cance at 100±110 min. For these reasons the
study time was limited to 90 min (30 min for establishing the bile ¯ow
and 60 min after injection of CLF and LLF). Time 0 represents the time
point at which LFCBAA were injected. Data presented as mean ^ SD,
n � 3±4. W, CLF; A, LLF; X, control.
3.1. Controls
No ¯uorescence was seen in the control sections obtained
before injection of CLF or LLF (Fig 5A).
3.2. CLF
Within 1 min following intravenous administration, CLF
was present in the lumen of bilary canaliculi, mainly in
acinar zones 2 and 3 (Table 1). Fluorescence was also
observed in bile duct epithelium at this time. Between 3
and 10 min CLF was present in canaliculi in a panacinar
distribution (Fig. 5B,C). Strong bile duct ¯uorescence was
also noted during this period (Fig. 5C). Thereafter the ¯uor-
escence in the canaliculi and bile duct steadily diminished.
No de®nite basolateral membrane or cytoplasmic hepato-
cyte ¯uorescence was observed at any time point during
the study (Fig. 5 A±D).
3.3. LLF
At 1 min following injection, LLF was present in hepa-
tocytes, in a predominantly canalicular distribution (Fig.
6A). Weak cytoplasmic ¯uorescence was observed at this
stage (Fig. 6A). The canalicular pattern of ¯uorescence
persisted throughout the study period (Fig. 6A±D). At 3
and 7 min cytoplasmic ¯uorescence was increased in inten-
sity and persisted at this level throughout the study period
(Fig. 6 B±D). In the later phases of the experiment hepato-
cellular ¯uorescence tended to be most marked in acinar
zone 3 (Fig. 6B and D).
Bile duct ¯uorescence was ®rst seen at 3 min following
the injection of LLF and persisted throughout the study
period. Intense bile duct ¯uorescence was observed between
10 and 30 min following injection (Fig. 6C±D).
One minute biopsies after retrograde injection of
LFCBAA showed that both ¯uorescent bile acids were
taken by cholangiocytes of bile ducts (Fig. 7A). A canali-
cular pattern of ¯uorescence was also seen at this time and
was present at each time point of the experiment (Fig. 7A±
C). Cytoplasmic ¯uorescence was initially weak at 1 min
but increased in intensity thereafter (Fig. 7B), with focal
¯uorescence of basolateral membranes also seen (Fig.
7C). The distribution of CLF and LLF did not differ during
the time of the study.
4. Discussion
Preliminary experiments by Buscher et al. [12], in which
bile acids conjugated with N-[7-(4 nitrobenzo-2-oxa-1,3-
diazol)] in the 3-beta position of the steroid ring were
P. Milkiewicz et al. / Journal of Hepatology 34 (2001) 4±106
Fig. 3. Biliary output of total bile salts in CLF group, LLF group and
controls. The procedure did not exert any signi®cant effect on this
parameter within 60 min. following injection of LFCBAA. Time 0
represents the time point at which LFCBAA were injected. Data
presented as mean ^ SD of biliary bile salt output before injection of
LFCBAA, measured at 10 min time intervals and normalized to 100%.
n � 3±4. W, CLF; A, LLF; X, control.
Fig. 4. Biliary excretion of CLF and LLF. Data presented as mean ^
SD, n � 3±4. W, CLF; A, LLF.
Table 1
Histological ®ndings in experiments with (A)CLF and (B) LLFa
(A) CLF
Time (min) Cytoplasmic Canaliculi Bile duct
0 2 2 2
1 2 11 111
3 2 111 111
5 2 111 111
7 2 111 111
10 2 111 111
30 2 2 1
45 2 1 1
60 2 1 1
(B) LLF
0 2 2 2
1 1 11 1
3 11 11 11
5 11 11 11
7 11 11 11
10 11 111 111
30 11 111 111
45 11 1 111
60 11 1 111
a The transport of LLF was clearly delayed with prolonged cytoplasmic
hepatocyte ¯uorescence. Both compounds were avidly taken by cholangio-
cytes, with LLF retaining signi®cantly longer within bile duct cells. Fluor-
escence intensity was scored semiquantitatively by two of the investigators
(S.G.H. and P.M.) as: 2, none; 1, mild; 11, moderate; 111, strong.
used, showed that ¯uorescent bile acids can be visualized in
the liver and intestine. We have synthesized LFCBAA
which are excreted into the bile in an intact form and in
which the 3-alpha hydroxy steroid group is intact and the
¯uorescein is attached to the side chain via a lysine bridge
[1,3]. As we recently demonstrated in the study on mutant
TR-/GY rats, the presence of an intact 3-alpha hydroxy
group determines whether ¯uorescent bile acids are recog-
nized by the bile salt export pump (BSEP), the transporter
involved in canalicular secretion of bile salts [11]. On the
contrary, conjugation of ¯uorescent bile acids in position 3
of the steroid ring with other moieties including glucuronide
or sulfate results in them becoming substrates for another
transporter called multidrug resistant protein 2 (mrp2),
which is non-speci®c and participates in canalicular secre-
tion of several different organic anions [11,13,14]. We have
previously demonstrated that the biliary clearance rates of
these LFCBAA are indistinguishable from those of their
naturally occurring analogues (14C-cholylglycine and 14C±
lithocholylglycine) and when sulfated in the 3-position
sulfated lithocholyl-lysyl ¯uorescein, like sulfated litho-
cholic acid becomes a mrp2 substrate [1,3,11].
The aim of this study was to establish a method for the
study of the time dependent distribution of ¯uorescent bile
acid derivatives of primary and secondary bile acids. We
used bile ®stula rats not only to monitor bile ¯ow, total
biliary bile salt output and accumulation of LFCBAA but
also to prevent their enterohepatic circulation, the phenom-
enon which has been recently observed by us (C.O. Mills
and T. Cardenas, November 1999, unpublished data). We
used CLF and LLF which are ¯uorescent analogues of
glycocholate and glycolithocholate respectively and have
been shown to be excreted into the bile in an intact form.
CLF and LLF were studied in order to demonstrate the
applicability of the technique to bile acid localization
within the liver during physiological states of bile secre-
tion. Our experiments con®rm that transport of bile acids
can be visualized at a cellular level within hepatocytes and
cholangiocytes by taking liver samples at different time
intervals following injection into the jugular vein of
LFCBAA.
Dramatic accumulation of CLF within canalicular
lumena within 1 min of its intravenous injection is in
marked contrast to previous studies with FITC-glycocholic
acid, where a 3±5 min delay was observed [15]. The rapidity
with which CLF is concentrated within the canalicular
lumen, together with the virtually complete absence of
hepatocellular ¯uorescence at any time following its intra-
venous administration, strongly support the notion that
hepatocellular transport of the conjugated primary bile
P. Milkiewicz et al. / Journal of Hepatology 34 (2001) 4±10 7
Fig. 5. Representative biopsies showing the transport of CLF within liver tissue. Liver tissue samples were taken before (control) and 1, 3, 5, 7, 10, 30,
45, 60 min after injection in CLF. They were then processed as described in Section 2. (A) Control (original magni®cation £400); (B) 3 min (original
magni®cation £100); (C) 5 min (original magni®cation £300); (D) 7 min (original magni®cation £200).
acids involves no hepatic sequestration. Uptake of CLF and
LLF by biliary epithelium after both intrajugular and retro-
grade bile duct injection is consistent with the ability of the
ileal bile acid transporter, known to occur on cholangio-
cytes, to transport these ¯uorescent bile acids. Recent
work has demonstrated expression of the ileal bile acid
transporter (IBAT) on biliary epithelial cells [16]. Since it
was recently reported that another ¯uorescent bile acid
analogue CGamF was not a substrate for this transporter
[17] LFCBAA appear to comprise a unique group of analo-
gues of primary and secondary bile acids which may parti-
cipate in a physiological cholehepatic circulation.
LLF in a similar physiological and apparently non-chole-
static concentration, showed a different pattern of distribu-
tion to that seen with CLF. It showed the characteristic,
prolonged cytoplasmic ¯uorescence and retarded canalicu-
lar secretion.
LLF, although injected in a physiological, non-cholestatic
dose showed characteristic retention and a lobular gradient,
with late retention being most marked in zone 3 hepatocytes
and least in zone 1. Interestingly, scanning electron micro-
scopy images of the liver in lithocholate cholestasis showed
distortion and dilatation of canaliculi, maximal in zone 3
[18]. These observations are compatible with the hypothesis
that zone 3 of the liver lobule is most susceptible to choles-
tasis caused by secondary bile acids, at least in part because
of the relatively diminished protective effect of primary bile
acids whose transport is occurring mostly via zone 1. Coin-
fusion of taurocholic acid has been shown to protect against
lithocholate-induced cholestasis further supporting the role
of primary bile acid ¯uxes in protection of the liver against
the hepatotoxic potential of more hydrophobic secondary
bile acids.
In this paper we describe a simple method permitting
visualization of bile acid transport across liver tissue in
the intact rat. This method can be further developed in
order to address speci®c issues related to the transport of
bile acids in physiological and pathological conditions.
Given that the physicochemical properties of LFCBAA
and their biological effects, such as in the ability of LLF
to induce cholestasis [3], are closely similar to those of
their natural counterparts it is reasonable to assume that
the distribution of the ¯uorescent bile acid will, in most
instances, be representative of the naturally occurring but
invisible moiety. We have also recently shown that CLF
clearance has the potential to be a useful test of liver func-
P. Milkiewicz et al. / Journal of Hepatology 34 (2001) 4±108
Fig. 6. Representative biopsies showing the transport of lithocholyl lysyl¯uorescein (LLF) within liver tissue. (A) 1 min (original magni®cation £400);
(B) 3 min (original magni®cation £200); (C) 10 min (original magni®cation £300); (D) 45 min (original magni®cation £300).
tion in man [19,20]. It may be speculated that combining
these techniques may in the future allow precise localiza-
tion of bile acids in human liver specimens taken during
various perturbations of bile formation and secretion.
Acknowledgements
The authors are indebted to Mrs Gill Muirhead for her
excellent technical assistance.
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