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: p.milkiewicz@bham.ac.uk (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.

References

[1] Mills CO, Rahman K, Coleman R, Elias E. Cholyl-lysyl¯uorescein:

synthesis, biliaryexcretion in vivo and during single-pass perfusion of

isolated perfused rat liver. Biochim Biophys Acta 1991;1115:151±

156.

[2] Baxter DJ, Rahman K, Bushell AJ, Mills CO, Elias E, Billington D.

Biliary lipid output by isolated perfused rat livers in response to

cholyl-lysyl¯uorescein. Biochim Biophys Acta 1995;1256:374±

380.

[3] Mills CO, Milkiewicz P, Molloy DP, Baxter DJ, Elias E. Synthesis,

physical and biological properties of lithocholyllysyl¯uorescein: a

¯uorescent bile salt analogue with cholestatic properties. Biochim

Biophys Acta 1997;1336:485±496.

[4] Mills CO, Milkiewicz P, Saraswat V, Elias E. Cholyllysyl¯uorescein

and related lysyl ¯uorescein conjugated bile acid analogues. Yale J

Biol Med 1997;70:447±457.

[5] Wilton JC, Matthews GM, Burgoyne RD, Mills CO, Chipman JK,

Coleman R. Fluorescent choleretic and cholestatic bile salts take

different paths across the hepatocyte: transcytosis of glycolithocho-

late leads to an extensive redistribution of annexin II. J Cell Biol

1994;127:401±410.

[6] Roman ID, Johnson J, Coleman R. S-adenosyl-l-methionine prevents

disruption of canalicular function and pericanalicular cytoskeleton

integrity caused by cyclosporin A in isolated rat hepatocyte couplets.

Hepatology 1996;24:134±140.

[7] El-Seaidy AZ, Mills CO, Elias E, Crawford JM. Lack of evidence for

vesicle traf®cking of ¯uorescent bile salts in rat hepatocyte couplets.

Am J Physiol (Gastrointest Liver Physiol) 1997;272(35):G298±

G309.

[8] Roma MG, Stone V, Shaw R, Coleman R. Vasopressin-induced

disruption of actin cytoskeletal organisation and canalicular function

in isolated rat hepatocyte couplets: possible involvement of protein

kinase C. Hepatology 1998;28:1031±1041.

[9] Milkiewicz P, Mills CO, Roma MG, Choudhury JA, Elias E, Coleman

R. Tauroursodeoxycholate and S-Adenosyl-l-Methionine exert an

additive ameliorating effect on taurolithocholate-induced cholestasis:

a study on isolated hepatocyte couplets. Hepatology 1999;29:471±

476.

[10] Iqbal S, Mills CO, Elias E. Biliary permeability during ethinyl estra-

diol induced cholestasis studied by segmented retrograde intrabiliary

injections in rats. J. Hepatol 1985;1:211±219.

[11] Mills CO, Milkiewicz P, Muller M, Roma MG, Havinga R, Coleman

R, Kuipers V, Jansen PLM, Elias E. Different pathways of canalicular

secretion of ¯uorescent bile acids: a study in isolated hepatocyte

couplets and TR- rats. J Hepatol 1999;31:678±684.

[12] Buscher HP, Gerok W, Kurz G, Schneider S. Visualisation of bile salt

transport with ¯uorescent derivatives. In: Paumgartner G, Stiehl A,

Gerok W, editors. Enteropatic circulation of bile acids and sterol

metabolism, MTP Press Limited, Lancaster, Boston, The Hague,

Dordrecht, 1985, pp. 243±247.

[13] Paulusma CC, Bosma PJ, Zaman GJ, Bakker CT, Otter M, Schef̄ er

GL, et al. Congenital jaundice in rats with a mutation in a multidrug-

resistance-associated protein gene. Science. 1996;271:1126±

1128.

[14] Muller M, Jansen PL. Molecular aspects of hepatobiliary transport.

Am J Physiol 1997;272:G1285±G1303.

[15] Sherman IA, Fisher MM. Hepatic transport of ¯uorescent molecules:

In vitro studies using intravital TV microscopy. Hepatology

1986;6:444±449.

[16] Lazaridis KN, Pham L, Tietz P, Marinelli RA, deGroen PC, Levine S,

Dawson PA, LaRusso NF. Rat cholangiocytes absorb bile acids at

their apical domain via the ileal sodium-dependent bile acid transpor-

ter. J Clin Invest 1997;100:2714±2721.

[17] Holtzinger F, Schteingart CG, Ton-Nu HT, Eming SA, Monte MJ,

Hagey LR, Hofmann AF. Fluorescent bile acid derivatives: relation-

P. Milkiewicz et al. / Journal of Hepatology 34 (2001) 4±10 9

Fig. 7. Biopsies showing the tissue distribution of LFCBAA after retro-

grade injection into biliary tree. (A) CLF1 min (original magni®cation

£200); (B) CLF 3 min (original magni®cation £200); (C) CLF 10 min

(original magni®cation £200).

ship between chemical structure and hepatic and intestinal transport in

the rat. Hepatology 1997;26:1263±1271.

[18] Layden TJ, Schwarz J, Boyer JL. Scanning electron microscopy of rat

liver. Studies of the effect of taurolithocholate and other model of

cholestasis. Gastroenterology 1975;69:724±738.

[19] Milkiewicz P, Baiocchi L, Mills CO, Ahmed M, Khalaf H, Keogh A,

Baker J, Elias E. Plasma clearance of cholyl lysyl ¯uorescein: a pilot

study in humans. J Hepatol 1997;27:1006±1009.

[20] Milkiewicz P, Saksena S, Cardenas T, Mills CO, Elias E. Plasma

elimination of cholyl lysyl ¯uorescein; a pilot study in patients with

liver cirrhosis. Liver 2000;20:330±334.

P. Milkiewicz et al. / Journal of Hepatology 34 (2001) 4±1010