Biliary secretion of trace elements and minerals in the rat

II?

Biliary secretion of trace elements and minerals in the rat Effects of bile flow variation and diurnal rhythms Marjan

Dijkstra. Folkert Kuipers, Egbert P. Smit, Jeanette I. de Vries, Rick Havinga and Roe1 J. Vonk Depnn,“c”r”,ne~,?rr,tl”l”crriry ofmmingm, Gmml”~P”. nlr hwwlandr (Rcccivcd 7Sepmmber 1990)

Effects of bile flow variations on bile secretion of trace elements were systematically evaluated by Proton-Induced X-ray Emission (PIXE) in the unanesthetized rat: (i) longitudinally for a 9 day-period of bile drainage, (ii) continuously foi a period of 24 h. to mclude circadian rhythm and (iii) during exogenous bite salt administration. Potassium and Ca were determined for comparative purposes. In rat bile, six trace elements could always be detected by PIXE (Fe, Zn, Cu, MO, Mn. Br): occasionally some V. Cr. ST and Pb was found. Se could not be detected in bile. Bite-plasma concentration ratios of the elements could arbitrarily be divided into three groups: Fe. Zn and Se < 1; Ca, K, Cu, Mo and Br approx. 1 and Mn >> After interruption of the enterohepatic circulation, bile flow and bile salt cnncentration dropped sharply within 4 h to 36% and 4% of thar m~t~al values, respectively. Output rates of K, Ca, MO, Br, Zn and Fe closely followed the decrease in bile flow; bile xcretion of Mn and Cu. on the other hand, was minimally atfected. A circadian rhythm was observed for all detectable elements; during the night period, secretion rates of Zn. Fe, Ca and K increased maximally by 60-80% and that of Mn. MO, Brand Cu by 30.50%. Bile salt output and bile flow increased maximally by 70% and SO%, respectively, in the same time interval. Infusion (106 pm&h) of tauracholate (TC), a micelte forming bile salt or dehydrocbolate (DHC). a non-micellar i,de salt, caused a maximal increase in bile flow by 67% and93%, respenively. Under these condi-

I.

tions, biliary secretion of K, Ca, MO. Br and Fe were stimulated by both types of bile salts, almost to B similar extent as bile tlow. In contrast. the secretion of Zn. Cu and Mn was not affected or eve? reduced. This study shows i:rzt most detectable endogenous trace elements show a strong bile flow-dependency in their biliary secretion, except Zn. Cu and Mn. ‘Tie ciiminatmn of Cu and Mn via bile, which represents the main route for their removal from the body, IS largely independent of acute changrs in bile flow, but does show a marked circadian rhythm.

In additmn 10 bile salts, lipids, proteins and pigments, bile contains a variety of minerals and trace elements. The biliary pathway is the main route for removal from the body for a number of these elements, e.g., for Cu (1.2). Metals which are predominantly eliminated via other pathways like Zn, Br, MO end Se can also be detected in bile (3). The mechanism(s) of biliary trace element secretion are largely unclear (see Ref. 4 for review). A role of bile pigments, glutathione and bile salts as metal complexing agents has been suggested (5-S). but not yet proven.

The aim of this study was to make an inventory of the effects of bile flow and bile salt variation on bile secretion of endogenous minerals and trace elements. For this purpose, secretion rates were determined during ‘physiological’ variations in bile flow, i.e., during depletion of the bile salt pool (9) and overnight to include the circadian rhythm in bile flow (IO). as well as during infusions of exegenous bile salts. The methodology of Proton Induced Xray Emission (PIXE) (I 1) allows simultaneous determination of all elements with a molecular mass 319 and an

II? ohundance exceeding 0.5 ,tgiml

in a single small sample.

2nd is ther_‘ore

for comparative

ideally aired

Rats with permanent

catheters

studies.

Twelve rats were anesthetized by an intraperitoneal jecnon of sodium pentobarbital

in bile duct, ducdenum monitorin

of

bile secretion rates under normal feeding conditions

and heart were used, to allow continuous

and

sia was maintained during

Body

temperature

with a heating pad (37.5-38

equipped wth Mtdland. Malrrialsand

by injection of small doses of rhe drug

the c‘*periment.

tained

without the interference of anesthetic agents (IO).

m-

(60 mg/kg) and anesthe-

‘0.

was main-

The

a silastic catheter (Silastic,

rats were

Dow Corning,

,MI. t.d. 0.5 mm. o.d. 0.94 mm) in the iuaular

vein and m the common bile d*xt. Infusions wele given at

Methods

a rate of 3 ml/h via the jugular vein by means of an infn-

Animals

sion pump. Saline was administered

Male Wistar rats (a-300

ad

g) were housed in individual

s-itched

over to taurocholate

for a period of 1 h,

(n = 6) or dehydmchol-

plexiglass cages (25 x 25 x 30 cm) on wood shaving at

ate fn = 6). both sodium salts (Calbiochem.

20 “C with a light/dark

CA),

Water

and pellet

(RMH-B.

regime of 12 h light and 12 h dark.

food

were available

Hope farms NV,

Woerden,

ad lib. The

minerals.

including 0.85%

K, and Fe, Mn,

Zn. Co and Se in concentrations

12, 63, 22.4 and 0.24 mglkg. res+vely. equipped

with permanent

catheters

LB lolla,

for 3 h. Infusions were pre-

of 0.5 ml (18;vnoi)

of the infused

bile salt m order to achieve constant bile flow and btlc Bait

The Netherlands)

contained 6.1%

at a rate of 106~moVh

ceded by a pnmmgdose

diet

Ca and 0.65%

secretion rates. The infused bile salt solutions contaiord no significant

of 195,

amountS of trace elements.

Bile was col-

lected in intervals of 30 min.

The rats were

in the bile duct and

scribed in detail elsewhere (9). The metal parts included

Analyses To prevent contamination,

in the original procedure were replaced by teflon to avotd

nulas were washed prior to use with concentrated

contamination

acid and hi-destilled water.

duodenum

and via the jugolar vein into the heart. as de-

of blood and bile samples. The bile duct

and duodenum

catheters

were immediately

with a piece of polyethylene rnterohepatic pry,

circulation

of bde salts. After

the rats had a recovery period of et teat

fore they were used for experiments. ing the experiments, to a polyethylene

connected

tubing which maintained

(EHC)

Total

the sur-

7 days be-

To collect bile dur-

the bile duct catheter was connected

biliary bile sait concentratron

an enzymaoc

fluonmetr#c

gaard and Co.. (Ca),

iron

(Mn),

seleninm

(Fe),

copper (Se),

(Cu),

were determined

washed, pre-weighed

tad by us in a previous paper (11).

test tohrs aid stored zt -20 “C xtil

test i’tbcs and centrifuged stored at-20

zinc

(Zn), (MO)

by

Nyecalcium

manganese and bromine

by the PIXE

merh-

od. The accuracy and detection limits of this method for tows element determination

samp!es were transferred

was determined

Potassium (K).

molybdenum

collector outside the cage. Bile samples were collected in analysis. Rlwd

nitric

assay (Srerognost-Flu,

Oslo. Norway).

(Br) concentrations

cannula. which Icd the bile to a fraction

all vials, teu tubes and an-

‘tore! vi, ;;Zkze

to heparinized

(10 min. SW0 rpm). Plasma was

ing to Gr,ff+h

in bile have described in de-

(GSH+ZGSSG)

was essayed accord-

(12).

“C until dnalysis.

Output rates of bile salts and trace elements were deter-

In six rate the EHC was interrupted

ing the first 6 h bile samples were collected continuously l-h fractions into pre-weighed 9 after intermotion

mined by multiplying

at 1l:oO a.m. Durin

test tubes. Al day 1.3, and

of the EHC.

a 15-min bile ~emole was

were taken to assess baseline plasma values: thereafter.

at noon)

sum test, p values co.05

were re-

Results Usmg ZlxE

Bile was collected continuously

rank

a

sampling.

Day/night rhythm (starting

Significance of differences was assessed by Student’s r-test or Wilcoxon’s

blood samples of 0.15 ml

blood sample was taken on the days oibile

period

af(9).

garded as sigc:ficant.

collected at 11:OO a.m. One day before and immediately

prior to the start of bile drainage,

bile flow and bile concentntlon,

ter correcticln for the dead space of the tubingsystem

from five rats for a 24-h

in 2-h fractions,

after

a bile

(Proton

Induced X-ray Emission) six trace

elements were always detectable in bile of bile-fist:& (Table

1). Occassionally

drainage period of 8 days to establish a new sready-state

(0).

strontium

in bile salt synthesis and bile flow (9).

cromolar

some vanadium

(v).

rats

chromium

(Sr) or lead (Pb) were detected in the mi-

range. The levels of K, an ion that is believed IO

logical situation for bile composition. bile ilow and plasma concentration. The biie to plasma conceoo ation ratios are shown in Table I. The obtained ratios can arbitranly be divided into three classes: the ratio of Fe. Zo and Se is smaller than 1: that of K. Ca, Br. Mo and Cu appron. 1 and that of h!n larger than 1. Se was below the detection level in bile but present in plasma. In contrast, Mo was not detectable in rat plasma but always found in bile. Fig. 1 shows bile flow, bile salt secretion and biliary secretiio of Ca, Cu. Fe and Mo immediately after interruption of the EHC. Within the first 4 h. bile flow and bile oalt excretion declined to, respectively, 36% and 4% of their initial values. wldch reflects exhaustion of the endogenous bile salt pool (9). Biliary GSH excretion decreased

_______--

-_-

Valuer are mczlns t s E.: n = (1. ““less otherwise rla,ed bewee” twactctr. n.d.. “0, dc,ecraiJc

to 35% of its initial

value (2.1 ~cmollh) ovet the same pe-

riod. The bepatic bile salt output rose slightly following

be freely peimeablc, and of Ca can also accurately be measured by PtXE (11); values for these minerals are in-

value at day 9. Biliary

of K and Ca declined strongly

and lapidly

and Zn fell to 55-G%.

i.e.. at 30 min. a blood sample was taken to allow calcula-

flow, whi-h suggests bile flow-dependency

tic? nf bile to plasma concentration ratios. The cooceotra-

tion. In mntrast,

drainage were measxed

aft?: the start of bile

in order to approach the physio-

and

secretion

to 30-35%

their initial value, Mo and Br dropped to 40-45%

cluded for comparative reasoo~. At the midpoint of the first hourly bile sample after interruption of the EHC.

tions in the first fraction lmmedlately

during the

days, doe to increased hepatic synthesis,

reached 12% of its initial

of

and Fe

Secretion of these elements stahi-

lized at these low levels in paralle! to the low rate of bile of their sene-

biliary secretion of Co and Mn was only

slightly changed by acute bite drainage; the decrease in bile flow was compensated for by increased bde concentrations of tlrese elements. Analysis of plasma concentrations, measured during the 9-&y period of bile diversion, showed a 28% decrease of the bromine concentration. Plasma levels of the other trace elements were not significantly affected by bile drainage. As a consequence. bde to plasma ratios of most elements remained unaffected, but that of Cu increased nearly Z-fold. A second physiological manipulation of bile flow was achieved by monitoring the circadian variations. During a 24-h period bile was collected in 2-h fractions. Diurnal variation in biliary secretion existed for all detectable elements, but its amplitude varied for the different elements. Bile salt secretion and bile flow were increasea by 70% and JO%, respectively. at midnight compared to noon. During the night period the secretion rate of Fe, Zn, K and Cs was maximally enhanced by m-80%, while Cu, Mo and Br excretion was elevated by 30-40% of the value measured at noon. Mn was also secreted during the night period at a higher rate than during the day in all animals studied, hut the inter-individual variations were very high for this element (data not shown). The course of the circadian rhythm for bile flow, bile salts, Cu and Fe is shown in Fig. 2. Table 2 shows the conelation coefftcients between bi!e flow and trace element secretion during the first 6 h after bile drainage and during the overnight period of bile collection. Bile salt. K, Ca, Zn, Fe, MO and Br secretion rates highly correlated with bile flow after bile drainage; their correlation coefficients range from r = 0.938 to r = 0.999. Mn and Cu secretion did not carrelate with bile flow; r = 0.197 and I = 0.424. respectively. However. during the overnight period excretion of the latter elements also significantly correlated with bile flow.

The preceding results were obtained during ‘phyriological’ variations of bile flow and bile salt output. In addition, we induced

choleresis

by infusion of taurocholate

(TC) and dehydrocholate (DHC) (106~nt1&). The maximal increase in bile flow was 67% for TC and 93% for DHC. No effects on biliary GSH were observed. as pm v*ously described by others (13.14). The effect of this choleresis on biliary excretion of trace elements IS shown in Fig. 3. During TC infusion, the biliary secretion of Mn decreased by 48%; on the other hand. during DHC infusion Mn secretion was not significantly affected. Cu secretion rate was not affected by TC but stimulated by 55% during DHC infusion. Zn secretion rewzked unchanged during administration of either bile salt. The secretion of K, Ca. Fe, Ma and Br was increased by both bile salts It shwld be noted that the larger bile flow stimulation by DHC did not result in a higher secretion rate of either element, when compared to that induced by TC.

II6

i

I Discussion The liver serves an important function in the maintenance of homeostasisof a number of irace elements. This is achieved by accumulation in the bver and subsequent release into the bloodstream or into the bile (2). The mechanism of bitiary metal secrerion is only Poorly defined. Among the factors which have been Put forward to be of importance is the rate of bite flow (15). However, most studies address bile secretion aRer intravenous injection of metals an&or treatment of animals with microsomal enzyme inducersto stimulate bile flow In the present study. we have systematically analyzed the effects of variations in bile flow on secretion of en_ dogenous metals. In order to get a mure complete wer Ucw, we employed PIXE analysis, a method which allows slmuhaneous determination of all detectable trace elements in a small bile sample. Details of this procedure have been published elsewhere (11). Bile flow was varied under three different experimentat conditions by altering bde sat, output. Bile salt secretion provides the main driving force fcr bite formation (4). After interruption of the EHC in rats, bile flow drops to low levels, due to depletion of the intestinal bile salt pool (9). After a period of

chronic biliary drainage under a rigid lighting schedule, a new steady-statein bile flow and bile salt output is established in which a remarkable circadian rhythm can be observed (10.16). Bile flow and biliary secretion of bile salts and also of related bile constituents such as cholesterol and phospholipids increase during night (10). Feeding behavior, thermogenesis,metabolic processeswd hormone Production (e.g., cortical hormones) also exhibit a clear circadian rhythm in the rat with enhanced activity during the night (17.18). Pharmacological manipulations of bile flow by the bile salts TC and DHC are accompanied by differential physical interactions in bile. TC readily forms micelles: its critical micellar concentration (CMC) has a value of 6 mM in 0.15 M NaCl(l9). TC inducesthe biliary secretion of iipids and bile pigments in rats (4,20,21). In contrast, DHC has a very high CMC value and is highly choleretic but appears to have no effect or lipid and bilirubin secretionin rats (22). Secretion pattern of a number of bilizry trace elements (MO, Br, Fe) closely followed bile flow under all Fondilions examined. As expected, a similar pattern was observed for K and also for Ca, in accordancewith recent studies in dogs, which demonstrated that Ca enters bile primarily by passiveconvectionnnd diffusion (23.24). For

117 all these elements,

with the exception of Fe, the bile to

plasma concentration Wia?

ratio

appuximated

a value of 1.

Ca *ecretion in relation to changes in bile flow has

recently extensively

been studied in dogs (L3.24).

Ca ~8% found to be mainly small d~~mar

;.,_.:u...

flow stimulation,

of canalicular origin

!! I’ 7 ;-o$?~:latc.’ t+i:

Biliay with

?‘::.lp

changes in bde flow. with the exception of DHC-induced choleresis whxh

resulted in a small increase in Cu output

a

bile

Fraction

I” bdiary

Co concentratton.

lism of Cu, conicosteroids

infusion infusion

resulted in a slighrly in spite of the higher

bile flow induced by the first the ma-

mcluding conicosteroids.

estrogens and tzsto~temne

alfrct

the metabo.

me able to increase the biliary

Co secretion (36). The circadian varimon cretion may therefore mid production

The main excretory route of BT. quantitatively

~ecrcooo wu

which may he due to Iar-

\everal hormones

are known be able to bmd Ca (25,. Our results are compathan TC

Cr

ger dlctary supp!y at night or to some kind of hormonal rcgulat~on thyroxin,

tible with these data: DHC

- ‘.‘.

However,

show a clear circadian variation.

(23). Bik: salt micelles as well as monomeric taurochoiaie

lower CB output

-

Changes m bde “nw a’**- :.

the increased canalicular Ca output can

be divided into an osmotic fraction and a mxe!lar

at present not clear. cu Fecretlo” did not respond to acute

in hilixy

Co se-

be cauwxl by increased corticaste-

during @ht

period (17.36).

Interesting-

ly, such a circadian variadon io Co secretion WE not ob-

jor trace element i;. plasma and bile. and also for Mo and

qerved in man m which the peak in bile salt secretion 15

Se appears to be via wine

sh,Red more to the day period / 5).

(3,26).

Nevertheless.

MO were present in bile in concentrations plasma concentrations. in bile. Brand

In contrast.

Br and

equalling their

Se was almost absent

MO showed a strong bile flow dependency

In contrast to Cu. Zn is excr&d quantitative

terms (37,3?7). ne

very poorly via bile in majority

of endogenous

Zn is excreted by the pattcrea~ and via the intestinal resulting

to disposition

of Zn

.*to the stools (39).

wall It has

and might be secreted via a paracellular route by mechanisms simltar to that proposed for Ca secretion (23.24). Elimination of Fe from the body under borneostatic condi-

been shown that bilisry

secre? ?a of Zn is highly depen-

dent upon GSH

Zn sc:rctian

tions

B bde flow dependency after inrcrmptioo

occws mainly

from

gastrointestinal

mucosal cells

with smaller amounts lost via bile, urine and skin (27). Association

of Fe with

been demonstrated cretion night However,

monomeric

bile salts has recently

in vitro (8); the mechanism of its se-

therefore

be panly

similar

to that of Ca.

it has also been shown that during Fc overload

(32.40).

not during bite salt-induced choierais. tr.n

may be related to simultaneous

cretion; the GSH

cho‘ereais induced by TC or DHC,

that under these conditions

other secretory

mecha-

It is well established that Cu homeortasis to a large extent by regularion which is emphasized

i.e.,

is maintained

of its biliary excretion (2):

by the observation

that ligation

of

se-

avalue com-

Mn

concentration

as was also reported by

in rat bile is more than

higher than in plasma (this study and Refs.

nisms are involved in Fe disposition.

but

parable to the decrease in bile flow. but did nof respond to ether authors (13.14).

hk

changes in GSH

of the EHC.

fhe most important

(28). It is parsi-

of the EHC,

This secretory pat-

output rate decreased to 35% of its ini-

tial value after interruption

biliary secretion mediated by hepatic lyaosomes becomes mute of its elimination

apparently showed

During EHC.

4.41

the decrease in bde flow after interruption Mn showed no correlation

wvation

IO-times and 42). of r!te

with bile flow. This

ob-

soppons the importance of the biliary route as

the bile duct leads to an excessive increase m hepatic cop-

excretory pathway for Mn, a process which is apparently

per content in rats (29). The mechanism of biliary Cu se-

carefolly

cretion has only partially

been defined. Hepatocelhdar

sosontes are claimed to be the origin

of biliary

ly-

Co (30).

regulated.

ministration TC

Inhibition

of Mn secretion after ad-

of TC might be due to competition

between

and Mn, probably as a complex, for a hepatobiliary

where it is present as a wmplex

with amino acids or low

transport

molecular peptides. Glutathione

may play a role in the se-

carrier is evidently higher than that of Mn (complex). The

cretion from liver to bile by formation plexes which are subsequently nalicular membrane (27.31). shown

that GSH

of Co-GSH

transparted However,

com-

across the ca-

we have recently

becomes of importance

only after an

exogenous Cu overload. while its presence is not required for transport to GSH.

of endogenour Cu in the rat (32). In addition

biliary Cu has been reported to be bound to a w-

riety of contpmxnts cluding proteins,

of high and low molecular weight, inbile salt micelles.

bile salf monomers,

peptides or amino acids and bile pigments Whether

(X6,33-35).

this binding is of importance for Co secretion 1s

system. In that case, the affinity

finding that DHC

pathways. However,

sible and further

for the

does uot affect Mn secretion would in

this case imply that TC and DHC different

of TC

are secreted into bile via

other explanations

are pos-

studies are warranted.

In this study. we were able to show a strong bile flow dependency for the secretion of Fe, MO and Br, comparable to that observed for Ca and K. This

bile flow dependency

was also found for rbese elements in a recent study from our laboratory

(41). in which UC stuched metal secretion

in GY rats. GY rats express a congenital defect in hepatobiliary

organic anion transport,

and show low bile flow

M. DIJKSTRA

II8 due IO n reductm” The wrrerory

nf the bile acid-independent

pa,h\wy

fraction.

,,

I’( i !, ,,/I/,

hcpatohiliary

seems to be carefully regulated led

mnechonismr. More

via other.

of

thaefore

““I yet idemi-

research is required

The authors thank Lodewijk for preparing

the bgures.

., ^ c’i’Whfrom .,i,:,,,,> ! ,//

,,

rou~c is lorpely indepcndem

bile flow or tx,~ salt secretion. Their secredo”

Ihlx

Acknowledgements

“f Fe, Mo and Br may therefore

IIIW,YC p;~racel,ul;w transport from plasma f” bile as “ITrl,h. ..~..__ w,.,l <. r_ (1’ 1:’ Tko Wl/pn, Tccrptil)” of

MnvuIhe

al.

Cf

Manij”

the Dutch Health

the J.K. de Cock Stichting.

and Bert Tebbes

This ctudy was suppmted Rrrearch

. Falkert

Fellow from the Royal Netherlands

by

I:!

<... _*,,&<,,t; c.:d Kuiperr

is a Research

Academy of Arts and

Sciences.

to unravel

proccr~cr

LR,

20 Engelking Gronwall R. Anwer MS. Effect of dehydrach”liC. cbenodeoiychabc. and taurwbolic aads a” tl.~ ?‘:cre,,o” of bibrubin. Am J Vet Rer ,980: 41: 355-a 2, Gonzalez J. Hid&o F. Lopez MA, Erteller A. 1”flue”ce of bile salts on the endoaenousexcreti”” of bxlepigmcnll. Rev Es11Fisic3 1983.39 69-k 2.2Crawford JM. G&n JL. Heparacyle corransport of taurwhclate h”d bilirubi” glucuranides:role of microtubulcs. Am J PhysIO, 1%.8:215:01*1-31. 23 Cummings SA. “ofma”” AF. pbvrialogiedcterminsntsa‘biliary calciumsecretionin ,he dog. Gartrcaterology ,9&I; 87: 664-73. 24 Rege RV. Dawes I.& faoorc EW. Biliary EidSi”r” secrelio” in the dog occurs p¶im*rit~ by passiveCO”WCLiD”and diffusion and islinkedtabile Uow. J LabClin Med ,990,llS: 593-662 2s Moore EW. Cetic L., Ostrmv JD. I”tEracliU”6 bEtwee” lonlred ca,ciu”l an* JOdiUrntaYmCM.IS: ue ra,tr are impomm ,aclDrp for preventi”” of calcium-mntaining gallstones. Gastroentw “logy ,982; 83: ,079~89. 26 Gregus z. luaasren CD. Disposition of metatS8” law a cornpar. ative study of feeat urinary and biliary ewedo” and tissuedir6 bution of eighteen metals. Toxicol Appl Pharmacol ,986: 85: ?A-,*

29 Owe” CA. Copper metabolirm afrer bitiary fislula. obstruction. or rtlam operation in rats Mayo Clinic Proc ,975; 50: 412-5. 30 Gross. 18. Myers BM, Kor, U. Kuntz SM. LaRusro NF. Biliary copperexcretionby hepamcytelysosomesI” the rat major ercretory pamway in expertme.ta, copper oucrlusd. ? Cl,0 l”YCIf 1989; gj: 30-9. 31 Alexanderl. AaselhJ. Bitiaryexcretionofcopperandunc the rat as >““uc”ccd by dierhylmaleate, ~bnile and dictbyldirbi”. carbamate.Biochem Pharmam, ,980; 29: 212943. 32 Houwe” R, Dijkstra M. Kuipers F, Smit EP, Havinga R. Vonk RJ Two pathways for biliary copper cxcretm” in the rat. The rote ofglutarhione BioEhemPhrrmrc., ,990; 39: 1039-M. 33 &comer ,. lnvolvemc”, ofmetat,othiooei” in the bepadc metabUbErnof copper. J NY” 1987; 117: 19-29. 34 aouan ,I_, *cucr D,. SI”d,SSon IhS oa.Iux and eXE,StiO”0,fbll. iarympper in ma”. Cli” Sci ,97n3;44 9-15. 35 Lewis KO. The “WTL‘ o: ih copper complexesI” bite and their retationrhip 10 the aborp”o” and excrcfm” of copper in “Irma, subjectsand in Wilson’sdisease.Gut 197373: 14: 22t-32 36 Cousins RI. Absorption. tramp”rt. and hepalic metabolism of copper and zinc: $xcial rcrerc”ce IO me,a,,oth,o”ein and ccruk,. plasmi”. Phyriol rlrr 1985:65.*x-309.

in

40 Alexander 1. Aawih. J. Rcfswk T. Excretion of nn m rat bile -