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The determination of concentration of bile acid in peripheral, portal and hepatic blood of cebus monkeys
ARC’HIVES
OF
RIOCHEMISTRY
ASI)
The Determination Portal
RIOPHTYICS
96, 516-523 (1962)
of Concentrations and
Hepatic
Recei\-ed
of Bile Acids
Blood
August
of Cebus
in Peripheral,
Monkeys’
7, 1961
:1 proccdurc for ihe determination of conccntrnt,ions of bitr salts in the peripheral, portal, and hcpat ic bloom! of Cebua monkeys is deskbed. The methods were bawd on dilution calculations dcrived from the specific activities of labeled compounds in bite after the administration of C”-rarboxyl-labeled bile salts. The Cebus monkeys appeared to have largely tnurocholic nnd taurochenodeoxycholic acids in bile and in blood, Deoxgcholic. acid, lvhich was formed in significant qu:mtitiw in the intestintr from chotic acid, was apparently reabsorbed t,o a minimum cxtcnt, since labeled taurodeoxychotate was not found in ttrf, bile after chotatc administ,ration and the montic! tivcr dors not ntlcl~ ndministerctt tl~oxychotnte. IKTRODUCTIOK
The question of the range of concentrations of bile salts in the peripheral circulation, and to a certain extent in the portal circulation, has been examined in a wide variet,y of studies reported over the past half century. A range of values from 0 to 50 mg. ‘:: has been reported for peripheral venous
sera.
X wrtain part of the diaagrecmc~nt over the normal mean values can, perhaps, be explained as resulting from differences in the times at which blood samples were drawn, particularly from differences in the intwvals after the last meal. Except in the rat, which lacks a gall bladder or other I)ile salt concentrat,ing organ, the concentrations of bile salt’s in the intestine, and presumably in the portal vein, are high after a fat-con’ This work was supported in part by grants-inaid from the National Heart Institute, Kational Instit,utes of Health. Bcthesdn, Maryland (Grant 30. H-136) ; Life Insurance McdicA Research Fund. Sew Tort;; John .I. Hartford Mcmorint Fund : and the Fund for Hescawh m(l Teaching, Harvard S-hoot of Public Health. ‘This work was done dllring the tenure of an Estnbtishcd Invcsiigntorahip of the American He:ll,t, Asocial ion.
taining meal and drop very precipitously as t’he bile salts are rapidly removed from the gut. Little information is available about the concentrations of bile salts in the portal blood except for the rat (1 ) , but the rapidity with which administered bile salts rcappear in the bile indicates that t’he conccntrations in the portal blood should also be highly variable for those species with gall bladders (in which the concentrations of bile salts in the gastrointestinal tract, arc highly variable). If bile salts in the periphera1 circulation are a reflect,ion of the bile salts not extracted from the portal circulation by the liver, their concentrations coultl also be highly variable. In spite of the possible biological \-ariability in bile salt concentrations, it is likely that, thaw reported values of bile salt, concentrations which are highest tend to reflect the measurement of materials that arc not bile acids. One of the more recent) studit:: of the lcrcls of l)ilv salt:: in normal human sera, that of Rutlm:m and Kendall (21, w ported a zero low1 for trihydroxyand clihydroxycholanict acids, although it is not clear what the minimum detectable conccntrations would be for the sample sizes uwd. ,516
BILE
ACIDS
Carey (3) has reported a mean trihydroxycholanic acid value for normal human serum of 0.14 mg.:% with a range of 0 (presumably less than 0.005 mg.%) to 0.34 His values for dihydroxycholanic rng. yi. acids ranged from 0 to 0.19 mg.76. Carey has also summarized the values for serum bile salts obtained for several species by a number of other investigators. It seems likely that no purely chemical met,hod is yet available which can measure wry small quantities of bile salts in blood with ccrtaint,y. This study describes t,hc detwnrination of concentrations of bile salts in pcriphcral, portal, and hcpatic scra of C&us monkeys using Cl+-carboxyl-labeled compounds. An attempt was also made t’o e~~aluate the effect of feeding a fat meal on the concentration of bile salts in the periphcral blood. The techniques used for cnlculation of thr strum conccnt,rations were based on total radioactirity in serum and specific nct,ivities of the various labeled bile salts in bile. The points that had to be established to determine the valiclit#y of the method used were the distribution of bile acids in bile, the fate of the radiolabeled components during the enterohepatic circulation and their itlent’ity in the bile, and the nature of the latwlccl proclucts in t’hc blood. METHODS Twenty-one Cebus monkeys were used in these studies. Apparently healthy animals which had been fed the standard laboratory purified diet (4) and were to be sacrificed for ot,her purposes were used. The labeled bile acids used in t,hcse studies were cholic acid-24-C” at either 2.0 or 4.5 q./mg., dcoxycholic acid-24-P at 3.5 ~c./mg., and chmodeoxycholic acid-24-C” at 6.9 pc./mg. The labeled cholic and deoxycholic acids were prepared according to the procedure of Bergstrom et nl. (5). The labeled chenodeoxpcholic acid was obtained from Kichcm, Inc. of Bethesda. Md. .A11 of the acids al,peared to be radiocl~romatograpl~ically pure (6, 7) and to have absorption spectra which mere comparable to authentic unlabeled samples (8. 9). At \-arying intervals after the intraperitonea] administration of labeled bile salts, samples of blood mere drawn from the arm veins and the monktys were ancsthetizetl Tvith intravrnous soditim
IS
BLOOD
317
Scmbutal. The abdomen was immediately opened and a sample of portal x-ein blood was obtained. Within 30 sec. the thorax was opened and a sample of hepatic vein blood was drawn. The animal was then largely exsanguinated from the right atrium. The gall bladder was isolated and its contents were drained into a culture tube. An aliquot of bile was transferred to an appropriate graduat,ed flask, allowing for a dilution \v-ith, at least,, 10 vol. of absolute ethanol. The flask was heated at 6O”C., cooled, and brought to the mark with Ethanol. Serum was obtained from the blood samples and either frozen or immediately added to a gradiiated flask to which 5 vol. acetone-alcohol 1:l 1.1.,‘v) were added. The flask was heated to boiling, cooled, and brotight to the mark. IJsually 2 ml. sorum was usetl for each prc~paration. .I mc~asuretl alicluot, of the acetonc-alcol~ol tiltratc rol)rcsenting most of the serum sample was taken t,o drynws under nitrogen in a tube with a ground-glass fitting. Ten milliliters of 70”; c~thanol was atltlecl and ndju~tctl to pH 2.0. The (‘1l~molwater phase wa:: cstracted three times with lo-ml. 1.01. pet~rolcum ether (ligroine). The ethanol wu wmovecl under a stream of nitrogen, and the water phase was mad<> II]> to 10 ml. The lvater phase was then extracted with ?l-butanol. The butanol cxtracts were dried untlrr nitrogen. and the residue was transferred to plan~hets with several alictiiots of rt,lianol. The avt’lnge of five mcasurcmc~nte with thin window gas-flow counter (efficiency 17%) preset to 1000 events was used to determine radionctivity. In some instances much larger clusntit.ies of pooled sera from monkeys injected with cl~olaie-C’l were used for the isolation of a bile acid frartion, and theac were subjected to column rhromatography according to the procedure of Mosbacli et (II. (7). Siwe this tat trr l~rocedu~~r is designed for free bile acids. the butanol extract, dcsc.rihed above was dried and suhjwled to strong alkaline hyclrolysis (3 S KOH. 125°C. for 2 hr.), :ic*itlihc,d, and extracted with ethyl ether. The ether, raxtracts wfw tlric~cl and uw(l for the r:rtliochrolll;ltog~~:~~~l~~. The bile tiltmtc~ werr analyzed using the papri chl.olll;ltngr:t])llj(’ 11roc.ccl111~csof Sjovall (6, IO). Spwific actirit>. cl(‘tc~l,lllinaCions were mad? 011 tile f~ll1:itf~s from t)ortionz of Ihc strips by determiuation of r:rdioac,tivity and intlivitlru~l bik salt caonwntrationi. The most useful l~rcparalion for thwe analyses eniplo.wtl W~atman 3 hlbl filter paper with the Ta systcn~ (stationary pha>c: 70%’ formic acid; moving phase : isoamyl wetate-helitanc 85: 15) foi unhydrolyzed bile, although I hc other five systems dewribcd by Sjovall (IO) Tvere used for cert:rin .wpnr:rtions. .~l~~ol~ol filtmt pi: c~quivalent to 5-10 P].
200 CHOLATE
- Cl4
100 r3 2 r a k F 2 z ”
0 100 50 0 100
DISTANCE
FROM
ORIGIN
(CM)
Fro. 1. Distribution of radioactivity (corrected for background) in gall bladder bile of three representative Cehus monkeys 3 days after the administration of trace doses (10 PC.) of cholic acid-24-C’: chcnodeoxycholit: acid-24-C’: or droxycholic acid-24-C” as the potassitml salts. TC, TD, and CC indicate the loci of iaurocholic, taurochenodeoxycholic, and glycochohc acids, respecti\-ely. Taurodeoxycholic arid overlaps taurochenodeoxycholic acid in part, but is aomcv;hnt closer to the solvent front. System is 1hat of Sjovall (10). Moving phase 85% (v/v) isoamyl acetate-heptane; stationary phase iOq# (\-/I-) formic acid; support Whatman 3 hlhI filter paper. of whole bile Iv-ere used. One strit) containing the bile sample and one with model mixtures of bile acids ~verc st,ained with the l~llosl,Ilomol?;bdic acid reagent. The stained strip containing gall bladder bile was cut into 0.5-cm. segments. and the segments were radioassayed directly (counting efficiency 1% on filter paper). The duplicate unstained strips of chromatographed bile were cut and eluted as described by Sjorall (10) The eluates were dried and subjected to strong alkaline hydrolysis (3 A’ KOH, 125°C. for 2 hr.), acidified, and extracted vv-ith ethyl ether. The washed ethyl ether extracts were dried under nitrogen and diluted with exactly 3 ml. ethanol. Chemical analyses were performed on aliquots using the 65% HSO, reagent (lo), and radionnalyscs vverc performed on other aliquots. The mean errors for the chemical determination of the major fractions (taurocholic and taurochenodeoxycholic) from gall bladder bile were about 5%. The usual analysis was carried out under conditions which would assure that any clearly separable bile acid which was present as 4% (by radioactivity determination) of the major peaks would be clearly demonstrable. A similar sensit,ivit,y for the chemical determination was obtained for acids that had molecular extinction coefficients in 65% H&O, equivalent to those of cholic or chenodeoxycholic acid.
RESULTS NATURE BILE
0~ RADIO.~~TIVE
COMPOUNDS
Ix
3 HOWE AA-D 3 D,il-s AFTER LABELED BILE SALT ADMINISTRATIOS
Figure 1 illust,rates the distribution (carrect’ed for background) of labeled compounds in bile 3 days after the intraperitoneal administration of 10 pc. of labeled cholate-24-C?“, deoxycholatc-24-Crl, or chenodeoxycholate-24-C14. Very similar patterns were obtained for bile samples of monkeys sacrificed 3 lrr. after administrat,ion of labeled bile salts. All of the rndioactivity in the biIes of the monkeys given labeled cholate appeared at the taurocholate locus. The radioactivity of the biles from monkeys given labeled deoxycholate appeared as t,aurodihydroxycholanic acid. This failure to hydroxylate dcoxycholate in the 70r or ,f3or 6,8 positions (11) supports the observation of Bergstrom and others that the appropriate hydroxylating enzyme is apparently absent in many species (121
BILE
ACIDS
The failure to find any labeled taurodeoxycholate in Ccbus monkey bile even as long as 5 days after the administration of labeled cholate and after the monkeys had for several days been excreting significant quantitics of labeled free deoxycholate into tlie feces indicates that under thcee cxpcrimental conditions deoxycholate is not, normally being reabsorbed very efficiently from the lower gastrointestinal tract. (In the monkeys administered radiocholate, a mean of 42q0 of the radioactivity (range 26-737; I in t,he feces was associated with frre dcoxycholate.) This premise also requires that the contention of Bergstrom that tleosycholate is formed exclusively from cholate hy microorganisms ciaj be correct. In no monkey supplied diets with adequnt8e l)yridoxinc was radioactivitv in bile found at loci characteristic of glycine-conjugated bile salts. Labeled taurochenodeoxycholic acid accounted for 97?;, of the radioactivity in the bile of the monkeys adminietcrcd chenodeoxycliolate-24-C14 3 hr. or 3 days prior to sacrificing. Thus the hydroxylations of rhenodeoxycholate in the 6,8 position described by Hsia et nl. (13) for rodcnt#s apparently did not occur extensively in the Cebus monkey under the existing cxpcrimcntal conditions. NATURE
OF BILE SALTS NORMALLY IS CEBUS &IOSKEY BILE
PRESENT
The values for concentrations of bile salts in Cebus monkey bile are indicated in Tahle I. The mean values for taurotrihydroxycholanic and for taurodihydroxycholanic acids were nearly equal. When the taurodihydroxycholanic acid fractions from some chromatograms were hydrolyzed and rerun on paper chromatography, it appeared t,hat the free acid was entirely or largely ( >96% 1 chenodeoxycholic acid. This would also bc anticipat,ed from the fact that administered labeled cholic acid did not appear as deoxycholic acid in the bile of Cebus monkeys, and that administered labeled chenodeoxycholate cont’inucs to circulate principally as taurochenodeoxycholate in the enherohepatic circulation. No glvcineconjugated bile salts were detertcd in the
IS
,519
BLOOD TABLE
I
Values are in mg./ml. of bile. S = 16. The methods are those of Sjovall ilO) as modified (see test) ; 0.5 ml. of gall bladder bile was obtained from almost all monkeys.
171 Q 17‘29 1739
1200
1140 1080 1743 1430 1769 1240 1769 1220 178C? 1100 1790 1090 181 Q 1270 182Q 12”O _ 183 Q 1310 186Q 1210 1879 1580 1923 1420 19:3c? 1610 161 Q 1410 Mean valuf3
19.0 20.6 8 6 10.2 16.1 22.5 8.2 l!l.i 15.2 20.4 I) .4 28. :3 17.5 14.8 13.2 15.1 16.16
12.2 7.0 14.; 11.9 16.0 34.1 20 0 14.4 12.2 19.7 21.8 23.6 14.1 25.0 17.1 24 .o 17.w
31 .P 27.6 23 .s 22.1 32.1 56.6 28.2 34.1 ‘7.4 40 0 31.2 51 .!I :31.ci 5!) .8 30.3 :{!I 1 34.16
(I The valurs for t:~urochenodeo.u~choli~ acid include any t aurodeosycholic acid present. Gther separat,ions [Fa system of Sjovall ilO) on hydrolyzed snmplcs] indicated that only traces of taurodeosycholic acid wcro present, ‘#The ages of these monke>-s were rmknoxn. Weights and gonads1 evaluation suggest that most were prepubertal.
gall bladder of normal Cebus monkeys. The bilcs of two pyridoxine-deficient monkcvs3 contained predominantly glycine-con;ugat’ed acids [a reversal of glycine-taurine ratios n-as seen in pyridoxine-deficient, rats by Doisy et crl. (1411. NATURE OF THE LABELED BILE ACIDS IS GALL BLADDER BILE AFTER THE ADMIMSTRATIOX OF 1\IEVALOSIc hCXD-2-C’”
sodium nlevalonate-2-C14 was injected intraperitoneally into a male Cebus monkey weighing 1440 g. (20 pc.; specific activit,y 3 Rile was supplied from long-term pyridoxinedeficient monkeys by Dr. S. K. Gcrshoff and Dr. 8. R. Anthus of this department.
520
PORTMAN II
TABLE
(20 pc dose-l
.24 mr ./mmole) Bile acid concentration ~--
Total radioactivity .___~~~.
,mg. bde acid~atl.
7
Nonsaponifiable Choliv Chenodcosycholica u Present
5.8 44.1 50.1
bile
1X.2 20.1
in bile :ts taurine
conjugates.
1.24 mc./mm~l~) 14 After 5 days, during which radioactivity nieasurcnicnts were made on various fractions of serum and feces, the monkey was sacrificed and the l~yclrolyzed bile (3 S KOH, 125°C. for 2 11r.j separated into saponifiable and acidic coluponents. The acidic component’s were then fractionated by t,he procedure of illosbarb et al. (7). The distribution of radioactivity between the chenodeoxycholic acid and cholic acid peaks was almost cvcn as was the clistribut,ion of total chenodeoxycholic and cholic acids (Table II). Thus it appeared t’hat there was unlikely to be any marked difference in the turnover rates for rholic and chenodeoxycholic acids. This latter conclusion is consistent with the observations of Lindstedt and Norman in rats (16). NATURE
OF
RADIOACTIVE
PERIPHERAL AFTER
ASD
PRODCCTS
PORTAL
ADMIKISTRATIOP~
IN
BLOOD OF
cHOL.4TE-24-c’4
Because the levels of radioactivity in blood, particularly in peripheral blood, were very low, large samples mere required to iclentify the nature of t.hc labeled coinpounds present. Pooled samples of periphoral ecra and of portal sera (20 ml. periphcral, 4 ml. portal) from two monkeys which had been administ,ered cholate-24-U” 3 days previously were used. The fractions containing bile acids \yerc subjected to ’ Mcvalonic acid was ohtnined from the X,X’dilwnzylethylene-diaminc salt according to the p~~oce~ure
of Hofiman
c,f al.
(15).
The
mr\-nlonic
from I hc Sew England clv:rr Corp.. Boston. Mass. nci~l
dt
was
ol,t:tinrtl
T\Tu-
AND SHAH
strong alkaline hydrolysis, and the free bile acid fractions tyere isolated. These fractions wcrc then subjcctcd to column chromatography by the procedure of nlosbach et ul. (7,1. As the radiochromatographs of in Fig. 2 indicate, most of tlic activity portal and peripheral blood behaved as a trihydroxycholanic acid. There was, nerertlwleas, some’ radioactivity in the dihydroxy regions of the serum cl~ron~atogran~s (16% of portal serum, 8% of peripheral serum I, suggesting that, tile bile acids from blood and bile might not be entirely homogcnous. If tlic: radioact,iviQ present in peripheral and portal blood as a dihvdraxy acid n-as tlcoxycholic acid, it. is d&cult to explain tlic failure to find labeled deoxycliolic acid in l)ilc after cholate administration. These observations on the nature of the metabolitcs of cllolatc-24-CY1 in gall bladtl~~r bile and in portal and peripheral blood suggest t,liat, thew anatomical sites may 1w considered as largely constituting a common pool of taurocholic acid. It is possiblr to tletcrniiw the specific activity of taurocllolate in bilr rclativcly easily and, using calculations based on that specific activity lnwwrcnlcnt, to determine blood taurocholato concentrations at various sites in thtr vascular system. 0~
COXCENTR.~~~ON IN
THE
BILE
SALTS
BLOOD
Table III lists the values for t#aurocholic acid (exprcsscd as cholic acid) in the samples of serum from thr arm vein, portal win, and hepatic vein. Except for monkcg 187, all samples were drawn from monkeys which had been subjected to an overnight fast. This lattw monkey, which had betw injert,rd intraperitoneally with cholate-24Cl-’ 3 days prior to sacrificing, was blctl from the arm win and then given a mcal of 25 ml. corn oil by stomach tuhc. Sixty minutw later the animal was again bl~l from the arm and sacrificed for other sariplw of blood and bile. Calculated tarlrocho1:~t.t: concentrations for the fasting and the post-fatty meal sample arc both listctl in the column labclcd “peripheral.” The calculat’ions of taurocholatc in blood XVW~ based on the assumption that the bile and blood pools were homogeneous. Since r:i(lio-
5
IO TUBE
20
15 NUMBER
FIG. 2. Distribution of radioactivity in pooled serum obtained from heart blood (peripheral) and in serum from portal vein blood of monkeys treated with cholic acid-24-C”. The isolated fractions containing the I-jile acids were hydrolyzed and the free bile acids were IYIU by the column chromatographic procedure of Mosbach et al. (7). The IPE 40 region is eluted with 40% (v/v) isopropyl ether-ligroine. Dihydroxycholnnic acids are found in the IPE 40 region, and trillytlros?-~holanic acids are fou1~1 in the IPE 60 region.
were not available on strum cllromatograms of all monkeys studied, a correction could not regularly be made for any inaccuracy of that
assumption.
In all cared, the concentrations of tnurocholatc were highest in the port’al serum. and in four of the six monkeys the ~~lucs for ~~criplieral serum and heart serum (ralucs for heart) serum not shown ) wrc greater than for hopatic vein serum. The lower yaluw
for
liel)atic
vein
serum
can
best
lw
ascribed to anesthesia changes and the fact that, this sample was drawn last in the sacrificing procedure. Since the Ircpatic~ win blood represents a mixture of portal win blood and hepatic arterial blood, tllo efficiency of liepatic clearance of bile salts is undoubtedly measured by the diffcrencc between some value intermediate bctw-ccn those for l~eripheral and portal blood and tllat for hepatic vein blood. If t,hc assumption is made that the hepatic vein blood iy derived four-fifths from portal vvin and one-fifth from licpatic artery (peripheral 1, the efficiency of the liver in removing taurocholate wit,11 each passage varies from 44 to 997; for these six monkeys. The gall bladder of these six nlonkcys contained taurochenodeoxycholate :U well
Concentration oi cholate in serum Peri~~beral
161 lil 172 176 186 1%
Portal Hepatic ~~~ ~~___ srg., 1011 nil.
Concentration of chenodeoxgcholate in serum Peripheral
lll~.iiliO ml.
0.15 0.64 0.30 0. 10 0. ‘25 0.004 0.11 0.44 0.046 0.12 2.25 0.15 0.19 0.33 0. 10 0.18 0.45 0.15 0.10 (before fat meal)
0 Serum cholic acid conrn. = counts/min./l00 ml. serum counts/min./mg. cholic acid bile h Serum rhenodcosycholic acid concn. mg. cheno/ml. bile mg. cholic/ml, bile -> x (mg. cholic/lOO ml. serum) mg. !I,
0.22 0.064 0.032 O.l!J 0.16 0.14
mg. ‘, c =
TABLE C~SC~I~:NTI~ATION Mosrtms OF
OF
1~11.13
DETI~RILIINET) LhREmo
11 SALTS
IS
FROILI
SER.*
OF
C;I~:BL.:,
:~PI’EhRAKCE
RIEARI.REMXNTS
T.\~~Ro(.I~ES~UPOSY(‘HO~,.~T~
OF’
BILE
PERIPHERAL
SALTS
lx
THE
CIRCPLATIOS
IN
It could be anticipated that after a fat meal and gall bladder contraction the conOSY(‘IIOI.ATI~~ AND ‘rAll-RO(‘FIOIATE (‘OScentrations of bile salts in the portal and, (‘ENTRATIOSS IS fiII,R” perhaps, in the peripheral circulation would Determinations were made afl.er the intraperibe elevated above those seen in the fasting tonral administration of chenodeoxycholic acidstate. The: effect of the int’ubation of a fat 24-C” (10 WLC.dose; 6.9 pc./mg.) as the potassium salt. meal on the concentration of labeled bile salts in the peripheral circulation was, concentration of C‘oncentration of chenot,hereforc, evaluated. cholate Monkey deoxycholate in serum in serum As Fig. 3 indicates, t’here was no connumber sistent effect of feeding a fatty meal on the Peripheral Hepatic Peripheral PClrtal concentration of radioactivity in the periplllng./lon ml. rrq.ilOO d. era1 sera of monkeys that had been prc0.20 0.33 0.09‘4 192 2.62 viously treated with labeled cholatc. In only 0.20 0.096 193 0.25 0.61 one t#rial was there a marked increase in t,he a Calculat,ions analogous to those l&cd with amount of radioactivity found in the pcTable III apply. ripheral blood after the administration of a fat meal. It would have undoubtedly been as t,aurocholate. If the same ratio of taurocholnte to taurochenodeoxycholate were present in serum as was found in the bile, a range of taurochenodeoxycholate conccn(expressed as chenodeoxycholic trations acid) of 0.032-0.223 mg.% was present. l3Ir.s
Asn
CALCULATIOK RILE
SALTS
ON
R.zTIo~
OF
SERIM
FROM
24-Cl’
OF
TAI-R~c’H~;~~I)E~
COZTCESTRAUOKS
OF
CHENODEOXTCHOLATESTUDIFS
JL
Since administered labeled chenodeoxycholate appeared to be largely stable as t,aurochcnodeoxycholate in the cntcrohepatic circulation of the monkey, it was possible to estimate the concentrations of chenodeoxycholate in blood from isotope dilution calculations based on the specific act’ivity of taurochenodeoxycholate in bile. The results of those calculations are indicated in Table IV. For the two monkeys evaluated, the concentrations of taurochcnodeoxycholate (expressed as chrnodeoxycholatei mere 0.33 and 0.25 mg.YG. The calculated values for taurocholate based on t’he distribution of cholic and chenodeoxycholic acids in bile were 0.20 mg.% for both monkeys. As was t’he case in the radiocholate experiment’s described above, there were very large differences between the concentrations for port,al and those for hepatic vein sera.
20 -
0
IO
20
I
30
40
TIME
Lminl
I
50
1 IA
60
120
FIG. 3. The effect on Cebus monkeys of administering a fat meal (25 ml. corn oil by stomach tube) on the roncentration of bile acids in the peripheral veinous circulation (arm rein). The monkeys had been treated 24 hr. previously with 10 pc. cholic acid-24-V as the potassium salt. The t,ime zero sample was drawn following an overnight fast and was immediately followed hy administration of the fat mwl.
RILT;
.\CIDS
much easier to evaluate the effect of emptying of the gall bladder on the peripheral concentration of bile salts if cholecystokinin had been available in lieu of t’hc fat meal to more precisely induce gall-bladder conBact.ion. DISCUSSIOS
Although it is extremely difficult to dctermine by purely chemical procedures the concentrations of-bile salts in-the peripheral blood of animals or humans, t.he use of labelcd bile salts with analyses of simultaneous samples of bile and blood makes thcsc determinations feasible. It is necessary to establish that the distribution of radioactivity between different bile salts is the same for serum and for bile. This latter determination was relatively easy in the Cebus monkey where all of the bile salts were taurine conjugated and where taurocholate and taurochenodeoxycholate were the principal bile salts present. Experiments carried out with labeled cholic and labeled deoxycholic acid indicated that the dcoxycholatc which was formed in the gastrointestinal tract was absorbed to a very small extent, since only labeled taurocholate appeared in the bile even 5 days after radiocholate administration. Administ,ered radiodeoxycholate was not converted to other compounds by the Cebus monkey liver. The evidence that cleoxycholatc was poorly absorbed from its site of formation is, however, indirect. In those situat.ions where it is possible to obtain samples of bile by duodenal intubat,ion, a relatively innocuous procedure for estimating bile salt concentration in peripheral blood should be available. Although it was impossible to estimate precisely the rate of clearance by the liver of bile salts from the portal blood because of lack of information about the proportion of the liver circulation supplied by the portal vein and that by the hepatic artery, it was possible to demonstrate very large differcncts in the concentrations of bile salt,:: in the portal and hepat,ic veins.
IS
,jL?:j
BLOOD
Since the preparation of this manuscript, the authors have seen the report of Grundy and Sjovall ( 17) on bile acids in rat, systcmic blood. These workers have based their calculations on the distribution of radioactivity bctn-ccn intestine and blood. The method hcrcin presented would bc applied with great difficulty in rats with their much 1110rc coluplcx liepatic intcrconr-rwions of bile acids.
T.. .4x1) SJOV.4l.L, J., .-lctn Physid. Scnd46,284 (1959). F. E.. J. (‘/in. 1% 2. RI-DMAS, D., -4s” IiESDALL, ITS/. 36, 530 (1057). J. B., ,JN.. J. c/h. In c~,.st. 37, 1494 3. CAREP, (1958). 4. OLI\~RO.\..~,
5. 6. 7. 8. 9.
10. 11.
kJOV4JJ.. R.~TLIFF,
15. A,
J., ilclri
(:h.
C&m.
I<.
4, 652 (1959).
I,., MATWHISEI~, JR.. Hs1.4, S. I,., THATER.
JV. H.. AXJ) 3133 (1959).
Domu.
E.
,I.,
J.
J.
T..
S. A.,
I)OISY, ELJ.IOTT,
Biol. Phcru. 234,
12.
J. T., 13. Hs1.4, S. I,., ELLIOTT, S. H., MATSCWKER, I>OISU, E. .I., Ja., TIIATEH. S. A., ASD I)OISY, E. --I., J. Bid. Chcna. 235, 1963 (1960). 14. Dorsr. E. .I., JR., DAKIELS, M., AXD hI~IERhlAN, hl. A., Fcclcrntion Pm-. 15, 243 (1956). C. H., ~~7.4GXJU~, ~1. F.. WILSOS. A. N., 15. Hmxci, n:.4LTOS, E. tiHC:NIC, c. H., WOLP, D. E., HOLLY.. F. W., AXID FOLKERS, I<., J. Attr. Chum. Soc.79,2316 (1957). 16.
17.
OF
RIOCHEMISTRY
ASI)
The Determination Portal
RIOPHTYICS
96, 516-523 (1962)
of Concentrations and
Hepatic
Recei\-ed
of Bile Acids
Blood
August
of Cebus
in Peripheral,
Monkeys’
7, 1961
:1 proccdurc for ihe determination of conccntrnt,ions of bitr salts in the peripheral, portal, and hcpat ic bloom! of Cebua monkeys is deskbed. The methods were bawd on dilution calculations dcrived from the specific activities of labeled compounds in bite after the administration of C”-rarboxyl-labeled bile salts. The Cebus monkeys appeared to have largely tnurocholic nnd taurochenodeoxycholic acids in bile and in blood, Deoxgcholic. acid, lvhich was formed in significant qu:mtitiw in the intestintr from chotic acid, was apparently reabsorbed t,o a minimum cxtcnt, since labeled taurodeoxychotate was not found in ttrf, bile after chotatc administ,ration and the montic! tivcr dors not ntlcl~ ndministerctt tl~oxychotnte. IKTRODUCTIOK
The question of the range of concentrations of bile salts in the peripheral circulation, and to a certain extent in the portal circulation, has been examined in a wide variet,y of studies reported over the past half century. A range of values from 0 to 50 mg. ‘:: has been reported for peripheral venous
sera.
X wrtain part of the diaagrecmc~nt over the normal mean values can, perhaps, be explained as resulting from differences in the times at which blood samples were drawn, particularly from differences in the intwvals after the last meal. Except in the rat, which lacks a gall bladder or other I)ile salt concentrat,ing organ, the concentrations of bile salt’s in the intestine, and presumably in the portal vein, are high after a fat-con’ This work was supported in part by grants-inaid from the National Heart Institute, Kational Instit,utes of Health. Bcthesdn, Maryland (Grant 30. H-136) ; Life Insurance McdicA Research Fund. Sew Tort;; John .I. Hartford Mcmorint Fund : and the Fund for Hescawh m(l Teaching, Harvard S-hoot of Public Health. ‘This work was done dllring the tenure of an Estnbtishcd Invcsiigntorahip of the American He:ll,t, Asocial ion.
taining meal and drop very precipitously as t’he bile salts are rapidly removed from the gut. Little information is available about the concentrations of bile salts in the portal blood except for the rat (1 ) , but the rapidity with which administered bile salts rcappear in the bile indicates that t’he conccntrations in the portal blood should also be highly variable for those species with gall bladders (in which the concentrations of bile salts in the gastrointestinal tract, arc highly variable). If bile salts in the periphera1 circulation are a reflect,ion of the bile salts not extracted from the portal circulation by the liver, their concentrations coultl also be highly variable. In spite of the possible biological \-ariability in bile salt concentrations, it is likely that, thaw reported values of bile salt, concentrations which are highest tend to reflect the measurement of materials that arc not bile acids. One of the more recent) studit:: of the lcrcls of l)ilv salt:: in normal human sera, that of Rutlm:m and Kendall (21, w ported a zero low1 for trihydroxyand clihydroxycholanict acids, although it is not clear what the minimum detectable conccntrations would be for the sample sizes uwd. ,516
BILE
ACIDS
Carey (3) has reported a mean trihydroxycholanic acid value for normal human serum of 0.14 mg.:% with a range of 0 (presumably less than 0.005 mg.%) to 0.34 His values for dihydroxycholanic rng. yi. acids ranged from 0 to 0.19 mg.76. Carey has also summarized the values for serum bile salts obtained for several species by a number of other investigators. It seems likely that no purely chemical met,hod is yet available which can measure wry small quantities of bile salts in blood with ccrtaint,y. This study describes t,hc detwnrination of concentrations of bile salts in pcriphcral, portal, and hcpatic scra of C&us monkeys using Cl+-carboxyl-labeled compounds. An attempt was also made t’o e~~aluate the effect of feeding a fat meal on the concentration of bile salts in the periphcral blood. The techniques used for cnlculation of thr strum conccnt,rations were based on total radioactirity in serum and specific nct,ivities of the various labeled bile salts in bile. The points that had to be established to determine the valiclit#y of the method used were the distribution of bile acids in bile, the fate of the radiolabeled components during the enterohepatic circulation and their itlent’ity in the bile, and the nature of the latwlccl proclucts in t’hc blood. METHODS Twenty-one Cebus monkeys were used in these studies. Apparently healthy animals which had been fed the standard laboratory purified diet (4) and were to be sacrificed for ot,her purposes were used. The labeled bile acids used in t,hcse studies were cholic acid-24-C” at either 2.0 or 4.5 q./mg., dcoxycholic acid-24-P at 3.5 ~c./mg., and chmodeoxycholic acid-24-C” at 6.9 pc./mg. The labeled cholic and deoxycholic acids were prepared according to the procedure of Bergstrom et nl. (5). The labeled chenodeoxpcholic acid was obtained from Kichcm, Inc. of Bethesda. Md. .A11 of the acids al,peared to be radiocl~romatograpl~ically pure (6, 7) and to have absorption spectra which mere comparable to authentic unlabeled samples (8. 9). At \-arying intervals after the intraperitonea] administration of labeled bile salts, samples of blood mere drawn from the arm veins and the monktys were ancsthetizetl Tvith intravrnous soditim
IS
BLOOD
317
Scmbutal. The abdomen was immediately opened and a sample of portal x-ein blood was obtained. Within 30 sec. the thorax was opened and a sample of hepatic vein blood was drawn. The animal was then largely exsanguinated from the right atrium. The gall bladder was isolated and its contents were drained into a culture tube. An aliquot of bile was transferred to an appropriate graduat,ed flask, allowing for a dilution \v-ith, at least,, 10 vol. of absolute ethanol. The flask was heated at 6O”C., cooled, and brought to the mark with Ethanol. Serum was obtained from the blood samples and either frozen or immediately added to a gradiiated flask to which 5 vol. acetone-alcohol 1:l 1.1.,‘v) were added. The flask was heated to boiling, cooled, and brotight to the mark. IJsually 2 ml. sorum was usetl for each prc~paration. .I mc~asuretl alicluot, of the acetonc-alcol~ol tiltratc rol)rcsenting most of the serum sample was taken t,o drynws under nitrogen in a tube with a ground-glass fitting. Ten milliliters of 70”; c~thanol was atltlecl and ndju~tctl to pH 2.0. The (‘1l~molwater phase wa:: cstracted three times with lo-ml. 1.01. pet~rolcum ether (ligroine). The ethanol wu wmovecl under a stream of nitrogen, and the water phase was mad<> II]> to 10 ml. The lvater phase was then extracted with ?l-butanol. The butanol cxtracts were dried untlrr nitrogen. and the residue was transferred to plan~hets with several alictiiots of rt,lianol. The avt’lnge of five mcasurcmc~nte with thin window gas-flow counter (efficiency 17%) preset to 1000 events was used to determine radionctivity. In some instances much larger clusntit.ies of pooled sera from monkeys injected with cl~olaie-C’l were used for the isolation of a bile acid frartion, and theac were subjected to column rhromatography according to the procedure of Mosbacli et (II. (7). Siwe this tat trr l~rocedu~~r is designed for free bile acids. the butanol extract, dcsc.rihed above was dried and suhjwled to strong alkaline hyclrolysis (3 S KOH. 125°C. for 2 hr.), :ic*itlihc,d, and extracted with ethyl ether. The ether, raxtracts wfw tlric~cl and uw(l for the r:rtliochrolll;ltog~~:~~~l~~. The bile tiltmtc~ werr analyzed using the papri chl.olll;ltngr:t])llj(’ 11roc.ccl111~csof Sjovall (6, IO). Spwific actirit>. cl(‘tc~l,lllinaCions were mad? 011 tile f~ll1:itf~s from t)ortionz of Ihc strips by determiuation of r:rdioac,tivity and intlivitlru~l bik salt caonwntrationi. The most useful l~rcparalion for thwe analyses eniplo.wtl W~atman 3 hlbl filter paper with the Ta systcn~ (stationary pha>c: 70%’ formic acid; moving phase : isoamyl wetate-helitanc 85: 15) foi unhydrolyzed bile, although I hc other five systems dewribcd by Sjovall (IO) Tvere used for cert:rin .wpnr:rtions. .~l~~ol~ol filtmt pi: c~quivalent to 5-10 P].
200 CHOLATE
- Cl4
100 r3 2 r a k F 2 z ”
0 100 50 0 100
DISTANCE
FROM
ORIGIN
(CM)
Fro. 1. Distribution of radioactivity (corrected for background) in gall bladder bile of three representative Cehus monkeys 3 days after the administration of trace doses (10 PC.) of cholic acid-24-C’: chcnodeoxycholit: acid-24-C’: or droxycholic acid-24-C” as the potassitml salts. TC, TD, and CC indicate the loci of iaurocholic, taurochenodeoxycholic, and glycochohc acids, respecti\-ely. Taurodeoxycholic arid overlaps taurochenodeoxycholic acid in part, but is aomcv;hnt closer to the solvent front. System is 1hat of Sjovall (10). Moving phase 85% (v/v) isoamyl acetate-heptane; stationary phase iOq# (\-/I-) formic acid; support Whatman 3 hlhI filter paper. of whole bile Iv-ere used. One strit) containing the bile sample and one with model mixtures of bile acids ~verc st,ained with the l~llosl,Ilomol?;bdic acid reagent. The stained strip containing gall bladder bile was cut into 0.5-cm. segments. and the segments were radioassayed directly (counting efficiency 1% on filter paper). The duplicate unstained strips of chromatographed bile were cut and eluted as described by Sjorall (10) The eluates were dried and subjected to strong alkaline hydrolysis (3 A’ KOH, 125°C. for 2 hr.), acidified, and extracted vv-ith ethyl ether. The washed ethyl ether extracts were dried under nitrogen and diluted with exactly 3 ml. ethanol. Chemical analyses were performed on aliquots using the 65% HSO, reagent (lo), and radionnalyscs vverc performed on other aliquots. The mean errors for the chemical determination of the major fractions (taurocholic and taurochenodeoxycholic) from gall bladder bile were about 5%. The usual analysis was carried out under conditions which would assure that any clearly separable bile acid which was present as 4% (by radioactivity determination) of the major peaks would be clearly demonstrable. A similar sensit,ivit,y for the chemical determination was obtained for acids that had molecular extinction coefficients in 65% H&O, equivalent to those of cholic or chenodeoxycholic acid.
RESULTS NATURE BILE
0~ RADIO.~~TIVE
COMPOUNDS
Ix
3 HOWE AA-D 3 D,il-s AFTER LABELED BILE SALT ADMINISTRATIOS
Figure 1 illust,rates the distribution (carrect’ed for background) of labeled compounds in bile 3 days after the intraperitoneal administration of 10 pc. of labeled cholate-24-C?“, deoxycholatc-24-Crl, or chenodeoxycholate-24-C14. Very similar patterns were obtained for bile samples of monkeys sacrificed 3 lrr. after administrat,ion of labeled bile salts. All of the rndioactivity in the biIes of the monkeys given labeled cholate appeared at the taurocholate locus. The radioactivity of the biles from monkeys given labeled deoxycholate appeared as t,aurodihydroxycholanic acid. This failure to hydroxylate dcoxycholate in the 70r or ,f3or 6,8 positions (11) supports the observation of Bergstrom and others that the appropriate hydroxylating enzyme is apparently absent in many species (121
BILE
ACIDS
The failure to find any labeled taurodeoxycholate in Ccbus monkey bile even as long as 5 days after the administration of labeled cholate and after the monkeys had for several days been excreting significant quantitics of labeled free deoxycholate into tlie feces indicates that under thcee cxpcrimental conditions deoxycholate is not, normally being reabsorbed very efficiently from the lower gastrointestinal tract. (In the monkeys administered radiocholate, a mean of 42q0 of the radioactivity (range 26-737; I in t,he feces was associated with frre dcoxycholate.) This premise also requires that the contention of Bergstrom that tleosycholate is formed exclusively from cholate hy microorganisms ciaj be correct. In no monkey supplied diets with adequnt8e l)yridoxinc was radioactivitv in bile found at loci characteristic of glycine-conjugated bile salts. Labeled taurochenodeoxycholic acid accounted for 97?;, of the radioactivity in the bile of the monkeys adminietcrcd chenodeoxycliolate-24-C14 3 hr. or 3 days prior to sacrificing. Thus the hydroxylations of rhenodeoxycholate in the 6,8 position described by Hsia et nl. (13) for rodcnt#s apparently did not occur extensively in the Cebus monkey under the existing cxpcrimcntal conditions. NATURE
OF BILE SALTS NORMALLY IS CEBUS &IOSKEY BILE
PRESENT
The values for concentrations of bile salts in Cebus monkey bile are indicated in Tahle I. The mean values for taurotrihydroxycholanic and for taurodihydroxycholanic acids were nearly equal. When the taurodihydroxycholanic acid fractions from some chromatograms were hydrolyzed and rerun on paper chromatography, it appeared t,hat the free acid was entirely or largely ( >96% 1 chenodeoxycholic acid. This would also bc anticipat,ed from the fact that administered labeled cholic acid did not appear as deoxycholic acid in the bile of Cebus monkeys, and that administered labeled chenodeoxycholate cont’inucs to circulate principally as taurochenodeoxycholate in the enherohepatic circulation. No glvcineconjugated bile salts were detertcd in the
IS
,519
BLOOD TABLE
I
Values are in mg./ml. of bile. S = 16. The methods are those of Sjovall ilO) as modified (see test) ; 0.5 ml. of gall bladder bile was obtained from almost all monkeys.
171 Q 17‘29 1739
1200
1140 1080 1743 1430 1769 1240 1769 1220 178C? 1100 1790 1090 181 Q 1270 182Q 12”O _ 183 Q 1310 186Q 1210 1879 1580 1923 1420 19:3c? 1610 161 Q 1410 Mean valuf3
19.0 20.6 8 6 10.2 16.1 22.5 8.2 l!l.i 15.2 20.4 I) .4 28. :3 17.5 14.8 13.2 15.1 16.16
12.2 7.0 14.; 11.9 16.0 34.1 20 0 14.4 12.2 19.7 21.8 23.6 14.1 25.0 17.1 24 .o 17.w
31 .P 27.6 23 .s 22.1 32.1 56.6 28.2 34.1 ‘7.4 40 0 31.2 51 .!I :31.ci 5!) .8 30.3 :{!I 1 34.16
(I The valurs for t:~urochenodeo.u~choli~ acid include any t aurodeosycholic acid present. Gther separat,ions [Fa system of Sjovall ilO) on hydrolyzed snmplcs] indicated that only traces of taurodeosycholic acid wcro present, ‘#The ages of these monke>-s were rmknoxn. Weights and gonads1 evaluation suggest that most were prepubertal.
gall bladder of normal Cebus monkeys. The bilcs of two pyridoxine-deficient monkcvs3 contained predominantly glycine-con;ugat’ed acids [a reversal of glycine-taurine ratios n-as seen in pyridoxine-deficient, rats by Doisy et crl. (1411. NATURE OF THE LABELED BILE ACIDS IS GALL BLADDER BILE AFTER THE ADMIMSTRATIOX OF 1\IEVALOSIc hCXD-2-C’”
sodium nlevalonate-2-C14 was injected intraperitoneally into a male Cebus monkey weighing 1440 g. (20 pc.; specific activit,y 3 Rile was supplied from long-term pyridoxinedeficient monkeys by Dr. S. K. Gcrshoff and Dr. 8. R. Anthus of this department.
520
PORTMAN II
TABLE
(20 pc dose-l
.24 mr ./mmole) Bile acid concentration ~--
Total radioactivity .___~~~.
,mg. bde acid~atl.
7
Nonsaponifiable Choliv Chenodcosycholica u Present
5.8 44.1 50.1
bile
1X.2 20.1
in bile :ts taurine
conjugates.
1.24 mc./mm~l~) 14 After 5 days, during which radioactivity nieasurcnicnts were made on various fractions of serum and feces, the monkey was sacrificed and the l~yclrolyzed bile (3 S KOH, 125°C. for 2 11r.j separated into saponifiable and acidic coluponents. The acidic component’s were then fractionated by t,he procedure of illosbarb et al. (7). The distribution of radioactivity between the chenodeoxycholic acid and cholic acid peaks was almost cvcn as was the clistribut,ion of total chenodeoxycholic and cholic acids (Table II). Thus it appeared t’hat there was unlikely to be any marked difference in the turnover rates for rholic and chenodeoxycholic acids. This latter conclusion is consistent with the observations of Lindstedt and Norman in rats (16). NATURE
OF
RADIOACTIVE
PERIPHERAL AFTER
ASD
PRODCCTS
PORTAL
ADMIKISTRATIOP~
IN
BLOOD OF
cHOL.4TE-24-c’4
Because the levels of radioactivity in blood, particularly in peripheral blood, were very low, large samples mere required to iclentify the nature of t.hc labeled coinpounds present. Pooled samples of periphoral ecra and of portal sera (20 ml. periphcral, 4 ml. portal) from two monkeys which had been administ,ered cholate-24-U” 3 days previously were used. The fractions containing bile acids \yerc subjected to ’ Mcvalonic acid was ohtnined from the X,X’dilwnzylethylene-diaminc salt according to the p~~oce~ure
of Hofiman
c,f al.
(15).
The
mr\-nlonic
from I hc Sew England clv:rr Corp.. Boston. Mass. nci~l
dt
was
ol,t:tinrtl
T\Tu-
AND SHAH
strong alkaline hydrolysis, and the free bile acid fractions tyere isolated. These fractions wcrc then subjcctcd to column chromatography by the procedure of nlosbach et ul. (7,1. As the radiochromatographs of in Fig. 2 indicate, most of tlic activity portal and peripheral blood behaved as a trihydroxycholanic acid. There was, nerertlwleas, some’ radioactivity in the dihydroxy regions of the serum cl~ron~atogran~s (16% of portal serum, 8% of peripheral serum I, suggesting that, tile bile acids from blood and bile might not be entirely homogcnous. If tlic: radioact,iviQ present in peripheral and portal blood as a dihvdraxy acid n-as tlcoxycholic acid, it. is d&cult to explain tlic failure to find labeled deoxycliolic acid in l)ilc after cholate administration. These observations on the nature of the metabolitcs of cllolatc-24-CY1 in gall bladtl~~r bile and in portal and peripheral blood suggest t,liat, thew anatomical sites may 1w considered as largely constituting a common pool of taurocholic acid. It is possiblr to tletcrniiw the specific activity of taurocllolate in bilr rclativcly easily and, using calculations based on that specific activity lnwwrcnlcnt, to determine blood taurocholato concentrations at various sites in thtr vascular system. 0~
COXCENTR.~~~ON IN
THE
BILE
SALTS
BLOOD
Table III lists the values for t#aurocholic acid (exprcsscd as cholic acid) in the samples of serum from thr arm vein, portal win, and hepatic vein. Except for monkcg 187, all samples were drawn from monkeys which had been subjected to an overnight fast. This lattw monkey, which had betw injert,rd intraperitoneally with cholate-24Cl-’ 3 days prior to sacrificing, was blctl from the arm win and then given a mcal of 25 ml. corn oil by stomach tuhc. Sixty minutw later the animal was again bl~l from the arm and sacrificed for other sariplw of blood and bile. Calculated tarlrocho1:~t.t: concentrations for the fasting and the post-fatty meal sample arc both listctl in the column labclcd “peripheral.” The calculat’ions of taurocholatc in blood XVW~ based on the assumption that the bile and blood pools were homogeneous. Since r:i(lio-
5
IO TUBE
20
15 NUMBER
FIG. 2. Distribution of radioactivity in pooled serum obtained from heart blood (peripheral) and in serum from portal vein blood of monkeys treated with cholic acid-24-C”. The isolated fractions containing the I-jile acids were hydrolyzed and the free bile acids were IYIU by the column chromatographic procedure of Mosbach et al. (7). The IPE 40 region is eluted with 40% (v/v) isopropyl ether-ligroine. Dihydroxycholnnic acids are found in the IPE 40 region, and trillytlros?-~holanic acids are fou1~1 in the IPE 60 region.
were not available on strum cllromatograms of all monkeys studied, a correction could not regularly be made for any inaccuracy of that
assumption.
In all cared, the concentrations of tnurocholatc were highest in the port’al serum. and in four of the six monkeys the ~~lucs for ~~criplieral serum and heart serum (ralucs for heart) serum not shown ) wrc greater than for hopatic vein serum. The lower yaluw
for
liel)atic
vein
serum
can
best
lw
ascribed to anesthesia changes and the fact that, this sample was drawn last in the sacrificing procedure. Since the Ircpatic~ win blood represents a mixture of portal win blood and hepatic arterial blood, tllo efficiency of liepatic clearance of bile salts is undoubtedly measured by the diffcrencc between some value intermediate bctw-ccn those for l~eripheral and portal blood and tllat for hepatic vein blood. If t,hc assumption is made that the hepatic vein blood iy derived four-fifths from portal vvin and one-fifth from licpatic artery (peripheral 1, the efficiency of the liver in removing taurocholate wit,11 each passage varies from 44 to 997; for these six monkeys. The gall bladder of these six nlonkcys contained taurochenodeoxycholate :U well
Concentration oi cholate in serum Peri~~beral
161 lil 172 176 186 1%
Portal Hepatic ~~~ ~~___ srg., 1011 nil.
Concentration of chenodeoxgcholate in serum Peripheral
lll~.iiliO ml.
0.15 0.64 0.30 0. 10 0. ‘25 0.004 0.11 0.44 0.046 0.12 2.25 0.15 0.19 0.33 0. 10 0.18 0.45 0.15 0.10 (before fat meal)
0 Serum cholic acid conrn. = counts/min./l00 ml. serum counts/min./mg. cholic acid bile h Serum rhenodcosycholic acid concn. mg. cheno/ml. bile mg. cholic/ml, bile -> x (mg. cholic/lOO ml. serum) mg. !I,
0.22 0.064 0.032 O.l!J 0.16 0.14
mg. ‘, c =
TABLE C~SC~I~:NTI~ATION Mosrtms OF
OF
1~11.13
DETI~RILIINET) LhREmo
11 SALTS
IS
FROILI
SER.*
OF
C;I~:BL.:,
:~PI’EhRAKCE
RIEARI.REMXNTS
T.\~~Ro(.I~ES~UPOSY(‘HO~,.~T~
OF’
BILE
PERIPHERAL
SALTS
lx
THE
CIRCPLATIOS
IN
It could be anticipated that after a fat meal and gall bladder contraction the conOSY(‘IIOI.ATI~~ AND ‘rAll-RO(‘FIOIATE (‘OScentrations of bile salts in the portal and, (‘ENTRATIOSS IS fiII,R” perhaps, in the peripheral circulation would Determinations were made afl.er the intraperibe elevated above those seen in the fasting tonral administration of chenodeoxycholic acidstate. The: effect of the int’ubation of a fat 24-C” (10 WLC.dose; 6.9 pc./mg.) as the potassium salt. meal on the concentration of labeled bile salts in the peripheral circulation was, concentration of C‘oncentration of chenot,hereforc, evaluated. cholate Monkey deoxycholate in serum in serum As Fig. 3 indicates, t’here was no connumber sistent effect of feeding a fatty meal on the Peripheral Hepatic Peripheral PClrtal concentration of radioactivity in the periplllng./lon ml. rrq.ilOO d. era1 sera of monkeys that had been prc0.20 0.33 0.09‘4 192 2.62 viously treated with labeled cholatc. In only 0.20 0.096 193 0.25 0.61 one t#rial was there a marked increase in t,he a Calculat,ions analogous to those l&cd with amount of radioactivity found in the pcTable III apply. ripheral blood after the administration of a fat meal. It would have undoubtedly been as t,aurocholate. If the same ratio of taurocholnte to taurochenodeoxycholate were present in serum as was found in the bile, a range of taurochenodeoxycholate conccn(expressed as chenodeoxycholic trations acid) of 0.032-0.223 mg.% was present. l3Ir.s
Asn
CALCULATIOK RILE
SALTS
ON
R.zTIo~
OF
SERIM
FROM
24-Cl’
OF
TAI-R~c’H~;~~I)E~
COZTCESTRAUOKS
OF
CHENODEOXTCHOLATESTUDIFS
JL
Since administered labeled chenodeoxycholate appeared to be largely stable as t,aurochcnodeoxycholate in the cntcrohepatic circulation of the monkey, it was possible to estimate the concentrations of chenodeoxycholate in blood from isotope dilution calculations based on the specific act’ivity of taurochenodeoxycholate in bile. The results of those calculations are indicated in Table IV. For the two monkeys evaluated, the concentrations of taurochcnodeoxycholate (expressed as chrnodeoxycholatei mere 0.33 and 0.25 mg.YG. The calculated values for taurocholate based on t’he distribution of cholic and chenodeoxycholic acids in bile were 0.20 mg.% for both monkeys. As was t’he case in the radiocholate experiment’s described above, there were very large differences between the concentrations for port,al and those for hepatic vein sera.
20 -
0
IO
20
I
30
40
TIME
Lminl
I
50
1 IA
60
120
FIG. 3. The effect on Cebus monkeys of administering a fat meal (25 ml. corn oil by stomach tube) on the roncentration of bile acids in the peripheral veinous circulation (arm rein). The monkeys had been treated 24 hr. previously with 10 pc. cholic acid-24-V as the potassium salt. The t,ime zero sample was drawn following an overnight fast and was immediately followed hy administration of the fat mwl.
RILT;
.\CIDS
much easier to evaluate the effect of emptying of the gall bladder on the peripheral concentration of bile salts if cholecystokinin had been available in lieu of t’hc fat meal to more precisely induce gall-bladder conBact.ion. DISCUSSIOS
Although it is extremely difficult to dctermine by purely chemical procedures the concentrations of-bile salts in-the peripheral blood of animals or humans, t.he use of labelcd bile salts with analyses of simultaneous samples of bile and blood makes thcsc determinations feasible. It is necessary to establish that the distribution of radioactivity between different bile salts is the same for serum and for bile. This latter determination was relatively easy in the Cebus monkey where all of the bile salts were taurine conjugated and where taurocholate and taurochenodeoxycholate were the principal bile salts present. Experiments carried out with labeled cholic and labeled deoxycholic acid indicated that the dcoxycholatc which was formed in the gastrointestinal tract was absorbed to a very small extent, since only labeled taurocholate appeared in the bile even 5 days after radiocholate administration. Administ,ered radiodeoxycholate was not converted to other compounds by the Cebus monkey liver. The evidence that cleoxycholatc was poorly absorbed from its site of formation is, however, indirect. In those situat.ions where it is possible to obtain samples of bile by duodenal intubat,ion, a relatively innocuous procedure for estimating bile salt concentration in peripheral blood should be available. Although it was impossible to estimate precisely the rate of clearance by the liver of bile salts from the portal blood because of lack of information about the proportion of the liver circulation supplied by the portal vein and that by the hepatic artery, it was possible to demonstrate very large differcncts in the concentrations of bile salt,:: in the portal and hepat,ic veins.
IS
,jL?:j
BLOOD
Since the preparation of this manuscript, the authors have seen the report of Grundy and Sjovall ( 17) on bile acids in rat, systcmic blood. These workers have based their calculations on the distribution of radioactivity bctn-ccn intestine and blood. The method hcrcin presented would bc applied with great difficulty in rats with their much 1110rc coluplcx liepatic intcrconr-rwions of bile acids.
T.. .4x1) SJOV.4l.L, J., .-lctn Physid. Scnd46,284 (1959). F. E.. J. (‘/in. 1% 2. RI-DMAS, D., -4s” IiESDALL, ITS/. 36, 530 (1057). J. B., ,JN.. J. c/h. In c~,.st. 37, 1494 3. CAREP, (1958). 4. OLI\~RO.\..~,
5. 6. 7. 8. 9.
10. 11.
kJOV4JJ.. R.~TLIFF,
15. A,
J., ilclri
(:h.
C&m.
I<.
4, 652 (1959).
I,., MATWHISEI~, JR.. Hs1.4, S. I,., THATER.
JV. H.. AXJ) 3133 (1959).
Domu.
E.
,I.,
J.
J.
T..
S. A.,
I)OISY, ELJ.IOTT,
Biol. Phcru. 234,
12.
J. T., 13. Hs1.4, S. I,., ELLIOTT, S. H., MATSCWKER, I>OISU, E. .I., Ja., TIIATEH. S. A., ASD I)OISY, E. --I., J. Bid. Chcna. 235, 1963 (1960). 14. Dorsr. E. .I., JR., DAKIELS, M., AXD hI~IERhlAN, hl. A., Fcclcrntion Pm-. 15, 243 (1956). C. H., ~~7.4GXJU~, ~1. F.. WILSOS. A. N., 15. Hmxci, n:.4LTOS, E. tiHC:NIC, c. H., WOLP, D. E., HOLLY.. F. W., AXID FOLKERS, I<., J. Attr. Chum. Soc.79,2316 (1957). 16.
17.