Biochimica et Biophysica Acta, 716 (1982) 383-390 383 Elsevier Biomedical Press

BBA 21143

DIRECT QUANTITATIVE ESTIMATION OF SEVERAL IODOTHYRONINES IN RAT BILE BY RADIOIMMUNOASSAY AND BASAL DATA ON THEIR BILIARY EXCRETION

ONDREJ FOLDES, PAVEL LANGER, KATARiNA STRAUSSOV.A., HANA BROZMANOV/k and KLAUDIA GSCHWENDTOV,h,

Institute of Experimental Endocrinology, Center of Physiological Sciences SA V, 833 06 Bratislava, (Czechoslovakia)

(Received June 22nd, 1981) (Revised manuscript received March 3rd, 1982)

Key words: Iodothyronine assay; Thyroid hormone," Biliary excretion; (Rat)

A method was developed for the hydrolysis of conjugated iodothyronines in bile with the aid of ~-glucuroni- dase/arylsulfatase and for subsequent direct estimation of total and free iodothyronines with the aid of specific radioimmunoassay. The amount of conjugated fraction could then be calculated from the difference. Thus, basal biliary excretion of several iodothyronines was measured in 31 normal, fed rats in which the bile duct was drained with polyethylene tubing under pentobarbiturate anesthesia and the bile was collected for 2 h. The free fraction of thyroxine, 3,5,3'-triiodothyronine and 3,3'-diiodothyronine was approx. 30% of total content, while that of 3,3',5'-triiodothyronine and 3,5-diiodothyronine was approx. 20% and that of 3',5'-di- iodothyronine was less than 10%. This suggests some considerable differences in the conjugation of individual iodothyronines in the liver. The concentration of T 4 in bile was about the same as in plasma, while that of other iodothyronines was about 3-8 times higher than in plasma. This shows close interrelations between the iodothyronine deiodinating pathway in liver cells in vivo and the spectrum of iodothyronine in bile. The average ratio of T 3/rT 3 as found in bile was about 4.

Introduction

The problems of biliary excretion and enter- ohepatic cycle at thyroid hormones have been excellently reviewed by van Middlesworth [1] and recently several reviews on peripheral metabolism of thyroid hormone appeared [2-4]. It is well- known that the liver is a most important place for the thyroid hormone deiodinating pathway and stores a considerable part of the extrathyroidal thyroid hormones. Moreover, the iodothyronines are conjugated predominantly in the liver, which results in the formation of glucuronides and sulfates. These compounds appear in the bile to- gether with a considerable amount of unchanged iodothyronines.

Practically all the present evidence on the excre-

0304-4165/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

tion of iodothyronines by bile is based on the data obtained with the aid of labelled compounds sep- arated by paper [5-13] or high performance liquid chromatography (HPLC) [14]. Most of these data were obtained only with labelled thyroxine. Flock et al. [8], however, used labelled triiodothyronine, and later detected 3,Y,5'-triiodothyronine and 3,3'-diiodothyronine generated from labelled thyroxine with the aid of two-dimensional paper chromatography [15]. Similarly, with the aid of HPLC, Takai et al. [14] recently detected several labelled free and conjugated iodothyronine gen- erated from labelled thyroxine.

The aims of this investigation were, first, to develop a method for direct estimation of iodothy- ronines in bile and, second, to obtain some quantitative data on the concentration of free and

384

conjugated iodothyronines in bile and on their biliary excretion under normal conditions.

Materials and Methods

Animals and operation procedure 31 male Wistar rats (each weighing about 350 g)

purchased from Velaz (Prague) were used. The animals were fed a normal pelleted diet and were maintained in a light (6.00-18.00 h)- and tempera- ture (26 --+ 1 °C) -conditioned room. During the last night before the operation, the animals had free access to food. On the day of experiment, they were anesthetized with 40 mg pentobarbiturate/kg body weight (Pentobarbital, Spofa) intraperitone- ally and, after a medial laparotomy, the bile duct was cannulated using PE 50 polyethylene tubing (Clay Adams, Parsipanny, N.J.) which was fixed by an external ligature as described previously in detail [13,17]. With the same tubing, the femoral artery and vein were also cannulated for obtaining blood samples after intravenous injection of 300 U heparin/kg body weight (Heparin, Spofa) as de- scribed previously [13,17]. The bile was collected for a period of 2 h in all animals between about 10.00 and 12.00 h and a sample of blood was taken at about 11.00 h. Six experiments of five to six animals were done and for the estimation of each iodothyronine, the samples from one to two ex- periments were measured in the same assay.

Estimation of iodothyronines in bile The bile from drained bile ducts was collected

into preweighed vials containing antioxidant (1 mg per vial methylmercaptoirnidazole, Tapazol Lilly) and the volume of bile was estimated by weighing, the specific weight of bile being found to be 1.0.

Estimation of total iodothyronines. 0.9 ml aliquots of bile were transfered into plastic tubes and Lnixed with 0.45 ml acetate buffer (0.5- mol 1- l, pH 5.0) containing 4500 Fishman U of fl-glucuron- idase /a ry lsu l fa tase Sigma (f l -o-glucuronide glucuronosohydrolase/arylsul fate sulfohydrolase, EC 3.2.1.31/EC 3.1.6.1) and 20 /~1 chloroform. The samples were then incubated for 2 h at 37°C in temperature-controlled oven with occasional shaking (approx. every 15 rain). The reaction was stopped by the addition of 0.45 ml glycine buffer (0.5 mol. l - i , pH 9.0). After mixing the samples

were kept for 2 h at 4°C and then centrifuged at 2000g for 20 min at 4°C. Aliquots of the clear supernatant were kept frozen at - 2 0 ° C until the individual iodothyronines were assayed. All the following estimations were run in duplicate.

Estimation of free and conjugated iodothyronines Aliquots of unprocessed bile were used for a direct estimation of the appropriate free compound with the aid of specific radioimmunoassay (see below). The conjugated fraction could be then estimated by a subtraction (free) fraction from the total amount of iodothyronine found in the enzyme treated sample.

Radioimmunoassay of iodothyronines in bile. Aliquots of unprocessed (20/~1 for T4, 50/xl for T~ and 3,5-T 2, 100 ~1 for other compounds) or en- zyme-treated bile (20/tl supernatant for T4, T 3 and 3,5-T 2 and 50 #1 for other compounds, each volume corresponding to a half of that of the original bile) were transfered into polystyrene Falcon type tubes. In each assay the enzyme blank was used contain- ing 500 Fishman U of fl-glucuronidase/arylsulfa- tase in 0.1 ml distilled water and subjected to the following procedure (the final values were close to zero). The volume was then made up to 300 ~l with barbiturate buffer (0.08 mol. l J, pH 8.6) containing 0.1% bovine serum albumin and the standards for a calibration curve were made in the same buffer. 100/~l of radioactive compound (all iodothyronines used were 125I-labelled and of high specific activity, i.e. more than 1.2 mCi. mg-1 and purchased from Medimpex, Budapest) diluted in the above buffer and 100~tl of specific rabbit anti- sera prepared in this laboratory (see below) diluted to the appropriate concentration with the above buffer were added. All authentic iodothyronines used were a gift from Henning, Berlin.

All samples were then mixed and incubated for 2 h at 37°C and then at 4°C overnight. After that 100/~1 y-globulin ( l l . 2g g. 1 -~ in distilled water; Sevac, Prague) and 500/zl 25% poly(ethylene gly- col) 6000 (Fluka) in distilled water were added. The samples were mixed and then centrifuged at 4°C and 3000g for 25 min. The supernatant was removed by aspiration and the radioactivity of the precipitate was counted using a Nuclear Chicago gamma spectrometer. The results were expressed either as a concentration of iodothyronines per unit of bile volume or as an amount excreted per unit of time (Table III).

Radioimmunoassay of iodothyronines in plasma Essentially the same procedure has been used as described above for bile with following modifica- tion: (1) 8-anilino-l-naphtholsulfonic acid (Sigma) was added to a solution of radioactive compound to a final concentration of 0.5 mg. ml-J, (2) for the estimation of T 4 20/xl of plasma were used, while 50/~1 were used for T 3 and 100/~1 for other iodothyronines, (3) all standards were diluted in iodothyronine free rat serum.

Specific rabbit antisera used for iodothyronine radioimmunoassay in plasma and bile The antisera used were prepared by long-term immunization of rabbits using authentic iodothyronines (kindly donated by Henning, Berlin) and conjugated to bovine serum albumin (Calbiochem) with carbodi- imide according to Siegel et al. [16]. Aliquots of conjugates were mixed with complete Freund's adjuvant (Difco) immediately before use. The cross reactions of these antisera are shown in Table I. It may be concluded that there was a considerable cross-reaction of anti-3,5-T2-serum with T 3 (i.e. 9.82%) and of anti-3',5'-T2-serum with rT 3 (i.e. 2.02%). The former cross-reaction was observed by calculating the data presented in Table III, but the latter was neglected. The data in Table I further show that the other cross-reactions were negligible. Table II shows satisfactory data on the detection threshold, recovery in plasma and bile and intra- and interassay variation of the antisera used which correspond to the average standard of radioim- munoassay methods.

385

Verification of some essential steps used for en- zymatic hydrolysis of conjugated iodothyronines

Degree of enzymatic hydrolysis of conjugated T 3 and T 4 The rats were injected 5 #Ci of 125I-labelled T4, 125I-labelled T 3 or 125I-labelled Y,5'-T2 into a venous cannula (see above) and their bile was collected for the subsequent 3 -4 h. Aliquots of bile were incubated with enzyme as described above. Corresponding amounts of unprocessed and di- gested bile were applied to Whatman 3 MM chro- matography paper strips and developed in an ascending system of n-butanol/acetic acid/water (4: 1:5, v/v). The chromatograms were cut into 1-cm strips and counted. It was found that the counts in a peak of representative iodothyronine conjugates left after digestion represent 5-10% of unprocessed bile as shown Ior T 4 in Fig. 1.

The concentration of enzyme As shown in Fig. 2, a maximum effect was found with enzyme con- centration of 2.5-10.0 Fishman U per/~l. A con- centration of 5.0 U-/~1-1 (i.e. 10 Fishman U per /xl of bile) was selected for routine use.

Time of incubation Maximum liberation of thyroxine was found after 2-h incubation under the conditions described above. No further in- crease of T 4 yield was found up to 6-h incubation.

Temperature and pH of incubation These factors are recommended by the manufacturer of enzyme. Nevertheless, it was tested whether such instruc- tions are valid also for conjugated iodothyronines. It was confirmed that the optimum conditions are at 37°C and pH 5.0.

TABLE I

CROSS REACTIONS OF ANTI-IODOTHYRONINE SERA USED

Indicated cross-reactivities were obtained as a ratio of concentrations of appropriate iodothyronine to individual tested iodothyronines necessary to produce 50% displacement.

Antibodies Cross-reactions (%)

to T 4 "1"3 rT3 3,3'-T2 3',5'-T2 3,5-T2

T 4 100 0.021 0.09 0.002 0.001 0.06 T 3 0.28 100 0.0001 0.32 0.0001 0.42 rT 3 0.092 0.004 100 0.001 0.036 0.001 3,Y-T 2 0.001 0.75 0.25 100 0.01 0.005 3',5'-T 2 0.05 0.03 2.02 0.03 100 0.0001 3,5-T 2 0.0001 9.82 a 0.0001 0.045 0.0001 100

a This cross reaction was observed by calculating the values of 3,5-T z presented in Table III

R e s u l t s

TABLE II

SOME QUALITATIVE CHARACTERISTICS OF THE METHODS USED FOR IODOTHYRONINE RADIO|MMUNOASSAY IN RAT PLASMA AND BILE

Values for recovery are the mean -+ S.E. taken from l0 estimations CV, coefficient of variation

Antigen Detection Recovery (%) Intraassay Interassay threshold variation variation per tube Plasma Bile (CV) (CV) (fmol)

T 4 41.4 97.7± 7.1 96.0± 6.2 7.2 13.0 T 3 66.3 92.4--+11.0 94.1- + 8.8 8.9 17.7 rT 3 62.5 93.7-+ 10.7 102.4± 7.2 8.0 14.2 3,Y-T 2 6.9 100.6-+ 7.0 93.3-- + 9.0 6.6 10.9 3',5'-T 2 5.7 96.2- + 5.2 100.3-- + 5.2 5.9 11.2 3,5-T 2 21.8 91.6-+ 6.2 89.9-+ 10.0 10.1 19.6

Serial dilutions of bile A n u m b e r of observat ions with various iodothyronines has been performed

using serial di lut ions of bile to check the l ineari ty

of assays. Practically in all cases a close interrela-

t ion between serial d i lut ion data and cal ibra t ion

curve was ob ta ined as demons t ra ted for 3,3'-T 2 in Fig. 3.

Bq

1000 ]

Table I I I shows the data ob ta ined by analyzing the level of several iodothyronines in p lasma and bile of 31 rats. The average bile flow was 1 . 2 8 - 0.08 ml . h - i (mean -+ S.E.).

500-

CT~

386

~.,..

~ " : " v'~ "~"~

1 5 10 15 20 25 (m

Fig. 1. Distribution of t2~l on paper chromatograms (for details see text). ( ), unhydrolyzed bile; ( - - - --), hydrolyzed bile; ( . . . . . ), -authentic t25I-labelled -1"4, CT 4 conjugated T 4, T 4 unconjugated T 4.

The highest amoun t of total iodothyronine de- tected in bile was that of T 4, while that of T 3 was

abou t four times less thus giving a T 4 / T 3 ratio abou t 4. The concent ra t ion of 3,5-T 2 was remarka-

bly high, being nearly a half of that of T 3 and

twice as high as that of rT 3. Finally, the concentra- t ion of rT3, 3,3'-T 2 and 3 ' ,5 ' -T 2 was about five

t imes less than that of T 3. The T3 / r T 3 ratio was about 4. These f indings differ considerably from

the relations between iodothyronines in plasma,

where T 4 / T 3 ratio was about 35 and T 3 / r T 3 ratio was about 2. The level of rT 3 and 3,5-T 2 was about

a half of that of T 3, bu t was about twice as high as that of 3,3'-T 2 and Y,5'-T 2.

The relations between the concentra t ions of free iodothyronines in bile were somewhat different

70'

~ 6 0 ,

E~50

~.0

o,1 o;s (o 2s slo ~o ~.o s~o [Utuf I ]

Fig. 2. Concentration of thyroxine (T4) in a pooled bile in- cubated with various concentrations of ~8-glucuronidase/aryl- sulfatase (U. /~ 1- i ).

387

.o .o

0 ~

~ 2

0 ~

~ o " ~

z ~ ~ . ~

0

~ .~ "~ "~

~< m_uo

i i.o.¢ o ~~ ~° ~.~ ~

t~

0

0

.i0

d Z

d Z

e ~

d Z

7

.2.

o d o o d d

0

0

~ 0 0 0 0 0

~ d d d d d

~ ~ .~~ m

388

Bq

150

100

I I |

0 1.0 2,0 3.0 pmol m1-1 I I

so 7's b,,e

Fig. 3. Comparison of calibration curve of 3,3'-T 2 with various amounts of 3,Y-T 2 in serial dilutions of bile. The aliquot of pooled bile was incubated with enzyme as described in the text. The amounts of bile shown in a scale indicate the volumes of original bile which means that a double volume of supernatant was used for each sample. The location of scale for bile volume was adjusted to the main scale for 3,3'-T 2 concentration accord- ing to the counts. Each point shows a mean of four parallel samples and S.E. O,bile; O, standard.

from these of total ones presented above, the differences being apparently due to a different degree of conjugation of individual compounds. Thus, by subtraction of free fraction from a total amount of the appropriate compound it may be seen that there was about 70% of conjugated T 4, T 3 and 3,3'-T 2, about 80% of conjugated rT 3 and 3,5-T 2 and about 90% of conjugated 3',5'-T 2. From this it may be suggested that some structural dif- ferences in a conjugation of iodothyronines may exist in a liver cell and that the absence of iodine in tyrosyl ring perhaps favors the conjugation pro- cess.

The ratio of T 4 concentration in bile to that in plasma ( B / P ratio in Table III) was close to 1.0, but that of other iodothyronines was several times higher, being about 8.0 for T 3 and 3,5-T 2 and about 4.0 to 6.0 for other compounds.

The amount of T 4 excreted by bile per hour corresponded to its content in about 1 ml of plasma, but that of T 3 and 3,5-T 2 corresponded to about 10 and 15 ml of plasma, respectively, which

means that a total amount of these compounds in plasma is excreted by bile in approx 1 h. For other iodothyronines this figure is equivalent to about 4 -7 ml of plasma.

Discussion

As presented, the standards of iodothyronines for a calibration curve were diluted in barbiturate buffer. This was because we did not succeed in obtaining iodothyronine free-bile even several weeks after surgical thyroidectomy followed by radioiodine administration which is in aggreement with recently published data [26] where about 20% of original triiodothyronine concentration was still found in rat liver 2 -4 months after thyroid abla- tion and, moreover, we did not choose the removal of iodothyronines from normal bile either with charcoal or with various resins or polydextrans since this would remove also a number of low molecular weight substances from the original bile. Actually, it should be pointed out that the final dilution of bile in an assay system used for various iodothyronines was 1:5 to 1:25 for free fraction and 1:16 to 1:50 for a total amount. In our opinion, such differences in bile content between the standards and unknown samples may be prac- tically neglected, although theoretically some in- fluence of this factor may be expected. This view seems to be supported by the data on the recovery from bile (Table II). The same data strongly sup- port our opinion that conjugated iodothyronines did not cross-react with the antisera used, al- though this was not directly checked. Finally, both the above discussed opinions may be further sup- ported by the data obtained in dilution experi- ments (e.g. Fig. 3).

The data obtained in this investigation show that, under the conditions used, 90-95% splitting of conjugated iodothyronines tested occurs. This means that the true values would be about 3.5-7% higher than indicated in Table III for total iodothyronines and these of conjugated ones 5-10% higher than indicated for this fraction. However, it may be assumed that considering the data on recovery and on intra- and interassay variation, these differences are apparently in the range of experimental error. From Table I, it may also be concluded that most of the cross-reactions

389

of the individual antisera did not contribute to the experimental error. In addition, it should be pointed out that using this method quantitative and absolute values may be obtained. In contrast, paper chromatography or HPLC give only quali- tative data, or indicate some relative distribution of individual iodothyronines or their conjugates in bile after the injection of labelled T 4 or T 3 . More- over, the HPLC system recently described [14] did not separate 3,Y-T 2 from 3',5'-T 2 and did not offer any data on 3,5-T 2. In contrast, our method could not distinguish the glucuronates from sulfates; this fact does not conflict with the conclusions of this report.

Another question to be discussed is whether the biliary disposal of various iodothyronines obtained in pentobarbiturate anesthetized, operated and heparin injected animals may be influenced by these interventions. We previously found a de- crease of plasma T 4 level at 30 and 60 min after a single intraperitoneal injection of pentobarbiturate [ 17,18], but we did not find any significant changes of plasma T 4 level after a single intraperitoneal injection of 800 and 2400 U of heparin per rat [18] which is about 3-8-times higher dose than that used in these experiments. Recently, Nicoloff et at. [19] described a reduction of T 4 uptake by liver slices after the addition of such a high amount of heparin as 1000 U per ml of incubation medium. Also, immobilization stress was found to decrease remarkably the level of T 4 and T 3 in rat plasma within 240 min, while that of rT 3 remained either unchanged or was slightly increased (Langer, P., Frldes, O., Kveti~ansk~, R., Culman, J. and Torda, M., unpublished data), which may be applied to operation stress. From these findings it appears that some effects of these factors on the observed data cannot be excluded. So far, there are only few observations showing that acute changes of blood level of thyroxine are rapidly reflected in the changes of its biliary disposal [13,20] or even on the disposal of T 3 [21] and other iodothyronines (Frldes, O., Langer, P., Straussovh, K. and Gschwendtov~ K., unpublished data).

Our findings of approx. 70% of conjugated T 4 are in agreement with those obtained several hours after the injection of labelled T 4 [13,14,22] and may be considered to represent a steady state. However, at intervals less than 2 -4 h after the

administration of tracer approx. 40-60% of the conjugated fraction was found [13,14,23].

The hepatic Clearance of T 4 was reported to be 0.7-2.0 m l . h - I [1,21,24,25]. From Table III, it may be concluded (by comparing the concentra- tion of T 4 in plasma and its excretion in bile) that the clearance of T 4 in our animals was about 1.0 m l - h - t , which agrees with the above data. In contrast, however, when comparing similar data for other iodothyronines the hepatic clearance would be 4-15 ml . h - i . Since there is apparently no special reason why the biliary clearance for various iodothyronines would differ so much from that of T 4, it may be suggested that the biliary excretion of these reflects the actual interrelations between individual steps of iodothyronine de- iodination in liver cells. From this it appears that, according to these data, the calculation of hepatic clearance of iodothyronines using simply a similar formula as used for a calculation of renal clearance of various substances, i.e. C = U. V / P (where C = clearance as volume of plasma per time, U = concentration of a substance in urine or bile, V = volume of urine or bile per time, P -- concentration of a substance in plasma) as reported by several authors [1,21,24,25] does not yield correct values, since, in a case of biliary excretion of iodothyron- ines, U does not depend only on V and P, but, in a considerable extent, either on metabolic degrada- tion (in a case of T4) or metabolic production (in a case of various tri- and diiodothyronines) in the liver. Finally, it should be mentioned that a direct estimation of actual changes of iodothyronine bil- iary excretion may be helpful to elucidate biliary disposal of iodothyronines an its relations to their deiodinating metabolism in the liver under in vivo conditions.

Acknowledgements

The authors wish to express their appreciation to Dr. E. Scheiffele and Dr. H. Steinmaus (Hen- ning, Berlin GmBH) for the generous gift of authentic unlabelled iodothyronines used in this study and to Dr. Grza T6th (Institute of Isotopes, Hungarian Academy of Sciences, Budapest) for preparing and supply of labelled diiodothyronines of high specific activity.

390

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