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Biliary excretion of 3,5,3′-triiodo-l -thyronine-125I by the brook trout, Salvelinus fontinalis (Mitchill)
GENERAL
AND
COMPARATIVE
Biliary
ENDOCRINOMGY
Excretion Brook
(1971)
of 3,5,3’-Triiodo-L-thyronine-1251 Trout,
J. G. EALES, Department
16, 169-175
LINDA
of Zoology,
Salvelinus
fontinalis
A. WELSH, University
AND
of Manitoba,
Received
July
by the (Mitchill)l
HO01
HAR
Winnipeg,
GHAN Manitoba
1, 1970
The percentage of administered radioactivity occurring in several organs and in protein-bound and non-protein-bound serum fractions was measured following intraperitoneal injection of 3,5,3’-triiodo-L-thyronine-“” I (Ta*), into brook trout acclimated at 10°C. The sequence of maximum liver uptake of 8.0% within 24 hours, followed by maximum gall bladder uptake including bile of 10.9% at 48 hours and by maximum intestinal uptake including contents of 20.7% at 96 hours suggested a biliary excretion route for T;*. By descending paper chromatography (butanol : acetic acid: HSO, 4: 1: 1) most bile radioactivity occurred as two adjacent peaks (RI 0.2-0.3) while approximately 15% corresponded to free thyronines. Bile ““I-levels were negligible. T,* loss from the serum was described by fast (tv = 6.9 hours) and slow (t”” = 65.4 hours) exponents. Ta* did not appear to bind as extensively as Td* (Eales, 1970) to serum proteins of brook trout, but both deiodination and biliary excretion seemed less pronounced for TX* than for Td*.
Biliary excretion of thyroid hormones principally as glucuronide or sulfate conjugates has been described for several mammals (reviewed by Tata, 1964; Bollman and Flock, 1965) and chickens (Hutchins and Newcomer, 1966). Biliary excretion of radiothyroxine has been demonstrated recently in teleosts by Osborn and Simpson (1969) for the plaice, Pleuronectes platessa, and by Eales (1969, 1970) for the brook trout, Salvelinus fontinalis. However, there are no data for fish on biliary excretion of triiodothyronines which in at least one salmonid, the rainbow trout, Salmo gairdneri, comprise a high percentage of circulating thyronines (Jacoby and Hickman, 1966). In this study, biliary excretion of intraperitoneally injected 3,5,3’-triiodo-L-thyronine-1251 (T3*) has been followed in the brook trout. ‘Supported by grants-in-aid the National Research Council by a Fisheries Research Board to the University of Manitoba Research Unit.
of
research from Canada and of Canada Grant Aquatic Biology of
169 @ 1971 by
Academic
Press,
Inc.
MATERIALS
AND
METHODS
Eighty Zyear-old brook trout (average weight, 142.9 g, range 75.2232.5 g; average length 23.8 cm, range 19.3-27.9 cm) obtained from the Province of Manitoba trout hatchery, West Hawk Lake, were acclimated in the laboratory at 10°C in running water. Ground beef liver was fed on alternate days until 1 day before injection, and food was then withheld for the duration of the experiment. Sampling procedures were almost identical to those used previously in radiothyroxine studies on brook trout (Eales, 1970). Trout were injected intraperitoneally with 0.25 PCi of 3,5,3’-triiodo-nthyronine-=“I (Nuclear Consultants; specific activity 16.56 &i/ug) and containing <3% inorganic radioiodide, in 0.1 ml of 50% propylene glycol. Eight fish were killed at each of 0, 1, 5.5, 17.5, and 28 hours, and 2, 3, 4, 6, and 8 days post injection (p.i.). Serum samples were collected and serum and carcasses were frozen at -22°C. The radioactivity in serum, individual organs thyroid, liver, gall bladder (including bile), stomach and intestine (including contents), and the whole body was expressed as the percentage of the injected dose. Serum protein-bound (Trichloroacetic acid-
170
EALEY,
\l’ELSH,
.lNl)
CH.\N
m
FIG. 1. Percent of total serum radioactivity occurring in the protein-bound fraction at variolls times after intraperitoneal injection of Ta*. Each point represents the mean ( k SE) for 8 brook trout.
precipitated) radioactivity (PBI-?C) and nonprotein-bound radioactivity percentages were cxpressed in a slightly modified form as: To injected radioactivity X body wt.(g) serum(m1) X 100 This expression corrects for the altered distribution volume of a standard dose being injected into fish of different sizes. Serum and bile radioactive materials were subjected to preliminary separation by descending paper chromatography (butanol:acetic acid:water, 4: 1: 1, v/v) using procedures already described (Eales, 1970). RESULTS Serum
At 1 hour p.i. 54% of the serum mdioactivity was protein-bound, and this proportion fell exponentially with time to 1570 at 8 days
(Fig.
1). Serum
radioactive
able proportion of the circulating thyronine (probably mostly T,“) is not precipitated with the proteins. After an initial rise presumably due to T,” uptake from coelom to blood, both bound and unbound lz51 fell in a multiexponential fashion (Figs. 3 and 4). This multiexponential decrease of serum radioactivity with time for both PBI-lZ”I and I-Kt fractions was analyzed in terms of a rapid iphase I) and a slow (phase II) exponent as follows. The line of best fit rlescribing phase II was calculated for values from 2 to 8 days and extrapolated to zero time. Values on this line corresponding to 5.5, 17.5, and 28 hours were subtracted from the recorded values at these times. The differences so obtained
ma-
terials were separated chromatographically for the fish killed at 1, 17.5, and 48 hours. At 1 hour p.i. 6.8 -t- 0.52 (SE) s, at 17.5 hours, 8.4 f 1.45%, and at 48 hours, 9.6 +5.0% of the serum radioactivity corresponded in migration to lZ51-, while most of the radioactivity corresponded to T,“. However, since this solvent system does not distinguish individual thyronines, the exact identity of the T,* was not certain. Other thyronines could be present. A typical serum radiochromatogram is shown in Fig. 2. Most of the unbound radioactivity is not 1251-and, by this particular FIG. 2. Radiochromatogram (butanol: acetic acid acid:H?O; 4: 1:l v/v) of serum from brook trout application of the trichloroacetic method to brook trout serum, a consider- killed 1 hour after intraperitoneal inject,ion of T,*.
BILIARY
FIG. 3. Decrease Ts*. Rate constants point represents the wt. (g)]/[serum (ml)
FIG. 4. Decrease T,*. Rate constants point represents the wt. (g)]/[serum (ml)
EXCRETION
OF
T3
BY
TROUT
171
in serum protein-bound radioactivity with time following intraperitoneal injection of (fraction/hour) were calculated as -0.10 for phase I and -0.011 for phase II. Each mean (&SE) for 8 brook trout. % dose/ml serum = [% injected radioactivity X body X 1001.
in serum non-protein-bound radioactivity with time after intraperitoneal injection of (fraction/hour) were calculated as -0.124 for phase I and -0.0033 for phase II. Each mean (*SE) for 8 brook trout. y0 dose/ml serum = [% injected radioactivity X body X 1001.
172
EALES,
WELSH,
TABLE BIOLOGICAL
HALF-LIVES FROM SERUM
(t+) ASU FIUCTIONS
RATE AND
AND
CHAN
1
CONSTA~VTS (k) FOR L)JSAPP~.~R~~CE TISSUES OF BROOK THOUT IKJECTED
fi
Tissue Serum
PBI-9
Serum
unbom~d
(fraction/hri
t:
Liver Gall bladder
6.94 65.4 5.6 208 60.1 6‘2.8
-0.100 -0.0110 -0.124 -0.0033 -0.0115 -0.0110
Intestine Enterohepatic Extraenterohepatic Thyroid Total body
88.1 264 182 214 210
-0.0079 -0.0025 -0.0038 -0.0032 -0.0033
(I) (11) I251 (I) (II)
-.
--
Time int,erval OII which t+ and k were based 5.5-28 2-8 5.5-28 2-8 5.5 hr-8 2-8
hr days hr days days days (less 4 days) 6-8 days 5.5 hr-8 days 1 hr-8 days 2-8 days 1 hr-8 days
Rate constants for unbound and bound lz51 were similar for phase I, presumably representing distribution of T,” to tissues. For phase II, bound and unbound lz51 rate
were replotted, and phase I was calculated from the line of best fit for these points. Half-lives and rate constants are given in Table 1. 60’~w,_ 5040-
OF R.~DIO.WTIVITY WITH T3*.a
--__ --w_ -4
30-
I -: 0.9 -0.8 -: 0.7 -!
-4
:
0.6 t
0.5 b
FIG. 5. Percent of the dose of radioactivity occurring in intact liver, gall bladder, and intestine at various times after intraperitoneal injection of Ta*. Each point represents the mean for 8 brook trout. Four of the fish at 4 days were unusual in having completely empty gall bladders.
BILIARY
EXCRETION
constants were different. This is expected. While the rate constant for bound lz51 presumably represents principally loss of T3+, that for unbound lz51 represents not only TS* loss but is also influenced by alteration in the level of lz51-. It is therefore difficult to interpret. The slower loss of unbound lz51 is probably due to the slow excretion of lz51- in this species (unpublished data, Dorey and Eales; Higgs and Eales) . Percent Uptake of Radioactivity Various Organs
by
Uptakes of radioactivity by the enterohepatic organs (liver, gall bladder, and in-
O-IL 0
I I
2
3
OF
T3
BY
173
TROUT
testine posterior to entrance of bile duct) are shown in Fig. 5. The sequence of temporary accumulation of radioactivity in the liver to a maximum of 80 k 0.46 within 24 hours, followed by maximum gall bladder uptake including bile of 10.9 -+ 3.6% at 48 hours and intestine with a maximum of 20.7 t 2.0% at 96 hours suggests loss of T,” from liver to bile to intestine. The intestinal peak in radioactivity at 5 hours is probably due to the initial uptake of T,* from the coelom where it was injected. Half-lives and rate constants were calculated for the exponential phases of radioactive decline in these tissues (Table 1). Uptake and loss of radioactivity by the
5
I
I
I
6
7
%
FIG. 6. Percent hepatic injection
of the dose of radioactivity occurring in total body, extraenterohepatic organs (liver, gall bladder, and intestine), stomach, and thyroid at various times of T3*. Each point represents the mean for 8 brook trout.
after
organs, enterointraperitoneal
17.5 HR
FIG.
17.5,
7. Radiochromatograms 48 and 144 hours after
(butanol:acetio acid:H>O, 4: 1: 1, v/v) intraperitoneal injection of Ta*.
stomach, the thyroid, the enterohepatic organs combined, the whole body, and the extra enterohepatic tissues, are shown in Fig. 6 and half-lives an,d rate constants in Table 1. Of particular interest is the low thyroid uptake of radioactivity, which showed a decline rather than a rise from 24 hours onward and never exceeded 1.5% of the injected dose. Since the thyroid tends to concentrate radioiodide, this confirms the low circulating lz51- levels obtained previously on radiochromatograms. The curve for stomach suggests that it is a prominent site for T,” absorption from the coelom after intraperitoneal injection.
of bile from
brook
trout
killed
at
occurred as two peaks at Rf 0.2-0.4. A peak corresponding to free T, accounted for 15.2 + 6.3% of the radioactivity on the chromatogram at 17.5 hours, 15.1 & 5.0% at 48 hours, and 12.3 + 3.0% at 144 hours. However, since this solvent system does not distinguish individual thyronines, the exact identity of T, was not certain. Other thyronines could be present. DISCUSSION
Little discussion is necessary since these results closely resemble in most respects those obtained after intraperitoneal injection of radiothyroxine (T4*) into brook trout acclimated at 10°C (Eales, 1970). Significant biliary excretion occurred for Chromatographic Separation of Bile both hormones and, as judged by a single Radioactive Materials paper chromatography solvent system, simIndividual chromatograms were run on ilar types of derivatives appeared in the bile for each of the 24 fish killed at 17.5, bile. From the work of &born and Simp48, and 144 hours pi. Typical chromat- son (1969) on plaice, the presence of gluograms are shown in Fig. 7. Negligible curonic acid and sulfuric acid conjugates of T, and T, in brook trout bile is anticilz51- was present, and most radioactivity
BILIART
EXCRETION
pated. Major thyroid hormone bile derivatives are in the process of being identified for the brook trout. This work will be reported elsewhere. However, in contrast to T,, T3 does not seem to bind so extensively to serum proteins as judged by our application of the trichloroacetic acid-precipitation test. Despite this lesser binding, a slower loss of radioactivity was recorded from the body and organs of the T,*-injected trout, indicating slower peripheral metabolism of T,. Serum radioiodide levels as judged from several lines of evidence were lower in T,*- than in T,“-injected trout suggesting less rapid deiodination of T,“. Furthermore, a greater uptake and a more rapid loss of liver and gall bladder radioactivity occurred following T,* injection, suggesting a more rapid biliary excretion of T,“. These differences probably represent real differences in the metabolism of the two thyroid hormones. However the Tn and T, experiments were carried out on fish of different ages and sizes and at a different season. The modifying effect of these and other variables cannot be overlooked. ACKNOWLEDGMENTS The authors wish to thank Mr. Schaldemose. Province of Manitoba Trout Hatchery, West
OF
T3
BY
TROUT
175
Hawk Lake, for providing the brook trout through the courtesy of Dr. K. Doan, Director of Fisheries, Province of Manitoba. REFERENCES J. L., AND FLOCK, E. V. (1965). The role of the liver in the metabolism of Y-thyroid hormones and analogues. In “The Biliary System” (W. Taylor, ed.), pp. 345-367. Blackwell, Oxford. EALES. J. G. (1969). Bi!e excretion of radiothyroxine by the brook trout, Salvelinus jontinalis (Mitchill). Gen. Camp. Endoc inol. 13, 502. EALES, J. G. (1970). Biliary excretion of radiothyroxine by the brook trout, Szlvelinus fontin&s (Mitchill). Gen. Comp. Endoctinol. 14, 385-395. HUTCHINS, M. O., .~ND NEWCOMER. W. S. (1966). Metabolism and excretion of thyroxine and triiodothyronine in chickens. Gen. Camp. EnBOLLMAN,
docrinol.
6,
239-248.
AND HICKMAN, C. P., JR. (1966). A study of the circulating iodocompounds of rainbow trout, Salmo gairdneri, by the method of isotopic equilibrium. Gen. Comp. Endocrinol. 7, 245-254. OSEORN. R. H. AND SIMPSON, T. H. (E969). Thyroxine metabolism in plaice (Pleuwwze&w platessa L.). Gen. Comp. Endocrinol. .:I’$. -524. TATA, J. R. (1964). Distribution and metabolism of thyroid hormones. In “The ThyroidGland” (R. Pitt Rivers and W. R. Trotter, cdsd. pp. 163-186. JACOBY,
G.
H.,
AND
COMPARATIVE
Biliary
ENDOCRINOMGY
Excretion Brook
(1971)
of 3,5,3’-Triiodo-L-thyronine-1251 Trout,
J. G. EALES, Department
16, 169-175
LINDA
of Zoology,
Salvelinus
fontinalis
A. WELSH, University
AND
of Manitoba,
Received
July
by the (Mitchill)l
HO01
HAR
Winnipeg,
GHAN Manitoba
1, 1970
The percentage of administered radioactivity occurring in several organs and in protein-bound and non-protein-bound serum fractions was measured following intraperitoneal injection of 3,5,3’-triiodo-L-thyronine-“” I (Ta*), into brook trout acclimated at 10°C. The sequence of maximum liver uptake of 8.0% within 24 hours, followed by maximum gall bladder uptake including bile of 10.9% at 48 hours and by maximum intestinal uptake including contents of 20.7% at 96 hours suggested a biliary excretion route for T;*. By descending paper chromatography (butanol : acetic acid: HSO, 4: 1: 1) most bile radioactivity occurred as two adjacent peaks (RI 0.2-0.3) while approximately 15% corresponded to free thyronines. Bile ““I-levels were negligible. T,* loss from the serum was described by fast (tv = 6.9 hours) and slow (t”” = 65.4 hours) exponents. Ta* did not appear to bind as extensively as Td* (Eales, 1970) to serum proteins of brook trout, but both deiodination and biliary excretion seemed less pronounced for TX* than for Td*.
Biliary excretion of thyroid hormones principally as glucuronide or sulfate conjugates has been described for several mammals (reviewed by Tata, 1964; Bollman and Flock, 1965) and chickens (Hutchins and Newcomer, 1966). Biliary excretion of radiothyroxine has been demonstrated recently in teleosts by Osborn and Simpson (1969) for the plaice, Pleuronectes platessa, and by Eales (1969, 1970) for the brook trout, Salvelinus fontinalis. However, there are no data for fish on biliary excretion of triiodothyronines which in at least one salmonid, the rainbow trout, Salmo gairdneri, comprise a high percentage of circulating thyronines (Jacoby and Hickman, 1966). In this study, biliary excretion of intraperitoneally injected 3,5,3’-triiodo-L-thyronine-1251 (T3*) has been followed in the brook trout. ‘Supported by grants-in-aid the National Research Council by a Fisheries Research Board to the University of Manitoba Research Unit.
of
research from Canada and of Canada Grant Aquatic Biology of
169 @ 1971 by
Academic
Press,
Inc.
MATERIALS
AND
METHODS
Eighty Zyear-old brook trout (average weight, 142.9 g, range 75.2232.5 g; average length 23.8 cm, range 19.3-27.9 cm) obtained from the Province of Manitoba trout hatchery, West Hawk Lake, were acclimated in the laboratory at 10°C in running water. Ground beef liver was fed on alternate days until 1 day before injection, and food was then withheld for the duration of the experiment. Sampling procedures were almost identical to those used previously in radiothyroxine studies on brook trout (Eales, 1970). Trout were injected intraperitoneally with 0.25 PCi of 3,5,3’-triiodo-nthyronine-=“I (Nuclear Consultants; specific activity 16.56 &i/ug) and containing <3% inorganic radioiodide, in 0.1 ml of 50% propylene glycol. Eight fish were killed at each of 0, 1, 5.5, 17.5, and 28 hours, and 2, 3, 4, 6, and 8 days post injection (p.i.). Serum samples were collected and serum and carcasses were frozen at -22°C. The radioactivity in serum, individual organs thyroid, liver, gall bladder (including bile), stomach and intestine (including contents), and the whole body was expressed as the percentage of the injected dose. Serum protein-bound (Trichloroacetic acid-
170
EALEY,
\l’ELSH,
.lNl)
CH.\N
m
FIG. 1. Percent of total serum radioactivity occurring in the protein-bound fraction at variolls times after intraperitoneal injection of Ta*. Each point represents the mean ( k SE) for 8 brook trout.
precipitated) radioactivity (PBI-?C) and nonprotein-bound radioactivity percentages were cxpressed in a slightly modified form as: To injected radioactivity X body wt.(g) serum(m1) X 100 This expression corrects for the altered distribution volume of a standard dose being injected into fish of different sizes. Serum and bile radioactive materials were subjected to preliminary separation by descending paper chromatography (butanol:acetic acid:water, 4: 1: 1, v/v) using procedures already described (Eales, 1970). RESULTS Serum
At 1 hour p.i. 54% of the serum mdioactivity was protein-bound, and this proportion fell exponentially with time to 1570 at 8 days
(Fig.
1). Serum
radioactive
able proportion of the circulating thyronine (probably mostly T,“) is not precipitated with the proteins. After an initial rise presumably due to T,” uptake from coelom to blood, both bound and unbound lz51 fell in a multiexponential fashion (Figs. 3 and 4). This multiexponential decrease of serum radioactivity with time for both PBI-lZ”I and I-Kt fractions was analyzed in terms of a rapid iphase I) and a slow (phase II) exponent as follows. The line of best fit rlescribing phase II was calculated for values from 2 to 8 days and extrapolated to zero time. Values on this line corresponding to 5.5, 17.5, and 28 hours were subtracted from the recorded values at these times. The differences so obtained
ma-
terials were separated chromatographically for the fish killed at 1, 17.5, and 48 hours. At 1 hour p.i. 6.8 -t- 0.52 (SE) s, at 17.5 hours, 8.4 f 1.45%, and at 48 hours, 9.6 +5.0% of the serum radioactivity corresponded in migration to lZ51-, while most of the radioactivity corresponded to T,“. However, since this solvent system does not distinguish individual thyronines, the exact identity of the T,* was not certain. Other thyronines could be present. A typical serum radiochromatogram is shown in Fig. 2. Most of the unbound radioactivity is not 1251-and, by this particular FIG. 2. Radiochromatogram (butanol: acetic acid acid:H?O; 4: 1:l v/v) of serum from brook trout application of the trichloroacetic method to brook trout serum, a consider- killed 1 hour after intraperitoneal inject,ion of T,*.
BILIARY
FIG. 3. Decrease Ts*. Rate constants point represents the wt. (g)]/[serum (ml)
FIG. 4. Decrease T,*. Rate constants point represents the wt. (g)]/[serum (ml)
EXCRETION
OF
T3
BY
TROUT
171
in serum protein-bound radioactivity with time following intraperitoneal injection of (fraction/hour) were calculated as -0.10 for phase I and -0.011 for phase II. Each mean (&SE) for 8 brook trout. % dose/ml serum = [% injected radioactivity X body X 1001.
in serum non-protein-bound radioactivity with time after intraperitoneal injection of (fraction/hour) were calculated as -0.124 for phase I and -0.0033 for phase II. Each mean (*SE) for 8 brook trout. y0 dose/ml serum = [% injected radioactivity X body X 1001.
172
EALES,
WELSH,
TABLE BIOLOGICAL
HALF-LIVES FROM SERUM
(t+) ASU FIUCTIONS
RATE AND
AND
CHAN
1
CONSTA~VTS (k) FOR L)JSAPP~.~R~~CE TISSUES OF BROOK THOUT IKJECTED
fi
Tissue Serum
PBI-9
Serum
unbom~d
(fraction/hri
t:
Liver Gall bladder
6.94 65.4 5.6 208 60.1 6‘2.8
-0.100 -0.0110 -0.124 -0.0033 -0.0115 -0.0110
Intestine Enterohepatic Extraenterohepatic Thyroid Total body
88.1 264 182 214 210
-0.0079 -0.0025 -0.0038 -0.0032 -0.0033
(I) (11) I251 (I) (II)
-.
--
Time int,erval OII which t+ and k were based 5.5-28 2-8 5.5-28 2-8 5.5 hr-8 2-8
hr days hr days days days (less 4 days) 6-8 days 5.5 hr-8 days 1 hr-8 days 2-8 days 1 hr-8 days
Rate constants for unbound and bound lz51 were similar for phase I, presumably representing distribution of T,” to tissues. For phase II, bound and unbound lz51 rate
were replotted, and phase I was calculated from the line of best fit for these points. Half-lives and rate constants are given in Table 1. 60’~w,_ 5040-
OF R.~DIO.WTIVITY WITH T3*.a
--__ --w_ -4
30-
I -: 0.9 -0.8 -: 0.7 -!
-4
:
0.6 t
0.5 b
FIG. 5. Percent of the dose of radioactivity occurring in intact liver, gall bladder, and intestine at various times after intraperitoneal injection of Ta*. Each point represents the mean for 8 brook trout. Four of the fish at 4 days were unusual in having completely empty gall bladders.
BILIARY
EXCRETION
constants were different. This is expected. While the rate constant for bound lz51 presumably represents principally loss of T3+, that for unbound lz51 represents not only TS* loss but is also influenced by alteration in the level of lz51-. It is therefore difficult to interpret. The slower loss of unbound lz51 is probably due to the slow excretion of lz51- in this species (unpublished data, Dorey and Eales; Higgs and Eales) . Percent Uptake of Radioactivity Various Organs
by
Uptakes of radioactivity by the enterohepatic organs (liver, gall bladder, and in-
O-IL 0
I I
2
3
OF
T3
BY
173
TROUT
testine posterior to entrance of bile duct) are shown in Fig. 5. The sequence of temporary accumulation of radioactivity in the liver to a maximum of 80 k 0.46 within 24 hours, followed by maximum gall bladder uptake including bile of 10.9 -+ 3.6% at 48 hours and intestine with a maximum of 20.7 t 2.0% at 96 hours suggests loss of T,” from liver to bile to intestine. The intestinal peak in radioactivity at 5 hours is probably due to the initial uptake of T,* from the coelom where it was injected. Half-lives and rate constants were calculated for the exponential phases of radioactive decline in these tissues (Table 1). Uptake and loss of radioactivity by the
5
I
I
I
6
7
%
FIG. 6. Percent hepatic injection
of the dose of radioactivity occurring in total body, extraenterohepatic organs (liver, gall bladder, and intestine), stomach, and thyroid at various times of T3*. Each point represents the mean for 8 brook trout.
after
organs, enterointraperitoneal
17.5 HR
FIG.
17.5,
7. Radiochromatograms 48 and 144 hours after
(butanol:acetio acid:H>O, 4: 1: 1, v/v) intraperitoneal injection of Ta*.
stomach, the thyroid, the enterohepatic organs combined, the whole body, and the extra enterohepatic tissues, are shown in Fig. 6 and half-lives an,d rate constants in Table 1. Of particular interest is the low thyroid uptake of radioactivity, which showed a decline rather than a rise from 24 hours onward and never exceeded 1.5% of the injected dose. Since the thyroid tends to concentrate radioiodide, this confirms the low circulating lz51- levels obtained previously on radiochromatograms. The curve for stomach suggests that it is a prominent site for T,” absorption from the coelom after intraperitoneal injection.
of bile from
brook
trout
killed
at
occurred as two peaks at Rf 0.2-0.4. A peak corresponding to free T, accounted for 15.2 + 6.3% of the radioactivity on the chromatogram at 17.5 hours, 15.1 & 5.0% at 48 hours, and 12.3 + 3.0% at 144 hours. However, since this solvent system does not distinguish individual thyronines, the exact identity of T, was not certain. Other thyronines could be present. DISCUSSION
Little discussion is necessary since these results closely resemble in most respects those obtained after intraperitoneal injection of radiothyroxine (T4*) into brook trout acclimated at 10°C (Eales, 1970). Significant biliary excretion occurred for Chromatographic Separation of Bile both hormones and, as judged by a single Radioactive Materials paper chromatography solvent system, simIndividual chromatograms were run on ilar types of derivatives appeared in the bile for each of the 24 fish killed at 17.5, bile. From the work of &born and Simp48, and 144 hours pi. Typical chromat- son (1969) on plaice, the presence of gluograms are shown in Fig. 7. Negligible curonic acid and sulfuric acid conjugates of T, and T, in brook trout bile is anticilz51- was present, and most radioactivity
BILIART
EXCRETION
pated. Major thyroid hormone bile derivatives are in the process of being identified for the brook trout. This work will be reported elsewhere. However, in contrast to T,, T3 does not seem to bind so extensively to serum proteins as judged by our application of the trichloroacetic acid-precipitation test. Despite this lesser binding, a slower loss of radioactivity was recorded from the body and organs of the T,*-injected trout, indicating slower peripheral metabolism of T,. Serum radioiodide levels as judged from several lines of evidence were lower in T,*- than in T,“-injected trout suggesting less rapid deiodination of T,“. Furthermore, a greater uptake and a more rapid loss of liver and gall bladder radioactivity occurred following T,* injection, suggesting a more rapid biliary excretion of T,“. These differences probably represent real differences in the metabolism of the two thyroid hormones. However the Tn and T, experiments were carried out on fish of different ages and sizes and at a different season. The modifying effect of these and other variables cannot be overlooked. ACKNOWLEDGMENTS The authors wish to thank Mr. Schaldemose. Province of Manitoba Trout Hatchery, West
OF
T3
BY
TROUT
175
Hawk Lake, for providing the brook trout through the courtesy of Dr. K. Doan, Director of Fisheries, Province of Manitoba. REFERENCES J. L., AND FLOCK, E. V. (1965). The role of the liver in the metabolism of Y-thyroid hormones and analogues. In “The Biliary System” (W. Taylor, ed.), pp. 345-367. Blackwell, Oxford. EALES. J. G. (1969). Bi!e excretion of radiothyroxine by the brook trout, Salvelinus jontinalis (Mitchill). Gen. Camp. Endoc inol. 13, 502. EALES, J. G. (1970). Biliary excretion of radiothyroxine by the brook trout, Szlvelinus fontin&s (Mitchill). Gen. Comp. Endoctinol. 14, 385-395. HUTCHINS, M. O., .~ND NEWCOMER. W. S. (1966). Metabolism and excretion of thyroxine and triiodothyronine in chickens. Gen. Camp. EnBOLLMAN,
docrinol.
6,
239-248.
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