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Thyroxine and triiodothyronine turnover in the chicken and the bobwhite and Japanese quail
GENER.&
AND
COMPARATIVE
Thyroxine
ENDOCRINOLOGY
9,
353-361
(1967)
and Triiodothyronine and the Bobwhite and
AJIT
SINGH,”
E.P. REINEKEp3
Received
May
Turnover Japanese ANI)
R.
in the uaill
Chicke
K. RIXGER*
18, 19Gi
Except for the data of Heninger and Newcomer (1964) on the tg of Tg and Ta in cardiac tissue of chickens and brief reports on the 1; of T, in blood plasma of chickens (Tata and Shellabarger, 1959; Hendrich and Turner, 1967) and Japanese quail (McFarland el nl.? 1966), no information has been found in the literature on the degradaiion of thyroid hormones in birds. In the present investigations, thyroxineand triiodothyronine-distribution spaces (TDS) and biological half lives (tg) of the two hormones are reported. The extrnthyroidal thyroxine(ETT) and thyroid-secretion rate (TSR) from thyroxine degradation have been calculated from Td turnover data in the chicken, bobwhite and Japanese quail. Protein-bound iodine values (PBI) in the three species of birds are also presented. In addition, the results of TSR by thyroxine degradation are compared in chickens receiving diets adequate or deficient in iodine. Thyroxine and triiodothyronine turnover was studied in the chicken, bobwhite quail and Japanese quail. The thyroid secretion rate (TSR) as determined by this method in 7-week normal chickens, 7-week goitrogen-treated chickens, 56-week normal chickens, bobwhite, and Japanese quail, averaged 2.03, 1.59, 1.02, 2.49, and 2.78 pg/lOO gm body weight/day, respectively. The TSR of iodine-deficient. chickens was found to be signnificantly decreased. The representative biological half lives of Tb in the blood of chickens, bobwhite, and Japanese quail were 3.25,4.60, and 5.50 hours, respectively. The half lifeof T3 in each species of birds tested is not significantly different. The representative thyroxinedistribution space (TDS) in ml/100 gm body weight of chickens, bobwhite, and Japanese quail, respectively, measured 29.39, 28.08, and 55.29. TDS/unit’ body weight of 56-week old chickens is significantly lower than in 7-week chickens. Ts-distribution spaces in all birds are higher than that of Tq. The representative protein-bound iodine of chickens, bobwhite, and Japanese quail were measured as 1.1’ 3, 1.76, and 1.26 fig, respectiveig. Increased plasma radioactivity was found in cardiac blood samples as compared i.o those taken by venous puncture. It is believed that this represents discharge of unchanged hormone from extrathyroidal tissues, probably the liver, in response to the withdrawal of a relatively large volume of blood. The possible role of this mechanism in regulating thyroid hormone levels is discussed. MATERIALS
1 Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article Number 4080. Supported in part by NIH grant No. AM 08513. 2 Presented by the senior author in partial fulfillment of the doctoral degree in the Department of Physiology, Michigan State University. 3 Professor, Department of Physiology, Michigan State University. 4 Professor, Departments of Poultry Science and Physiology, Michigan P:;ate University.
AND
METHQ)DS
IMale white Leghorn chickens of varying ages, sexually mature male bobwhite quail (CC&W virginianus) and sexually mature male Japanese quail (Coturnix coturnix jrcpo?al:ca) were used in the thyroxine and t,riiodothyronine degradation experiments. Part of the chickens were fed on 3, die? deficient in iodine (SR-21, while all other birds were on a diet adeqilate in iodine (Michigan Stats University 63-S chick start.er crumbles, standard poult.ry mash, or quail rations. respectively). 353
354
SINGH,
REINEKE,
The chickens were housed in standard growing batteries, exposed to natural daylight through windows in the room. The environmental temperature ranged from 70 to 75°F. Both species of quail were housed at 72 + 2°F with 16 hours of light and 8 hours of darkness per day until the day of the experiments. Methimazole (I-methyl-2-mercaptoimidazole or Tapazole) was dissolved in drinking water with the aid of a minimum quantity of NaOH at the level of 0.025$&. The goitrogen-treated birds were started on Tapazole water a day before the experiment. Radioactive thyroxine and triiodothyronine labeled with 1’31 were obtained from the Abbott Laboratories as 50% propylene glycol solutions. These were diluted with 0.9 gm To NaCl solution containing a drop of chicken plasma. Addition of plasma was found to be necessary to prevent adherence of the labeled hormone to the glassware. The solution was administered intravenously in doses of 10~~ per chick or bobwhite quail and 5 PC per Japanese quail. Radioactivity of all samples was measured in a well counter (2-inch crystal) connected to a radiation analyzer and scaler. The standards, containing &a to xl5 of the dose in the same volume of fluid, were counted in plastic vials similar to those used for the plasma samples. The standards were counted at the same geometry each time plasma samples were counted. After making correction for the background count, percent of the injected dose per unit of plasma was calculated. Standards were based on the amount of proteinprecipitable radioactive material in the labeled hormones. Gregerman et al. (1962) reported that approximately 90% of the 1131 label in T4 solutions was actually combined with T4. In the present study also, it was observed that the recovery of counts after TCA precipitation of the plasma containing labeled hormone in eight trials was 91.9 rt 0.52% of the initial radioactivity. Since the impure component would probably be eliminated much more rapidly than the hormone, the counts on standards were corrected by using the factor 0.919 to avoid overestimation of the T.l space (TDS) measurements. In general, the methods used for man (Ingbar and Freinkel, 1955; Sterling and Chodos, 1956; Gregerman et al., 1962) were applied to birds. The blood samples were taken from the brachial vein opposite to the one used for administration of labeled hormones. Sufficient blood was drawn every third, sixth, ninth, and twelfth hour to yield 0.5 cc and 0.1 cc plasma in case of chicks and quail, respectively. In some preliminary experiments on chickens, blood samples were collected 24 hours after administration of the tagged hormone, but appreciable countIs could not be detected at that
AND
RINGER
time. Thus samples were collected at 3-hour intervals. The last or la-hour blood samples in many cases were collected by heart puncture. These samples were large enough for use both for counting radioactivity and for PBI determination. Under the conditions of these experiments it was. not feasible to draw more than four serial blood samples. Especially in the two species of quail the loss of blood would have been so great that interpretations would be questionable. Also, this is about, the maximum number of samples that can be taken from peripheral veins during the course of a day in these small birds. In certain birds, especially quail, it was noticed that the samples obtained by heart puncture contained more radioactivity than the earlier samples obtained from the brachial vein. A similar type of apparent discharge was particularly well marked when the last samples were taken by decapitation of quail in certain earlier experiments, not included for collection of these data. In view of this finding, the last samples in Ta-degradation experiments with quail were taken both by venous and heart punctures and the percent radioactivity compared in the two. From this, percent discharge was calculated. In case of the subjects where fourth samples were collected only by heart puncture, percent increase in radioactivity was calculated from the difference between this point and that of the curve extrapolated from the earlier samples. In quail, only the first three plasma samples could be used to estimate the slopes for Td-degradation because of the marked increase in counts in the final heart puncture sample. Four samples per bird were usable for the T3-degradation studies in quail because final samples were taken by wing vein followed by heart puncture. For the same reasons only the first three plasma samples could be used to slopes in establish Td-degradation goitrogentreated and 56-week-old normal chickens. The percent injected dose per unit of plasma as calculated for each sample was plotted against time on semilogarithmic graph paper. With the exceptions already noted (heart puncture samples) the plasma radioactivity showed very little deviation from a straight line. However, too few points were available for valid statistical treatment of the curves on individual birds. Therefore, each curve was fitted by inspection and extrapolated to zero time to obtain an estimate of the maximum plasma radioact,ivity, thyroxineor triiodothyronine-distribution space (TDS), and biological half life (t+) (Fig. 1). Statistical comparisons between the pertinent groups and treatments were made later by analysis of variance, including the New Multiple Range Test (Li, 1964). The following equations were used to calculate T4 degradation:
THYROXINE
AKD
TRIIODOTHYRONINE
Daily
2.b
lzcr*
time
intercept
space,
TDS = 7. dose at 0 ti:‘iu Im-Tr
turnover
rate
1 ml plasma
(‘)
(2) Fractional
t’urnover: K = l-e-z
(3)
where e is the base of natural logarithms, and z is the value derived from Equation No. 2; em’ is most easily obtained from a table of descending exponentials.” Extrathyroidal weight : ETT (The factor equivalent)
thyroxine = TDS/lOO 1.53
is used
hrucine (Faulkner
pool,
fig/100
gm X PBI/ml to convert
gm
body
X 1.53 PBI
to its
(4) Td
j Tables of the Exponential Function ez. National Bureau of Standards Applied Mathematics Series 14, 4th Ed., 1969. U. S. Gov’t. Printing Office, Washington, D.C.
X li
: X 21
(5’
et al., X%1), and t.rar~anit&r~cte
in a Coleman at a wave length
RESULTS THYROID
constant:
= ETT
(TSR)
In order to study uptake of T, or Ta by avian erythrocytes, a drop each of 1131-labelecl T, and T, was added to two chicken blood samples of eqluai volume. Whole blood was counted and then incubated at, the chicken body temperature (41.6%) for 2 hours. Plasma was separated from the red cells after centrifugation. The cells were washed thre(’ times with normal saline solution, centrifuged, and the wash mixed each time with the aupernatam solution. The cells and the diluted plasma were the>! counted and necessary corrections made for geometry arrd decay. The percent of the hormones hours: to red blood cells was calculated. PBI was determined hy the alkaline ashing procedure of Barker and Humphrey (1950) except, that the protein was first precipitated with triehloroacetie acid, resuspended in glass-dist.illed water, reprecipitated with zinc hvdroxide, and washed by the usual procedure. In View of the low I331 values in chicken plasma, t,he sensitivity of the method was increased by diluting two parts of the ceric ammonium sulfat,e (Hyeei prepared reagent) with three parts of glass-distilled water and eonducting the final react,ion at 50°C. After exactly 211 minutes, the reaction was stopped by adding was measured trophotometer
ml:
355
degradation TSR
in the text. distribution
thyroxine
=1.2~~
FIG. 1. Thyroxine degradation curve for a 610 gm cockerel. As fitted by least squares there is a correlation between log plasma Im and time of 0.9998 (p < 0.01). In our usual procedure the line was fitted by inspection and the zero-time intercept and t+ were read from this line. Using these values, results were then calculated by use of Equations i-5
Thyroxine
TURSOVER
AND
Universal of 480 rn+
Spec-
DISCUSSION
SECRETION
RATE
Details of the estimation of daily thyroxine utilization of chickens derived from data in this study will be presented in a subsequent8 paper in comparison with values for TSR o’btained by three other methods. However, it is of interest to report at this time (Tabk 1) the average TSR irn several categories of birds studied. TSR showed a twofold decrease in mature chickens as compared to growing birds. It was reduced t,o some extent 'by Tapazole treatment and to a very pronounced degree by iodine deficiency. Interspecies comparisons are complicated by differences in body size and physiological age. The bobwhite quail matures at about the same age as the &i&en. Even at 10 weeks of age the Japanese quail are sexually mature wMe the only mature
chickens are the 56-week group. TSR ~6~s
SINGH, REINEKE,
356
TABLE THYROXINE
Species
No. birds
Chickens Chickens Chickens Chickens Chickens Bobwhite quail Japanese quail Note.-Results
(TSR)
UTILIZATION
6 8 9 6 6 8 8
IN
MALE
AND RINGER 1
CHICKENS,
Age, we&s
7.0 5.5 6.5 7.0 56.0 56-68 10.0
BOBWHITE, TSR
1.59 0.73 2.03 2.03 1.02 2.49 2.78
AND
JAPsNESE
ZIZ SE
QUAIL Treatment
3~ 0.116 2 0.087 5 0.150 2~ 0.178 5 0.242 + 0.493 + 0.349
Tapazole Iodine deficient Normal Normal Normal Normal Normal
are expressed as fig Tq utilized per 100 gm body weight.
for the two groups of quail do not differ significantly from normal immature ehickens, but are significantly higher than those of the adults. In T,-degradation methods for TSR estimation it is assumed that endogenous hormone reaching the peripheral tissues is in equilibrium with the PBI and that it is distributed and metabolized in the same manner as a tracer dose of labeled hormone. Secondly, the amount of hormone degraded daily by the extrathyroidal tissues is CODsidered to be equal to that released by the thyroid. It can be seen from Equations 1 to 5 that the TSR in this method is calculated from three parameters: the biological half life (tg), the thyroxine-distribution space (TDS) and protein-bound iodine (PBI). The lower TSR values for the goitrogen-treated, iodine deficient, and 56-week-old normal birds is accounted for by their lower TDS/lOO gm body weight. In these cases there was simply less thyroid hormone available for metabolism, and this in turn resulted in lower TSR values. At this time it is not possible to account with any certainty for the proportions of Ts and Tq that are actually metabolized. Wentworth and Mellen (1961) reported that the T3:T4 ratio was 40 : 60 in the blood of chickens, turkeys, and ducks. Vlijm (1958) reported that 24 hours after P31administration TO and Tq comprised 37& and 20%, respectively, of the radioiodinated compounds in the adult chicken thyroid. A further complication is that, although TZ and Tq have similar degradation rates, the
distribution spaces for the two compounds are significantly different. In the present work, TSR values were calculated only for the T, degradation studies and the entire PBl fraction was treated as T4 iodine. Although these cannot be taken as absolute values, the errors involved in the calculation are constant and results of different experiments using Tq as a tracer should be comparable. The 4, TDS and PBI were also determined in the T3-degradation experiments, but no attempt was made to calculate TSR becauseof the considerations noted above. BIOLOGICAL
HALF
LIVES
(53
The biological half lives of T, and Ta are not significantly different in bobwhite and Japanese quail (Table 2). The apparent differences are due to an interaction between the two hormones and the two species of quail. In the case of chickens, only the tg of Ta in 56-week normal chickens was significantly greater than in all other groups of chickens treated by both T3 and Tq (Table 3). The interaction between the three groups of chickens and the two hormones is significant. It is interesting to note that the biological half lives of Tq and TS are relatively much shorter in birds than in man or other mammals. Heninger and Newcomer (1964) reported mean half lives of 4.9 and 3.9 hours for Tq and Tat respectively, in the cardiac tissue of chickens. These values are close to the tg of Tq and T3 in the chicken plasma observed in this study. In contrast to these results Tata and Shella’barger reported
$HYROXINE
AKD
TRIfODOTIIYROXt’iSt
TAE&E STATISTICAL ANALYSIS OF THYROXINE AND JAFAXESE QUAIL. TWO-WAY Distribution SOWW
qpace ml/l00 Mean
0
Quail T4 and TB internct~ion Error
1 1 1 32
AND
2
TRIIODOTHYRONINE
ANALYSIS
-
T4 TS
IN BOBWWTE BEPLIICATS?
F
squares
64.49” 109.43” 7.216
-
t3 (hours) IV
?&an
046.56 .02083 .I4350 .01535
1
1 1 32
I--F
sq.lares
3 .03:< 1.357 9.34P.* __-
raz~ge te%c
c
B
A
1.96276 91.76 4.3
1.81428 65.21 7.3
1.74673 55.81 5.4
1.44891 28.18 4.7
OF OBSERVED MINUS ADJUSTED Two GROWS OF QUAIL AND Distribution
Hormonee
NINE
D
xeans
TABLE
WITII
weight
New mukipk
Log. means-TDSd Means-TDS Mean--/a (houi:s)
TURNOVER
OF VARIANCE
gm body
4482 .7606 .0501 00695
351
IURn’OVER
MEANS ~~IOWISG IKTERACTIOS THE Two THYIXOID HOXIUOKES
BETWEEK
qxce
Bobwhite
Japanese
Bobwhite
-_ 0373 ,0373
.0373 p.0373
-.0631 .0631
.0631 -. 0631 l__-
a Significant at 0.005 level. h Significant at 0.025 level. c Means underscored by the same line are not significantly different at the 0.05 level. d Log transformation of the data was used to obtain variance homogeneity by Bartlett’s test. &onps A and B are bobwhite and Japanese quail in Td degradation experiment. Groups C and D are bobwhite and Japanese quail, respectively, in Ta degradation experiment.
mean t+ values for both Ta and Tq in chickens of 22.5 hours. In chickens exposed to varying environmental conditions, Hendrich and Turner (1967) reported I+ averages ranging from 7.0 to 14.8 hours. It seemspossiblethat t,he difference between these and our results may be due to differences in blood sampling procedures. McFarland et al. (1966) reported turnover rates of Tq in Japanese quail, which when expressed as 4 were approximately 18.4 hours at 21’C and 30.4 hours at 32°C. These values are much higher than those in the present data,. However, all blood samples in the earlier study on quail were taken by heart puncture. As will be shown later, sampling by this method results in increased radioactivity in the blood and an overestjimation of tg values.
THYROID-H•
RMONE-DISTRIBUTXON
SFACZ
(TDS)
The distribution spacesfor T, are sign&. cantly higher than for Tq in both chickens and quail (Tables 2 and 3). Among t,ho three groups of chickens treated with. T3, there are no significant differences in their distribution spaces,but in the caseof T4, the distribution spacesin 7-week normal chickr are significantly higher than in 56week normal chickens. The TDS of both T, and T3 is significantly higher in Japanese quail than in bobwhite quail. The decreasedTIM of Tb in older chickens could be an age effect. Gregerman at al. (1962) reported a decrease of TDS with aging in man after decade six and he suggested t,hat the decrease of metabolic
358
SINGH,
REINEKE,
AND
TABLE STATISTICAL
ANALYSIS
OF THYROXINE
Distribution SOIXCX
space ml/100 Mean
df
Chickens Ta and T, Interaction Error
2 1 2 30
AND gm body
TABLE
T4
weight Mean
df
2.12 209.55” 2.93 multiple
CHICKENS
t2 (hours)
F
149.07 14697.93 205.65 70.14
IN
2 1 2 30 range
squares
F
40.49 49.47 22.345 1.547
26.17” 31.98” 14.44=
t&b
F
D
A
B
C
F
E
C
D
A
B
63.87
61.35
60.46
29.39
20.53
14.52
9.35
3.86
3.86
3.76
3.23
2.85
THE
T3
TURNOVER
E
OF OBSERVED
H0rlWXW
3
TRIIODOTHYKONINE
squares
New
Means<
RINGER
MINUS THREE
ADJUSTED
GROUPS 7 weeks
MEANS
OF CHICKENS Normal
,905 -.905
FOR
t+ VALUES
AND
TIME
Two
SHOWING TI~YROID
7 weeks Goitrogen-treated
.664 -.664
INTERACTION
BETWBN
HORMONES 56 weeks
Nomud
-1.569 1.569
Note.-Two-way analysis of variance with six replicates. a Significant at 0.005 level. b Means underscored by the same line are not significantly different at the 0.05 level. c Seven weeks normal, 7 weeks goitrogen-treated, and 56 weeks normal groups of chickens from T4 degradation experiment are respectively termed as A, B, and C. Similar groups from TO degradation experiment are termed D, E, and F. Each group contained six birds.
mass with age could influence the distribution space. Contrarily, Oddie et al. (1966) observed that the distribution space is independent of age and height in man. The above mentioned workers also reported a positive correlation between the distribution space and the body weight in man. In the case of 6- to 7-week-old chickens regression of the total thyroxine-distribution space on body weight was not found to be significant. The relationship between the total triiodothyronine-distribution space and body weight of ‘i-week normal and goitrogentreated and 56-week normal chickens was tested by the Spearman rank correlation coefficient. This showed a significant positive correlation with values of rs = 0.9023 and p < 0.04 (two-tailed analysis). The TDS per unit body weight for Ta is about twice that for Tq. Thus, after the initial distribution period of 3-4 hours, the percent injected dose for Ta in the plasma is only about one-half that for Tq. However,
the I+ of the two hormones is not significantly different, indicating that their rate of metabolism and excretion is similar. This is contrary to the interpretation of Hutchins and Newcomer (1966) who stated, on the basis of data obtained only during the first 4 hours after labeled-hormone administration, that T3 is metabolized and excreted at a faster rate than Td in chickens. In an in v&o experiment on the uptake of T, and TX in avian erythrocytes, it was observed that the red blood corpuscles took up 1.11% of P31-T4as compared to 4.71y. of 1131-T3after 2 hours incubation of the chicken blood mixed with 1131-labeledhormones. Qualitatively similar results regarding the differential uptake of the two hormones by avian red blood cells were reported by Heninger and Newcomer (1964)) although percent uptake of the two hormones varied in their experiments. A relatively larger uptake of T3 by red blood cells may account in part for the
THYROXINE
AND
TRIIODOTHYRONINE
higher TDS for T3 than for Tq. This view is supported by the observation that the Tadistribution spaces/100 gm body weight in sexually immature and sexually mature chickens are not significantly different (Table 2) while the TDS for T, dechnes. Sexually mature males have a higher number of erythroeytes and the androgens are believed to be responsible for the difference (Sturkie, 1965). Thus, more red blood cells in older birds take up more P3’-Tz, which may result in decreased radioactivity in the plasma and an increase in the calculated TDS. The Japanese quail have higher T, and Tz-distribution spaces than chickens and bobwhite quail. The tg for T, and T, turnover of this quail is, however, not significantly different from that of the other species studied. INCREASED TO
PLASMA CARDIAC
DUE
RADIOACTIVITY SAMPLING
During the course of plasma sampling it was noticed that in some birds their 12-hour samples contained higher radioactivity than some of their earlier samples (Table 4). This increased radioactivity was (1) more pronounced in I131-T, than P31-Ta turnover experiments; (2) greater in Japanese quail than in bobwhit,e quail, (3) greater in goitrogen-treated and normal older birds than in the normal younger birds, and also seemed to be related to the amount of the blood drawn from the bird. By far the greatest increase was seen in Japanese quail, followed in turn by bobwhite quail. PrePERCENT
TABLE INCREASE OF RADIOACTIVITY 1%HOUR SAMPLE TAKEN
TURNOVER
sumably the increased radioactivity in blood plasma after cardiac puncture was due to discharge of previously stored hormone from the liver although possible releasefrom other tissues must also be considered. In some of our experimems, not. to be reported in detail, high counts were found in livers of chickens after both P-T3 and P-‘is4 administration. In studies on rats, dogs and men, Taurog et nl. (1951, 1952) and Albert and I-keating (1949) observed t,hat thyroxine concentrates in the liver even when it is administered intravenously. They thought that T, probably undergoes an enterohepatic ~jr~~la~,~~~l. Recently, German et aZ. (1966) isolated and perfused livers of rats at I- to 26-hour intervals following administration of the thyroid hormones. They observed that unchanged Tq was released to the perfusing blood until an equilibrium was reached. They also reportBed that the livers of rats given labeled T, released only a small amount of the hormone to the perfusing blood. These differences between T, and ‘I’,$ together with a lower affinity of T, for the plasma proteins may account for t’he higher discharge seen in the U-hour cardiac samplesin the T, than in the T3-degradation experiment. It is obvious that additional research will be needed to explairl this unexpected phenomenon. PROTE:IN-BOUND
IODINE
Iiormsi~
4 IN BLOOD BY HEART
AS CAIAXLATED PUNCTURE
Goitrogen-treated
i-iO?Illai
Bobwhite
JZLpWM?
11” (56-58p
06 11op --~-
-
66 (7)C
6b (7)C
66 (5EF
PI-T4
8.72 k4.86 3.25 21.76
23.08 k6.86 6.38 k2.46
25.35 +10.34 4.81 F3.31
a Mean + standard error. * Number of birds in group. c Age of birds in weeks.
IS THE Quail
Isotope
p-T,
(PBI)
The results of PBI analyses are given in Table 5. There is little overall difference in
Chickens
-
359
33.23 ki.17 24.68 ts.03
134.94 +19.34 -67.93 i18.20 ----
360
SINGH,
PROTEIN-BOUND
Birds
TABLE 5 IODINE (PG PER 100 ML PL,sM~) IN CNICRENS AND QUAIL
“II;;;;
of
Age (weeks)
Iodine content of diet
Ghickens
43
5-7
Adequate
-Chickens
18
5-6
Deficient
Chickens
11
56
Adequate
Bobwhite quail
18
56-68
Adequate
17
PBIa 1.1226 F .0367 1.0135 i .0483 1.2840 ?!z .0975 1.7572 I .0637 1.4770”
106
Japanese
REINEKE,
10
Adequate
quail
1.2603
+ .1320 0 9344b
lob a Mean + standard error. b PBI of pooled plasma from
10 other
quail.
PBI levels of different groups of chickens and Japanese quail. The bobwhite quail, however, show higher PBI, both in analyses of individual samples and in the pooled plasma. The authors are not aware of any earlier reports about PBI of quail. The results obtained in this study are in agreement with those reported by Bumgardner and Shaffner (1957) and Mellen and Hardy (1957) for the chicken. The values reported by Rosenberg et al. (1964) were relatively lower. The chickens on a diet deficient in iodine had slightly lower levels of PBI than those on adequate iodine diet. Similar nonsignificant differences were noted by Rosenberg et al. (1964). COMMENT
From the observations reported in this paper together with those of other investigators, it seems clear that the control of thyroid function in the chicken and quail differs in several important respects from that of mammals. PBI levels are considerably lower, probably due to the lack of a specific thyroxine-binding alpha globulin in avian blood (Tata and Shellabarger, 1959). This is counterbalanced by more rapid utilization of the smaller quantity of thyroid hormones in the extrathyroidal thyroxine pool, as evidenced by their short
AND
RINGER
ta of 3-4 hours. The tg for Tq in mammalian species ranges from 19 hours in the rat (Feldman, 7 957) to 6.7 days in man (Sterling and Chodos, 1956). In accord with the short t+ for thyroid hormones in the chicken, thyroprotein-fed birds showed an increase in metabolic rate for only the first few hours of fasting (Mellen, 1958). In our experiments (Singh, 1966), chickens have shown only a transitory increase in metabolic rate after the administration of T8 and T+ singly or in combination. With the information now at hand it is not possible to assess precisely the part that the storage and discharge of thyroid hormones from extrathyroidal tissues may play in regulating the effective level of circulating thyroid hormones in birds. It is well established that the principal route of excretion for both Ta and T, is via the bile in chickens (Hutchins and Newcomer, 1966) as well as in mammals. Our unexpected finding that significant amounts of radioactive substance were released to the blood during sampling of relatively large quantities may indicate that the liver or other extrathyroidal tissues play a role in regulating the circulating thyroid hormone level. The liver perfusion experiments of Gorman et al. (1966) support the view that such a mechanism may exist. Although the thyroid hormone regulating mechanisms of birds seem to differ from those of mammals in several ways, the total activity, as shown by daily thyroid hormone secretion rate (Ringer, 1965) overlaps that of mammals (Reineke, 1959). ACKNOWLEDGMENTS Grateful acknowledgement is expressed to Mrs. Judianne Anderson for her assistance with the iodine analyses reported in this paper and to Dr. W. E. Cooper, Department of Zoology, Michigan State University, for advice on the statistical analyses. Thanks are also due to the Rockefeller Foundation for a Graduate Fellowship granted to Ajit Singh. REFERENCES ALBERT, A., AND KEATING, F. R. (1949). Metabolic studies with I”‘-labelled thyroid compounds. J. Clin. Endocrinol. 9, 1406-1421. BARKER, S. B., AND HUMPHREY, M. J. (1950).
THYROXINE
AND
TRIIODOTHYRONINE
Clinical determination of protein-bound iodine in plasma. J. Cl&. E&ocri~&. 10, 11361141. BUMGARDNER, H. L., .4xn SHAFFNER, C. S. (1957). Protein-bound iodine levels of the chick. PO& try Sci. 36, 207-208. FAT;LXNER, L. W., LEVY, R. P., ASD LEONAKDS, 6. R. (1961). Simplified t.echnique for the determination of serum protein-bound iodine. Clin. Chem. 7, 637-645. FELDMAN, J. D. (1957). Effect of estrogen on peripheral utilization of thyroid hormones. Am. J. Physiol. 188, 30-34. GREGERMAK, R. I., G.~FFP;ET, G. W., A?;D SHOCK, N. W. (1962). Thyroxine turnover in euthyroid man with special reference to changes with age. J. C%z. Invest. 41, 2065-2074. GORMAN, C. A., FLOCX: E. V., OWEN, C. A., JR., AND PARIS, J. (1966). Factors affecting exchange of thyroid hormones between liver and blood. Endocri~zology 79, 391-405. HENDRICK, C. E., AND TURSER, C. W. (1967). A comparison of the effects of environmental temperature changes and 4.4”C cold on the biological half life (tl/z) of thyroxine 1131 in fowls. Poultry Sci. 46, 3-5. HENIKGER, R. W., AND NEWCOMER, W. S. (1964). Plasma protein binding, half life and erythroeyte uptake of thyroxine and triiodothyronine in chickens. Proc. Sac. Exptl. Biol. Med. 116, 624-62s. HIJTCI~IXS, M. O., AND ~SERCOMER, W. S. (1966). Met.abolism and excretion of thyroxine and triiodothyronine in chickens. Gen. Camp. E,ndocrirwl. 6, 239-248. INGBAR, S. H., AND FREINKEL, N. (1955). Simultaneous estimation of rates of thyroxine degradation and thyroid hormone synthesis. J. C&. Invest. 34, 808-819. Er, 9. C. R. (1964). “Statistical Inference I.” Edwards Brothers, Inc., Ann Arbor, Mich. MCFARLAND, L. z., YOTJSEF, M. K., AND WILSOX, W. 0. (1966). The influence of ambient temperature and hypothalamic lesions on the disappearance rates of thyroxine I’“’ in the Japanese quail. Life S&zses 5, 309315. MELLEN, W. J. (1958). Duration of effect of thyroxine and thiouracil in young chickens. Poultry Sci. 37, 672-679.
TCRSOVER
3AI
W. J ,, .~ND HARDY, L. B. (1957). Blood protein-bound iodine in the fowl. E~crlocGoloq~~ 60, 547-551. ODDIE, T. II., ME.~DE, J. H., JR., AZXD FISHER, D. ‘1. (1966). An analysis of published data on thyrosine turnover in human subjects. J. Cl&. E/>u’:,crinol. 26, 425436. REIA-EKE: E. P. (1959). Thy-roid function in ce-, (1 era1 species of animals wit,h special referrnce to environment and body size. U.S. A!. Eneqy Commiss. I’lD 75’38, 87-96. RINGER, R. K. (1965). In lLAvian Physiology,” P. D. Sturkie, cd. Comstock Pub!. Aseocs. 2nd Ed., p. 614. ROSENBERG, 1,. L., GOLDMAN, M., LA Rocan, C.; AND DIXCII;, M. Ii. (1964). Thyroid function in rats and chickens. Eqcilibration of injected iodide with exist,ing thyroidal iodine in LongEvans rats and White Leghorn thicker:;;. Blidocrinology 74, 212-225. ~INGH, A. (1966). Studies of thyroid function in the chicken. Bound doctoral thesis. Michigan State Univ. Sl’ERLIKG, K., AKD CHODOs, R. B. (19%). Radiothyroxine turnover studies in myxedema, thyrotoxicosis and hypermetaboliem without andocrine discaae. J. CS,L, Invest. 35, 806-813. STUHBIE, P. D. (1965). “Avian Physiology,” Comstock P&l. Assoc., 2nd Ed., p. 2. T.4~.4, J R., AKD SHELLABAROER, C. 9. (1959). An explanation for the difference between the responses of mammals and birds to t,hyroxine and triiodothyronine. Biochem. J. 72, 60X-618,
MELLEN,
TAUROG,
,1.,
BRIGGS,
F.
r\‘.,
AND
CR.~TKOFF,
1.
L.
(1951). I’“‘-labelled n-thyroxine. I. An unidentified excretion product in bile. J. Biol. C&m. 191, 29-34. T~UROG, A., BRIGGS, F. x., AP*‘D CHAIHOY~F, 4. 1;. (1952). P3’-labelled L-thyroxine. II. Kature of the excretion products in bile. J. Z&l. C’hem. 194, 655668. VLIJM, L. (195X). On the production of hormones in the thyroid glands of birds. A quantitative study with the help of radioiodine and antithyroid drugs. AI&. Ne’erland. Zool. 12, 467631. WEXTVITORTH, B. C., AKD MELLES, W. 3. (1961). Circulating thyroid hormones in domestic birds. Poultry Sci. 40, 12751276.
AND
COMPARATIVE
Thyroxine
ENDOCRINOLOGY
9,
353-361
(1967)
and Triiodothyronine and the Bobwhite and
AJIT
SINGH,”
E.P. REINEKEp3
Received
May
Turnover Japanese ANI)
R.
in the uaill
Chicke
K. RIXGER*
18, 19Gi
Except for the data of Heninger and Newcomer (1964) on the tg of Tg and Ta in cardiac tissue of chickens and brief reports on the 1; of T, in blood plasma of chickens (Tata and Shellabarger, 1959; Hendrich and Turner, 1967) and Japanese quail (McFarland el nl.? 1966), no information has been found in the literature on the degradaiion of thyroid hormones in birds. In the present investigations, thyroxineand triiodothyronine-distribution spaces (TDS) and biological half lives (tg) of the two hormones are reported. The extrnthyroidal thyroxine(ETT) and thyroid-secretion rate (TSR) from thyroxine degradation have been calculated from Td turnover data in the chicken, bobwhite and Japanese quail. Protein-bound iodine values (PBI) in the three species of birds are also presented. In addition, the results of TSR by thyroxine degradation are compared in chickens receiving diets adequate or deficient in iodine. Thyroxine and triiodothyronine turnover was studied in the chicken, bobwhite quail and Japanese quail. The thyroid secretion rate (TSR) as determined by this method in 7-week normal chickens, 7-week goitrogen-treated chickens, 56-week normal chickens, bobwhite, and Japanese quail, averaged 2.03, 1.59, 1.02, 2.49, and 2.78 pg/lOO gm body weight/day, respectively. The TSR of iodine-deficient. chickens was found to be signnificantly decreased. The representative biological half lives of Tb in the blood of chickens, bobwhite, and Japanese quail were 3.25,4.60, and 5.50 hours, respectively. The half lifeof T3 in each species of birds tested is not significantly different. The representative thyroxinedistribution space (TDS) in ml/100 gm body weight of chickens, bobwhite, and Japanese quail, respectively, measured 29.39, 28.08, and 55.29. TDS/unit’ body weight of 56-week old chickens is significantly lower than in 7-week chickens. Ts-distribution spaces in all birds are higher than that of Tq. The representative protein-bound iodine of chickens, bobwhite, and Japanese quail were measured as 1.1’ 3, 1.76, and 1.26 fig, respectiveig. Increased plasma radioactivity was found in cardiac blood samples as compared i.o those taken by venous puncture. It is believed that this represents discharge of unchanged hormone from extrathyroidal tissues, probably the liver, in response to the withdrawal of a relatively large volume of blood. The possible role of this mechanism in regulating thyroid hormone levels is discussed. MATERIALS
1 Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article Number 4080. Supported in part by NIH grant No. AM 08513. 2 Presented by the senior author in partial fulfillment of the doctoral degree in the Department of Physiology, Michigan State University. 3 Professor, Department of Physiology, Michigan State University. 4 Professor, Departments of Poultry Science and Physiology, Michigan P:;ate University.
AND
METHQ)DS
IMale white Leghorn chickens of varying ages, sexually mature male bobwhite quail (CC&W virginianus) and sexually mature male Japanese quail (Coturnix coturnix jrcpo?al:ca) were used in the thyroxine and t,riiodothyronine degradation experiments. Part of the chickens were fed on 3, die? deficient in iodine (SR-21, while all other birds were on a diet adeqilate in iodine (Michigan Stats University 63-S chick start.er crumbles, standard poult.ry mash, or quail rations. respectively). 353
354
SINGH,
REINEKE,
The chickens were housed in standard growing batteries, exposed to natural daylight through windows in the room. The environmental temperature ranged from 70 to 75°F. Both species of quail were housed at 72 + 2°F with 16 hours of light and 8 hours of darkness per day until the day of the experiments. Methimazole (I-methyl-2-mercaptoimidazole or Tapazole) was dissolved in drinking water with the aid of a minimum quantity of NaOH at the level of 0.025$&. The goitrogen-treated birds were started on Tapazole water a day before the experiment. Radioactive thyroxine and triiodothyronine labeled with 1’31 were obtained from the Abbott Laboratories as 50% propylene glycol solutions. These were diluted with 0.9 gm To NaCl solution containing a drop of chicken plasma. Addition of plasma was found to be necessary to prevent adherence of the labeled hormone to the glassware. The solution was administered intravenously in doses of 10~~ per chick or bobwhite quail and 5 PC per Japanese quail. Radioactivity of all samples was measured in a well counter (2-inch crystal) connected to a radiation analyzer and scaler. The standards, containing &a to xl5 of the dose in the same volume of fluid, were counted in plastic vials similar to those used for the plasma samples. The standards were counted at the same geometry each time plasma samples were counted. After making correction for the background count, percent of the injected dose per unit of plasma was calculated. Standards were based on the amount of proteinprecipitable radioactive material in the labeled hormones. Gregerman et al. (1962) reported that approximately 90% of the 1131 label in T4 solutions was actually combined with T4. In the present study also, it was observed that the recovery of counts after TCA precipitation of the plasma containing labeled hormone in eight trials was 91.9 rt 0.52% of the initial radioactivity. Since the impure component would probably be eliminated much more rapidly than the hormone, the counts on standards were corrected by using the factor 0.919 to avoid overestimation of the T.l space (TDS) measurements. In general, the methods used for man (Ingbar and Freinkel, 1955; Sterling and Chodos, 1956; Gregerman et al., 1962) were applied to birds. The blood samples were taken from the brachial vein opposite to the one used for administration of labeled hormones. Sufficient blood was drawn every third, sixth, ninth, and twelfth hour to yield 0.5 cc and 0.1 cc plasma in case of chicks and quail, respectively. In some preliminary experiments on chickens, blood samples were collected 24 hours after administration of the tagged hormone, but appreciable countIs could not be detected at that
AND
RINGER
time. Thus samples were collected at 3-hour intervals. The last or la-hour blood samples in many cases were collected by heart puncture. These samples were large enough for use both for counting radioactivity and for PBI determination. Under the conditions of these experiments it was. not feasible to draw more than four serial blood samples. Especially in the two species of quail the loss of blood would have been so great that interpretations would be questionable. Also, this is about, the maximum number of samples that can be taken from peripheral veins during the course of a day in these small birds. In certain birds, especially quail, it was noticed that the samples obtained by heart puncture contained more radioactivity than the earlier samples obtained from the brachial vein. A similar type of apparent discharge was particularly well marked when the last samples were taken by decapitation of quail in certain earlier experiments, not included for collection of these data. In view of this finding, the last samples in Ta-degradation experiments with quail were taken both by venous and heart punctures and the percent radioactivity compared in the two. From this, percent discharge was calculated. In case of the subjects where fourth samples were collected only by heart puncture, percent increase in radioactivity was calculated from the difference between this point and that of the curve extrapolated from the earlier samples. In quail, only the first three plasma samples could be used to estimate the slopes for Td-degradation because of the marked increase in counts in the final heart puncture sample. Four samples per bird were usable for the T3-degradation studies in quail because final samples were taken by wing vein followed by heart puncture. For the same reasons only the first three plasma samples could be used to slopes in establish Td-degradation goitrogentreated and 56-week-old normal chickens. The percent injected dose per unit of plasma as calculated for each sample was plotted against time on semilogarithmic graph paper. With the exceptions already noted (heart puncture samples) the plasma radioactivity showed very little deviation from a straight line. However, too few points were available for valid statistical treatment of the curves on individual birds. Therefore, each curve was fitted by inspection and extrapolated to zero time to obtain an estimate of the maximum plasma radioact,ivity, thyroxineor triiodothyronine-distribution space (TDS), and biological half life (t+) (Fig. 1). Statistical comparisons between the pertinent groups and treatments were made later by analysis of variance, including the New Multiple Range Test (Li, 1964). The following equations were used to calculate T4 degradation:
THYROXINE
AKD
TRIIODOTHYRONINE
Daily
2.b
lzcr*
time
intercept
space,
TDS = 7. dose at 0 ti:‘iu Im-Tr
turnover
rate
1 ml plasma
(‘)
(2) Fractional
t’urnover: K = l-e-z
(3)
where e is the base of natural logarithms, and z is the value derived from Equation No. 2; em’ is most easily obtained from a table of descending exponentials.” Extrathyroidal weight : ETT (The factor equivalent)
thyroxine = TDS/lOO 1.53
is used
hrucine (Faulkner
pool,
fig/100
gm X PBI/ml to convert
gm
body
X 1.53 PBI
to its
(4) Td
j Tables of the Exponential Function ez. National Bureau of Standards Applied Mathematics Series 14, 4th Ed., 1969. U. S. Gov’t. Printing Office, Washington, D.C.
X li
: X 21
(5’
et al., X%1), and t.rar~anit&r~cte
in a Coleman at a wave length
RESULTS THYROID
constant:
= ETT
(TSR)
In order to study uptake of T, or Ta by avian erythrocytes, a drop each of 1131-labelecl T, and T, was added to two chicken blood samples of eqluai volume. Whole blood was counted and then incubated at, the chicken body temperature (41.6%) for 2 hours. Plasma was separated from the red cells after centrifugation. The cells were washed thre(’ times with normal saline solution, centrifuged, and the wash mixed each time with the aupernatam solution. The cells and the diluted plasma were the>! counted and necessary corrections made for geometry arrd decay. The percent of the hormones hours: to red blood cells was calculated. PBI was determined hy the alkaline ashing procedure of Barker and Humphrey (1950) except, that the protein was first precipitated with triehloroacetie acid, resuspended in glass-dist.illed water, reprecipitated with zinc hvdroxide, and washed by the usual procedure. In View of the low I331 values in chicken plasma, t,he sensitivity of the method was increased by diluting two parts of the ceric ammonium sulfat,e (Hyeei prepared reagent) with three parts of glass-distilled water and eonducting the final react,ion at 50°C. After exactly 211 minutes, the reaction was stopped by adding was measured trophotometer
ml:
355
degradation TSR
in the text. distribution
thyroxine
=1.2~~
FIG. 1. Thyroxine degradation curve for a 610 gm cockerel. As fitted by least squares there is a correlation between log plasma Im and time of 0.9998 (p < 0.01). In our usual procedure the line was fitted by inspection and the zero-time intercept and t+ were read from this line. Using these values, results were then calculated by use of Equations i-5
Thyroxine
TURSOVER
AND
Universal of 480 rn+
Spec-
DISCUSSION
SECRETION
RATE
Details of the estimation of daily thyroxine utilization of chickens derived from data in this study will be presented in a subsequent8 paper in comparison with values for TSR o’btained by three other methods. However, it is of interest to report at this time (Tabk 1) the average TSR irn several categories of birds studied. TSR showed a twofold decrease in mature chickens as compared to growing birds. It was reduced t,o some extent 'by Tapazole treatment and to a very pronounced degree by iodine deficiency. Interspecies comparisons are complicated by differences in body size and physiological age. The bobwhite quail matures at about the same age as the &i&en. Even at 10 weeks of age the Japanese quail are sexually mature wMe the only mature
chickens are the 56-week group. TSR ~6~s
SINGH, REINEKE,
356
TABLE THYROXINE
Species
No. birds
Chickens Chickens Chickens Chickens Chickens Bobwhite quail Japanese quail Note.-Results
(TSR)
UTILIZATION
6 8 9 6 6 8 8
IN
MALE
AND RINGER 1
CHICKENS,
Age, we&s
7.0 5.5 6.5 7.0 56.0 56-68 10.0
BOBWHITE, TSR
1.59 0.73 2.03 2.03 1.02 2.49 2.78
AND
JAPsNESE
ZIZ SE
QUAIL Treatment
3~ 0.116 2 0.087 5 0.150 2~ 0.178 5 0.242 + 0.493 + 0.349
Tapazole Iodine deficient Normal Normal Normal Normal Normal
are expressed as fig Tq utilized per 100 gm body weight.
for the two groups of quail do not differ significantly from normal immature ehickens, but are significantly higher than those of the adults. In T,-degradation methods for TSR estimation it is assumed that endogenous hormone reaching the peripheral tissues is in equilibrium with the PBI and that it is distributed and metabolized in the same manner as a tracer dose of labeled hormone. Secondly, the amount of hormone degraded daily by the extrathyroidal tissues is CODsidered to be equal to that released by the thyroid. It can be seen from Equations 1 to 5 that the TSR in this method is calculated from three parameters: the biological half life (tg), the thyroxine-distribution space (TDS) and protein-bound iodine (PBI). The lower TSR values for the goitrogen-treated, iodine deficient, and 56-week-old normal birds is accounted for by their lower TDS/lOO gm body weight. In these cases there was simply less thyroid hormone available for metabolism, and this in turn resulted in lower TSR values. At this time it is not possible to account with any certainty for the proportions of Ts and Tq that are actually metabolized. Wentworth and Mellen (1961) reported that the T3:T4 ratio was 40 : 60 in the blood of chickens, turkeys, and ducks. Vlijm (1958) reported that 24 hours after P31administration TO and Tq comprised 37& and 20%, respectively, of the radioiodinated compounds in the adult chicken thyroid. A further complication is that, although TZ and Tq have similar degradation rates, the
distribution spaces for the two compounds are significantly different. In the present work, TSR values were calculated only for the T, degradation studies and the entire PBl fraction was treated as T4 iodine. Although these cannot be taken as absolute values, the errors involved in the calculation are constant and results of different experiments using Tq as a tracer should be comparable. The 4, TDS and PBI were also determined in the T3-degradation experiments, but no attempt was made to calculate TSR becauseof the considerations noted above. BIOLOGICAL
HALF
LIVES
(53
The biological half lives of T, and Ta are not significantly different in bobwhite and Japanese quail (Table 2). The apparent differences are due to an interaction between the two hormones and the two species of quail. In the case of chickens, only the tg of Ta in 56-week normal chickens was significantly greater than in all other groups of chickens treated by both T3 and Tq (Table 3). The interaction between the three groups of chickens and the two hormones is significant. It is interesting to note that the biological half lives of Tq and TS are relatively much shorter in birds than in man or other mammals. Heninger and Newcomer (1964) reported mean half lives of 4.9 and 3.9 hours for Tq and Tat respectively, in the cardiac tissue of chickens. These values are close to the tg of Tq and T3 in the chicken plasma observed in this study. In contrast to these results Tata and Shella’barger reported
$HYROXINE
AKD
TRIfODOTIIYROXt’iSt
TAE&E STATISTICAL ANALYSIS OF THYROXINE AND JAFAXESE QUAIL. TWO-WAY Distribution SOWW
qpace ml/l00 Mean
0
Quail T4 and TB internct~ion Error
1 1 1 32
AND
2
TRIIODOTHYRONINE
ANALYSIS
-
T4 TS
IN BOBWWTE BEPLIICATS?
F
squares
64.49” 109.43” 7.216
-
t3 (hours) IV
?&an
046.56 .02083 .I4350 .01535
1
1 1 32
I--F
sq.lares
3 .03:< 1.357 9.34P.* __-
raz~ge te%c
c
B
A
1.96276 91.76 4.3
1.81428 65.21 7.3
1.74673 55.81 5.4
1.44891 28.18 4.7
OF OBSERVED MINUS ADJUSTED Two GROWS OF QUAIL AND Distribution
Hormonee
NINE
D
xeans
TABLE
WITII
weight
New mukipk
Log. means-TDSd Means-TDS Mean--/a (houi:s)
TURNOVER
OF VARIANCE
gm body
4482 .7606 .0501 00695
351
IURn’OVER
MEANS ~~IOWISG IKTERACTIOS THE Two THYIXOID HOXIUOKES
BETWEEK
qxce
Bobwhite
Japanese
Bobwhite
-_ 0373 ,0373
.0373 p.0373
-.0631 .0631
.0631 -. 0631 l__-
a Significant at 0.005 level. h Significant at 0.025 level. c Means underscored by the same line are not significantly different at the 0.05 level. d Log transformation of the data was used to obtain variance homogeneity by Bartlett’s test. &onps A and B are bobwhite and Japanese quail in Td degradation experiment. Groups C and D are bobwhite and Japanese quail, respectively, in Ta degradation experiment.
mean t+ values for both Ta and Tq in chickens of 22.5 hours. In chickens exposed to varying environmental conditions, Hendrich and Turner (1967) reported I+ averages ranging from 7.0 to 14.8 hours. It seemspossiblethat t,he difference between these and our results may be due to differences in blood sampling procedures. McFarland et al. (1966) reported turnover rates of Tq in Japanese quail, which when expressed as 4 were approximately 18.4 hours at 21’C and 30.4 hours at 32°C. These values are much higher than those in the present data,. However, all blood samples in the earlier study on quail were taken by heart puncture. As will be shown later, sampling by this method results in increased radioactivity in the blood and an overestjimation of tg values.
THYROID-H•
RMONE-DISTRIBUTXON
SFACZ
(TDS)
The distribution spacesfor T, are sign&. cantly higher than for Tq in both chickens and quail (Tables 2 and 3). Among t,ho three groups of chickens treated with. T3, there are no significant differences in their distribution spaces,but in the caseof T4, the distribution spacesin 7-week normal chickr are significantly higher than in 56week normal chickens. The TDS of both T, and T3 is significantly higher in Japanese quail than in bobwhite quail. The decreasedTIM of Tb in older chickens could be an age effect. Gregerman at al. (1962) reported a decrease of TDS with aging in man after decade six and he suggested t,hat the decrease of metabolic
358
SINGH,
REINEKE,
AND
TABLE STATISTICAL
ANALYSIS
OF THYROXINE
Distribution SOIXCX
space ml/100 Mean
df
Chickens Ta and T, Interaction Error
2 1 2 30
AND gm body
TABLE
T4
weight Mean
df
2.12 209.55” 2.93 multiple
CHICKENS
t2 (hours)
F
149.07 14697.93 205.65 70.14
IN
2 1 2 30 range
squares
F
40.49 49.47 22.345 1.547
26.17” 31.98” 14.44=
t&b
F
D
A
B
C
F
E
C
D
A
B
63.87
61.35
60.46
29.39
20.53
14.52
9.35
3.86
3.86
3.76
3.23
2.85
THE
T3
TURNOVER
E
OF OBSERVED
H0rlWXW
3
TRIIODOTHYKONINE
squares
New
Means<
RINGER
MINUS THREE
ADJUSTED
GROUPS 7 weeks
MEANS
OF CHICKENS Normal
,905 -.905
FOR
t+ VALUES
AND
TIME
Two
SHOWING TI~YROID
7 weeks Goitrogen-treated
.664 -.664
INTERACTION
BETWBN
HORMONES 56 weeks
Nomud
-1.569 1.569
Note.-Two-way analysis of variance with six replicates. a Significant at 0.005 level. b Means underscored by the same line are not significantly different at the 0.05 level. c Seven weeks normal, 7 weeks goitrogen-treated, and 56 weeks normal groups of chickens from T4 degradation experiment are respectively termed as A, B, and C. Similar groups from TO degradation experiment are termed D, E, and F. Each group contained six birds.
mass with age could influence the distribution space. Contrarily, Oddie et al. (1966) observed that the distribution space is independent of age and height in man. The above mentioned workers also reported a positive correlation between the distribution space and the body weight in man. In the case of 6- to 7-week-old chickens regression of the total thyroxine-distribution space on body weight was not found to be significant. The relationship between the total triiodothyronine-distribution space and body weight of ‘i-week normal and goitrogentreated and 56-week normal chickens was tested by the Spearman rank correlation coefficient. This showed a significant positive correlation with values of rs = 0.9023 and p < 0.04 (two-tailed analysis). The TDS per unit body weight for Ta is about twice that for Tq. Thus, after the initial distribution period of 3-4 hours, the percent injected dose for Ta in the plasma is only about one-half that for Tq. However,
the I+ of the two hormones is not significantly different, indicating that their rate of metabolism and excretion is similar. This is contrary to the interpretation of Hutchins and Newcomer (1966) who stated, on the basis of data obtained only during the first 4 hours after labeled-hormone administration, that T3 is metabolized and excreted at a faster rate than Td in chickens. In an in v&o experiment on the uptake of T, and TX in avian erythrocytes, it was observed that the red blood corpuscles took up 1.11% of P31-T4as compared to 4.71y. of 1131-T3after 2 hours incubation of the chicken blood mixed with 1131-labeledhormones. Qualitatively similar results regarding the differential uptake of the two hormones by avian red blood cells were reported by Heninger and Newcomer (1964)) although percent uptake of the two hormones varied in their experiments. A relatively larger uptake of T3 by red blood cells may account in part for the
THYROXINE
AND
TRIIODOTHYRONINE
higher TDS for T3 than for Tq. This view is supported by the observation that the Tadistribution spaces/100 gm body weight in sexually immature and sexually mature chickens are not significantly different (Table 2) while the TDS for T, dechnes. Sexually mature males have a higher number of erythroeytes and the androgens are believed to be responsible for the difference (Sturkie, 1965). Thus, more red blood cells in older birds take up more P3’-Tz, which may result in decreased radioactivity in the plasma and an increase in the calculated TDS. The Japanese quail have higher T, and Tz-distribution spaces than chickens and bobwhite quail. The tg for T, and T, turnover of this quail is, however, not significantly different from that of the other species studied. INCREASED TO
PLASMA CARDIAC
DUE
RADIOACTIVITY SAMPLING
During the course of plasma sampling it was noticed that in some birds their 12-hour samples contained higher radioactivity than some of their earlier samples (Table 4). This increased radioactivity was (1) more pronounced in I131-T, than P31-Ta turnover experiments; (2) greater in Japanese quail than in bobwhit,e quail, (3) greater in goitrogen-treated and normal older birds than in the normal younger birds, and also seemed to be related to the amount of the blood drawn from the bird. By far the greatest increase was seen in Japanese quail, followed in turn by bobwhite quail. PrePERCENT
TABLE INCREASE OF RADIOACTIVITY 1%HOUR SAMPLE TAKEN
TURNOVER
sumably the increased radioactivity in blood plasma after cardiac puncture was due to discharge of previously stored hormone from the liver although possible releasefrom other tissues must also be considered. In some of our experimems, not. to be reported in detail, high counts were found in livers of chickens after both P-T3 and P-‘is4 administration. In studies on rats, dogs and men, Taurog et nl. (1951, 1952) and Albert and I-keating (1949) observed t,hat thyroxine concentrates in the liver even when it is administered intravenously. They thought that T, probably undergoes an enterohepatic ~jr~~la~,~~~l. Recently, German et aZ. (1966) isolated and perfused livers of rats at I- to 26-hour intervals following administration of the thyroid hormones. They observed that unchanged Tq was released to the perfusing blood until an equilibrium was reached. They also reportBed that the livers of rats given labeled T, released only a small amount of the hormone to the perfusing blood. These differences between T, and ‘I’,$ together with a lower affinity of T, for the plasma proteins may account for t’he higher discharge seen in the U-hour cardiac samplesin the T, than in the T3-degradation experiment. It is obvious that additional research will be needed to explairl this unexpected phenomenon. PROTE:IN-BOUND
IODINE
Iiormsi~
4 IN BLOOD BY HEART
AS CAIAXLATED PUNCTURE
Goitrogen-treated
i-iO?Illai
Bobwhite
JZLpWM?
11” (56-58p
06 11op --~-
-
66 (7)C
6b (7)C
66 (5EF
PI-T4
8.72 k4.86 3.25 21.76
23.08 k6.86 6.38 k2.46
25.35 +10.34 4.81 F3.31
a Mean + standard error. * Number of birds in group. c Age of birds in weeks.
IS THE Quail
Isotope
p-T,
(PBI)
The results of PBI analyses are given in Table 5. There is little overall difference in
Chickens
-
359
33.23 ki.17 24.68 ts.03
134.94 +19.34 -67.93 i18.20 ----
360
SINGH,
PROTEIN-BOUND
Birds
TABLE 5 IODINE (PG PER 100 ML PL,sM~) IN CNICRENS AND QUAIL
“II;;;;
of
Age (weeks)
Iodine content of diet
Ghickens
43
5-7
Adequate
-Chickens
18
5-6
Deficient
Chickens
11
56
Adequate
Bobwhite quail
18
56-68
Adequate
17
PBIa 1.1226 F .0367 1.0135 i .0483 1.2840 ?!z .0975 1.7572 I .0637 1.4770”
106
Japanese
REINEKE,
10
Adequate
quail
1.2603
+ .1320 0 9344b
lob a Mean + standard error. b PBI of pooled plasma from
10 other
quail.
PBI levels of different groups of chickens and Japanese quail. The bobwhite quail, however, show higher PBI, both in analyses of individual samples and in the pooled plasma. The authors are not aware of any earlier reports about PBI of quail. The results obtained in this study are in agreement with those reported by Bumgardner and Shaffner (1957) and Mellen and Hardy (1957) for the chicken. The values reported by Rosenberg et al. (1964) were relatively lower. The chickens on a diet deficient in iodine had slightly lower levels of PBI than those on adequate iodine diet. Similar nonsignificant differences were noted by Rosenberg et al. (1964). COMMENT
From the observations reported in this paper together with those of other investigators, it seems clear that the control of thyroid function in the chicken and quail differs in several important respects from that of mammals. PBI levels are considerably lower, probably due to the lack of a specific thyroxine-binding alpha globulin in avian blood (Tata and Shellabarger, 1959). This is counterbalanced by more rapid utilization of the smaller quantity of thyroid hormones in the extrathyroidal thyroxine pool, as evidenced by their short
AND
RINGER
ta of 3-4 hours. The tg for Tq in mammalian species ranges from 19 hours in the rat (Feldman, 7 957) to 6.7 days in man (Sterling and Chodos, 1956). In accord with the short t+ for thyroid hormones in the chicken, thyroprotein-fed birds showed an increase in metabolic rate for only the first few hours of fasting (Mellen, 1958). In our experiments (Singh, 1966), chickens have shown only a transitory increase in metabolic rate after the administration of T8 and T+ singly or in combination. With the information now at hand it is not possible to assess precisely the part that the storage and discharge of thyroid hormones from extrathyroidal tissues may play in regulating the effective level of circulating thyroid hormones in birds. It is well established that the principal route of excretion for both Ta and T, is via the bile in chickens (Hutchins and Newcomer, 1966) as well as in mammals. Our unexpected finding that significant amounts of radioactive substance were released to the blood during sampling of relatively large quantities may indicate that the liver or other extrathyroidal tissues play a role in regulating the circulating thyroid hormone level. The liver perfusion experiments of Gorman et al. (1966) support the view that such a mechanism may exist. Although the thyroid hormone regulating mechanisms of birds seem to differ from those of mammals in several ways, the total activity, as shown by daily thyroid hormone secretion rate (Ringer, 1965) overlaps that of mammals (Reineke, 1959). ACKNOWLEDGMENTS Grateful acknowledgement is expressed to Mrs. Judianne Anderson for her assistance with the iodine analyses reported in this paper and to Dr. W. E. Cooper, Department of Zoology, Michigan State University, for advice on the statistical analyses. Thanks are also due to the Rockefeller Foundation for a Graduate Fellowship granted to Ajit Singh. REFERENCES ALBERT, A., AND KEATING, F. R. (1949). Metabolic studies with I”‘-labelled thyroid compounds. J. Clin. Endocrinol. 9, 1406-1421. BARKER, S. B., AND HUMPHREY, M. J. (1950).
THYROXINE
AND
TRIIODOTHYRONINE
Clinical determination of protein-bound iodine in plasma. J. Cl&. E&ocri~&. 10, 11361141. BUMGARDNER, H. L., .4xn SHAFFNER, C. S. (1957). Protein-bound iodine levels of the chick. PO& try Sci. 36, 207-208. FAT;LXNER, L. W., LEVY, R. P., ASD LEONAKDS, 6. R. (1961). Simplified t.echnique for the determination of serum protein-bound iodine. Clin. Chem. 7, 637-645. FELDMAN, J. D. (1957). Effect of estrogen on peripheral utilization of thyroid hormones. Am. J. Physiol. 188, 30-34. GREGERMAK, R. I., G.~FFP;ET, G. W., A?;D SHOCK, N. W. (1962). Thyroxine turnover in euthyroid man with special reference to changes with age. J. C%z. Invest. 41, 2065-2074. GORMAN, C. A., FLOCX: E. V., OWEN, C. A., JR., AND PARIS, J. (1966). Factors affecting exchange of thyroid hormones between liver and blood. Endocri~zology 79, 391-405. HENDRICK, C. E., AND TURSER, C. W. (1967). A comparison of the effects of environmental temperature changes and 4.4”C cold on the biological half life (tl/z) of thyroxine 1131 in fowls. Poultry Sci. 46, 3-5. HENIKGER, R. W., AND NEWCOMER, W. S. (1964). Plasma protein binding, half life and erythroeyte uptake of thyroxine and triiodothyronine in chickens. Proc. Sac. Exptl. Biol. Med. 116, 624-62s. HIJTCI~IXS, M. O., AND ~SERCOMER, W. S. (1966). Met.abolism and excretion of thyroxine and triiodothyronine in chickens. Gen. Camp. E,ndocrirwl. 6, 239-248. INGBAR, S. H., AND FREINKEL, N. (1955). Simultaneous estimation of rates of thyroxine degradation and thyroid hormone synthesis. J. C&. Invest. 34, 808-819. Er, 9. C. R. (1964). “Statistical Inference I.” Edwards Brothers, Inc., Ann Arbor, Mich. MCFARLAND, L. z., YOTJSEF, M. K., AND WILSOX, W. 0. (1966). The influence of ambient temperature and hypothalamic lesions on the disappearance rates of thyroxine I’“’ in the Japanese quail. Life S&zses 5, 309315. MELLEN, W. J. (1958). Duration of effect of thyroxine and thiouracil in young chickens. Poultry Sci. 37, 672-679.
TCRSOVER
3AI
W. J ,, .~ND HARDY, L. B. (1957). Blood protein-bound iodine in the fowl. E~crlocGoloq~~ 60, 547-551. ODDIE, T. II., ME.~DE, J. H., JR., AZXD FISHER, D. ‘1. (1966). An analysis of published data on thyrosine turnover in human subjects. J. Cl&. E/>u’:,crinol. 26, 425436. REIA-EKE: E. P. (1959). Thy-roid function in ce-, (1 era1 species of animals wit,h special referrnce to environment and body size. U.S. A!. Eneqy Commiss. I’lD 75’38, 87-96. RINGER, R. K. (1965). In lLAvian Physiology,” P. D. Sturkie, cd. Comstock Pub!. Aseocs. 2nd Ed., p. 614. ROSENBERG, 1,. L., GOLDMAN, M., LA Rocan, C.; AND DIXCII;, M. Ii. (1964). Thyroid function in rats and chickens. Eqcilibration of injected iodide with exist,ing thyroidal iodine in LongEvans rats and White Leghorn thicker:;;. Blidocrinology 74, 212-225. ~INGH, A. (1966). Studies of thyroid function in the chicken. Bound doctoral thesis. Michigan State Univ. Sl’ERLIKG, K., AKD CHODOs, R. B. (19%). Radiothyroxine turnover studies in myxedema, thyrotoxicosis and hypermetaboliem without andocrine discaae. J. CS,L, Invest. 35, 806-813. STUHBIE, P. D. (1965). “Avian Physiology,” Comstock P&l. Assoc., 2nd Ed., p. 2. T.4~.4, J R., AKD SHELLABAROER, C. 9. (1959). An explanation for the difference between the responses of mammals and birds to t,hyroxine and triiodothyronine. Biochem. J. 72, 60X-618,
MELLEN,
TAUROG,
,1.,
BRIGGS,
F.
r\‘.,
AND
CR.~TKOFF,
1.
L.
(1951). I’“‘-labelled n-thyroxine. I. An unidentified excretion product in bile. J. Biol. C&m. 191, 29-34. T~UROG, A., BRIGGS, F. x., AP*‘D CHAIHOY~F, 4. 1;. (1952). P3’-labelled L-thyroxine. II. Kature of the excretion products in bile. J. Z&l. C’hem. 194, 655668. VLIJM, L. (195X). On the production of hormones in the thyroid glands of birds. A quantitative study with the help of radioiodine and antithyroid drugs. AI&. Ne’erland. Zool. 12, 467631. WEXTVITORTH, B. C., AKD MELLES, W. 3. (1961). Circulating thyroid hormones in domestic birds. Poultry Sci. 40, 12751276.