THE EFFECT OF THYROID ACTIVITY ON CERTAIN ANABOLIC PROCESSES STUDIED WITH ...

13 Pages·2003·0.65 MB·English

THE EFFECT OF THYROID ACTIVITY ON CERTAIN
ANABOLIC PROCESSES STUDIED WITH
THE AID OF DEUTERIUM*
BY ADELE KARPt AND DEWITT STETTEN, Ja.1
(From the Department of Biochemistry, College of Physicians and Surgeons,
Columbia University, New York)
(Received for publication, January 18, 1949)
In the study of the metabolic defects of hypo- and hyperthyroidismp
great stress has been placed upon the alterations of the total metabolic
rate, as measured by gaseous exchange, which in turn represents the
summation of many simultaneous processes, among which the catabolic
ones predominate. The isotope technique has provided a convenient
method for the estimation of the rates at which certain tissue constituents
are being synthesized and deposited in the intact animal body. By re-
placement of a portion of the body water by deuterium oxide and ob-
servation of the rates at which stably bound deuterium appeared in tissue
fatty acids (l), cholesterol (2), and glycogen (3), it has been possible to
estimate the rates of synthesis of these several tissue constituents under
various circumstances. It was considered of interest to apply this tech-
nique to the study of the anabolic rates of various tissue constituents of
experimental animals at different levels of thyroid activity.
To this end, a group of rats was rendered thyrotoxic by the addition
to their diet of desiccated thyroid gland.’ Hypothyroidism was pro-
duced in a second series of rats by the administration of thiouracil (4);*
and a third group of rats, receiving no medication, served as controls.
Immature rats were selected as experiment.al animals in view of their
favorable and relatively rapid response to thiouracil (4).
EXPERIMENTAL
Animals and Diet-Immature female rats of the Sherman strain were
used throughout. At the age of 22 days, rats were placed in metabolism
* This work was carried out with the aid of grants from the Nutrition Foundation,
Inc., and the Josiah Macy, Jr., Foundation.
t Submitted by Adele Karp in partial fulfilment of the requirements for the degree
of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. Pres-
ent address, The Public Health Research Institute of The City of New York, Inc.,
New York.
$ Present address, The Public Health Research Institute of The City of New York,
Inc., New York.
1 Thyroid, U. S. P., Parke, Davis and Company.
2 The authors wish to express their gratitude to Lederle Laboratories Division,
American Cyanamid Company, for their gift of Deracil powder.
819
This is an Open Access article under the CC BY license.
820 EFFECT OF THYROID ACTIVITY
cages, two rats per cage, and divided into three groups, litter mate con-
trols being used as far as possible. The basal diet comprised 60 parts
of corn starch, 22 parts of casein, and 6 parts each of yeast powder, rough-
age (Celluration), and salt mixture (5). Initially four rats were em-
ployed for the determination of each experimental point in each of the
three groups, but as the mortality rate among the animals receiving
thyroid substance was high, supplementary rats were added to furnish
sufficient material. A total of 82 rats was employed in the entire ex-
periment. Food and water were allowed ad libitum and the quantity
consumed was estimated daily. Weekly supplements of vitamin A and
D concentrate were administered to all rats, and additional small sup-
plements of cod liver oil and linseed oil were given from time to time
to those rats receiving thyroid gland.
The first group of rats served as normal controls. To the drinking
water of the animals of the second group was added 0.1 per cent of
thiouracil. The thiouracil consumed increased from an initial value of
about 5 mg. to about 10 mg. per rat per day at the end of the experiment.
With the diet of the third group was admixed 1 per cent of desiccated
thyroid powder. Except for retarded growth (cf. Fig. l), no untoward
symptoms were noted among the animals receiving thiouracil. After
about 2 weeks the rats fed thyroid gland exhibited all the expected signs
of toxicity: hyperactivity, tremor, tachycardia, and cachexia. The mor-
tality in this group was 47 per cent and the animals which were sub-
jected to autopsy showed marked congestion of all internal organs. The
weight curve of this group is distinctly subnormal (cf. Fig. 1). The aver-
age daily food consumption of the three groups during the last week of
life was 12 gm. for the normal rats, 12 gm. for the rats receiving thiour-
acil, and 22 gm. per 100 gm. of body weight per day for the rats receiv-
ing thyroid substance.
All thyroid glands were studied grossly and histologically. The glands
from those animals which had received thiouracil were grossly enlarged,
hyperemic, firm, and readily separable from the tracheas. Histologi-
cally, the epithelium lining the follicles was hyperplastic and hy-
pertrophic, and there was great loss of colloid and invagination of the
lumen of the follicles. The thyroid glands from the rats which had been
fed thyroid substance were much smaller than normal, pale, and not
firm. In many cases they were distinguishable with difficulty from
surrounding tissues, even under the dissecting microscope. Histologi-
cally, the epithelium lining the follicles was flattened and, in many areas,
atrophied; the follicles were small and filled with dense colloid. No
difficulty was encountered in classifying the microscope slides according
to the treatment which the animals had received during life. The histo-
A. KARP AND D. STETTEN, JR. 821
logical results were confirmed by Dr. H. C. Stoerck and Dr. D. G. Wor-
cester of the Department of Pathology, College of Physicians and Surgeons.
Deuterium Oxide Administration-Subgroups of at least four animals
each, from each of the three groups, were given D20 for periods of 4, 1,
2, 4, and 8 days. The initiation of the isotope regimen was so adjusted
that all experiments were terminated on the 45th day of life. At the
appropriate time, each rat received a subcutaneous injection of 1 ml.
per 100 gm. of body weight of 99.5 per cent D20 containing 0.9 per cent
AGE-DAYS
FIG. 1. Growth curve of animals. The average weights of the total number of ani-
mals within each group are plotted against age throughout the period of medication.
The hollow blocks (right corner) represent the duration of the isotope regimens.
NaCl. From this time on, drinking water was replaced by 1.7 per cent
DzO, and this solution was also used to moisten the food. This pro-
cedure served to establish and maintain D20 concentrations of 1.1 to
1.2 per cent in the body water of the animals.
At exactly 45 days of age, each rat was killed by the intraperitoneal
injection of nembutal.
Isolations and Analyses-The tissues from the animals of each sub-
group were pooled for analysis. Livers were rapidly removed, weighed,
and ground with sand in a volume of cold 10 per cent trichloroacetic
acid approximating the weight of the tissue. This mixture was filtered
by suction and the solid residue extracted twice again with cold 5 per
cent trichloroacetic acid. From the combined filtrates glycogen was
immediately precipitated by the addition of ethanol to a final concen-
822 EFFECT OF THYROID ACTIVITY
tration of 60 per cent (6). Purification of the glycogen was effected
by successive reprecipitations with ethanol, once from 15 per cent KOH
solution, and twice from water. The white product was washed by
centrifugation with increasing concentrations of ethanol and then acetone,
was air-dried, and finally dried in vacua over Pz06.
The residue from the trichloroacetic acid extractions was further ex-
tracted by boiling successively with 95 per cent ethanol, ethanol-ether
mixture (3:1), and twice with ether. The ether was evaporated from
the combined lipide extract and the remaining ethanolic solution made
alkaline by the addition of 5 per cent of solid KOH. The solution was
boiled under a reflux for 3 to 4 hours, diluted with an equal volume of
water, and the non-saponifiable portion exhaustively extracted with
petroleum ether. From a solution of the non-saponifiable fraction in
80 per cent ethanol, sterol digitonides were precipitated and isolated.
The aqueous-ethanolic solution of saponifiable products was acidified
to Congo red paper, and the liberated fatty acids were extracted with
petroleum ether. The combined petroleum ether solution of fatty acids
was washed with water, dried over Na$Oc, evaporated to dryness, and
the residue dried in vucuo over PzO5 and paraffin.
The residual liver tissue which remained after the foregoing extrac-
tions was dried overnight at 90”, ground thoroughly with the sand with
which it was contaminated, and aliquot portions taken for micro-Kjeldahl
nitrogen determinations. This insoluble fraction has been designated
“liver protein,” and its quantity estimated from the nitrogen determination.
The eviscerated carcasses of the rats from each subgroup were ground
and digested in hot aqueous 30 per cent KOH, the volume of the reagent
approximating the mass of tissue. The alkaline digest was filtered
through glass wool and glycogen was precipitated from the filtrate by
the addition of 1.2 volumes of ethanol. The glycogen was collected
by sedimentation and centrifugation and was purified by successive
precipitations with ethanol, once from 10 per cent aqueous trichloro-
acetic acid, and twice from water.
The isolations of carcass sterols and carcass fatty acids were conducted
on a weighed aliquot portion of the aqueous-ethanolic alkaline supernatant
solution from which glycogen had been precipitated. The steps were
similar to those described above in the treatment of liver.
The pooled hearts, lungs, spleens, and kidneys from each subgroup were
used for the isolation of a sample of body water (7). The segment of
trachea to which the thyroid gland adhered was removed from each
animal, and the thyroid gland was subsequently freed of adjacent tissues
under the dissecting microscope. The gland was fixed in Bouin’s solu-
tion, imbedded in paraffin, sectioned, and stained with hematoxylin and
A. KARP AND D. STETTEN, JR. 823
eosin. The elapsed time between the killing of the animal and the dis-
position of the carcass was 5 to 6 minutes. All deuterium analyses were
carried out by the falling drop technique (8).
DISCUSSION
That the animals receiving thyroid substance exhibited the expected
toxic response was shown by the development of the symptoms of thyro-
toxicosis, the high mortality, and the inhibition of gain in weight despite
polyphagia (9). Whereas the normal rats, during the last 12 days of the
experiment, gained an average of 0.33 gm. in weight per gm. of food eaten,
TABLE I
Carcass and Liver Weights; Deuterium Content of Body Water
T T
Normal Thiouracil Thyroid
-
T
Duration L!ver Body i c,a rca. Liver ‘C :arcass Liver
;;“p,‘, Hz0 ZreeIr i ghrta t ;wK eTi ghrat t I wxre ighrat t vheerig hrta t
oblnr otooms
gm. b-m. czwccee nst s gm. m. ?cfizcmGn le ss !tcZieeCrn Cl SS
D D D
4 108 4.88 1.u 105 4.86 1.2: 88 7.03 1.22
4 127 5.46 1;li 109 5.61 1.x 79 5.28 1.21
4 111 5.24 1.21 80 3.92 1.1; 81 5.23 1.11
4 120 5.06 1.01 90 4.13 1.u 80 5.55 1.16
4 106 5.19 1.18 100 4.40 1.11 86 6.57 1.14
~__ -~
Weighted av-
erage. . . . . . . . 114 5.16 96 4.50 83 6.00
- - -
the rats receiving thyroid gland gained only 0.14 gm. The complete
absence of glycogen from the livers of the animals receiving thyroid
gland was a uniform observation in the present series and is in accord
with numerous reports of this effect of thyroid intoxication (10). Rela-
tive, as well as absolute, hepatomegaly was noted (Table I); liver weight
per 100 gm. of body weight averaged 4.53 gm. for the normal animals,
and 7.23 gm. for the rats receiving thyroid gland, a finding which has
previously been described (11). The grossly evident cachexia was re-
flected in the analytically determined diminution in the quantity of
depot fat; the rats receiving thyroid gland contained almost 25 per cent
less depot fat per 100 gm. of body weight than did the normal control
series. The histological changes observed in the thyroid glands of these
animals were in accord with the expected response to thyroid admin-
istration (12, 13).
824 EFFECT OF THYROID A-
The evidence that thiouracil had exerted its inhibitory effect in our an-
imals rests largely upon the gross and histological appearance of the thy-
roid gland itself, which was identical with the descriptions in the lit-
erature (14, 15). The correlation between this appearance of the gland
and lowered metabolic rate has been demonstrated by others (16, 17).
The moderate retardation of growth in response to thiouracil (cf. Fig.
1) was also in accord with the reports of others (14, 15). No signifi-
cant deviation from normal food intake was noted.
Glycogen-No trace of glycogen was recovered from the livers of any
of the rats receiving thyroid gland. The deuterium concentrations in
the successive samples of liver glycogen obtained from the normal and
LIVER GLYCOGEN
NORMAL THlOURACll
l l
.
.
. 0
.
.
I I I I I I I I I
OO 2 4 6 a
DAYS
FIQ. 2. Deuterium concentrationa in liver glycogen samples. The deuterium
concentrations, expressed ae percentage of deuterium in body water, have been
plotted against duration, in days, of the deuterium oxide regimen.
thiouracil-treated rats varied widely (cf. Fig. 2), a finding which has
been previously observed (3).
Comparison of the deuterium concentrations in the samples of carcass
glycogen from the normal and the thiouracil-treated rats (Fig. 3) reveals
no striking differences. On the other hand, point for point, the samples
of carcass glycogen from the rats receiving thyroid gland were richer in
deuterium than those from the other two groups. The three factors
which determine the rate of increase in deuterium of a tissue constituent
in such an experiment are the amount synthesized each day, the amount
of preexisting non-isotopic tissue constituent with which this is diluted,
and the actual isotope concentration in the newly synthesized product.
This last quantity may be evaluated experimentally by estimation of
the asymptotic value which the isotope concentrations appear to be
approaching. From Fig. 3 it would appear that this asymptote lies in
the neighborhood of 35 to 40 atoms per cent D for the glycogen in the
thyroid-treated animals, and at 15 to 20 atoms per cent D for the glyco-
A. KARP AND D. BT, JR. 325
gen in the other two groups. This suggests that when thyroid sub-
stance was fed, approximately twice as much stably bound hydrogen
in the newly synthesized glycogen had arisen from the body water as
was the case in the other two groups. Similar observations of variation
in the value of the asymptote, referred to as i,,,=., have been made in
diabetic (18) and in thiamine-deficient (19) animals, and also in animals
fed lactate in place of glucose (20). In each of these cases, i,,. was
higher than in the normal control.
Fatty Acids--The rises in deuterium concentration observed in the
fatty acids both of liver and of carcass show certain common character-
istics (cf. Figs. 4 and 5). In every case the thyroid-fed animals gave
samples richer in deuterium than did those of the other two groups. From
CCAARRCCAASSSS GGLLYYCCOOGGEENN
NORMAL 0 TTHHYYRROOllDD 00 TTHHllOOUURRAACCllLL l l
4400-
I
FIG. 3. Deuterium concentrations in carcass glycogen samples. The deuterium
concentrations, expressed as percentage of deuterium in body water, have been
plotted against duration, in days, of the deuterium oxide regimen.
an inspection of the curves it appears that in the thyroid-fed animals
the isotope concentrations approach a maximal value of about 45 to 50
atoms per cent D. On the other hand, in the cases of the normal con-
trols and thiouracil-treated animals, an i,,. of about 35 atoms per cent
D is approached. Estimation of the half life times in the case of the
liver fatty acids leads to a value of about 1 day when thyroid was ad-
ministered, and about 13 days for the normal and thiouracil-treated
groups. As expected, the fatty acids of the carcasses in each group of
animals approached the same maximal isotope concentrations as those
of the livers, but at a lower rate. In the case of the carcass fatty acids,
half the maximal isotope concentration is reached in about 23 days under
thyroid administration and in about 44 days in the other two groups.
With use of these figures together with the quantities of depot fatty acids
recovered from each of the three groups of animals (Table II), it has
been estimated that 1.2, 1.1, and 1.0 gm. of depot fatty acids per rat
826 EFFECT OF THYROID ACTIVITY
were replaced daily by newly synthesized fatty acids in the normal, the
hypothyroid, and the thyrotoxic animals, respectively.
The more rapid rise in deuterium concentration of the fatty acids of the
carcasses of the rats receiving thyroid substance is thus ascribable to the
higher value of i,,, and the paucity of depot fat. The diminutions in
LIVER FATTY ACIDS
NORMAL 0 THYROIDS THlOURAClL l
DAYS
FIQ. 4. Deuterium concentrations in liver fatty acid samples. The deuterium
concentrations, expressed as percentage of deuterium in body water, have been
plotted against duration, in days, of the deuterium oxide regimen.
CARCASS FATTY ACIDS
50 c NORMAL 0 THYROlD 0
THlOURAClL l
40-
*
I
DAYS
FIG. 5. Deuterium concentrations in carcass fatty acid samples. The deuterium
concentrations, expressed as percentage of deuterium in body water, have been
plotted against duration, in days, of the deuterium oxide regimen.
the quantity of depot fat which followed the administration of thyroid
gland, in spite of a virtually normal rate of deposition of newly synthesized
fatty acids, must therefore be attributed to accelerated degradation of
body fat. This is in contrast to the mechanism of diminution in the
quantity of body fat observed in diabetes (19), undernutrition, and thiamine
deficiency (20). In all of these cases, a striking diminution in the rate of
lipogenesis was observed.
A. KARP AND D. STETTEN, JR. 327
SteroLs-Whereas the level of blood cholesterol, both in man (21) and
in experimental animals (22), is known to be influenced by the activity
of the thyroid gland, no significant variations in the quantities of sterol
recovered from the livers and carcasses of the three groups of animals
here studied were noted (Table II). Consideration of the concentra-
tions of deuterium found in the sterol digitonides of the livers of the
TABLE II
Average Weights of Tissue Constituents
Liver constituents are reported as per cent of wet liver weight, carcass constit-
uents as per cent of total body weight.
Thiourscil Thyroid
--
per cent per cent )CI ccnl
Liver glycogen ................ ...... 3.26 3.90 0.00
Carcass glycogen .............. ...... 0.148 0.109 0.107
Liver fatty acids .............. ...... 2.04 2.04 2.40
Carcass fatty acids ............ ...... 5.23 5.79 4.00
Liver sterol digitonides ........ ...... 0.72 0.69 0.68
Carcass sterol digitonides. .... ...... 0.48 0.51 0.57
Liver “protein”. .............. ...... 16.1 13.3 17.4
- ~~--~
20
LIVER STEROL D/G/ TON/DES
I5 t
0
*
I .
-* :” :
5 . NORMAL 0 THYROID-
THlOURAClL l
I I I I I I I I
2 4 6 8
OO
DAYS
FIG. 6. Deuterium concentrations in liver sterol digitonide samples. The deu-
terium concentrations, expressed as percentage of deuterium in body water, have
been plotted against duration, in days, of the deuterium oxide regimen.
three groups of rats reveals the same tendency that has previously been
noted in the other tissue constituents (Fig. 6). The isotope concentra-
tions in the materials from the rats receiving thyroid gland are higher than
normal, while the thiouracil-treated rats appear to give slightly lower
values. However, the quantitative differences here are not as striking
as with the glycogen and fatty acids. The curves for the liver sterols
proper, corrected for the presence of digitonin (23), appear to approach
828 EFFECT OF THYROID ACTIVITY
maximal values between 45 and 60 atoms per cent D. A half life of 3 to
4 days is here estimated.
In view of the relatively long half life of carcass sterols (2), the low
deuterium analyses obt,ained on the carcass sterol digitonides are not
surprising. The resultant higher per cent analytical error would in part
account for the wide scattering of these points (Fig. 7), which precludes
any satisfactory interpretation.
Liver “Protein” -The isotope concentrations of the liver protein frac-
tions are plotted in Fig. 8. It is recognized that the protein mixture
analyzed is far from homogeneous and that the processes leading to the
CARCASS STEROL DIGITONIDES
NORMAL TWROID THIOURACIL
l l l :
I 1 I I I I I 1 I
OO 2 4 6 8
DAYS
FIG. 7. Deuterium concentrationa in carcase sterol digitonide samplea. The
deuterium concentrations, expressed as percentage of deuterium in body water, have
been plotted against duration, in daye, of the deuterium oxide regimen.
biological incorporation of deuterium into the protein molecule are nu-
merous and cannot at present be exactly described. Among these an im-
portant one probably is the introduction into the proteins of deuterio-
amino acids which have been synthesized in a medium of heavy water;
and in so far as this is true, the rise in deuterium concentration of the
liver protein should bear some relationship to the rate of protein synthesis.
Study of Fig. 8 reveals a tendency toward higher isotope concentrations
in the liver proteins of the thyroid-fed animals, when compared with the
other two groups. The thiouracil-fed animals do not deviate signifi-
cantly from the normal. Here again the differences are not marked
enough to allow a quantitative comparison. Under these experimental
conditions an i mm. of between 20 and 25 atoms per cent D appears to be
approached. From these figures the half life of the process which we are
measuring would be 2 to 3 days.