Iodine Requirements of Rats during Growth, Reproduction, and Lactation

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PU R D U E UNIVERSITY

THIS IS T O CERTIFY T H A T T H E THESIS P R E P A R E D U N D E R M Y SUPERVISION

by

Herbert Edmund Parker

ENTITLED

IODINE REQUIREMENTS OF RATS DURING GROWTH,

REPRODUCTION, AND LACTATION

COMPLIES W I T H T H E UNIVERSITY R E G U L A T I O N S O N G R A D U A T I O N T H E S E S

A N D IS A P P R O V E D B Y M E A S FULFILLING THIS P A R T O F T H E R E Q U I R E M E N T S

FOR THE DEGREE OF

Doctor of Philosophy

• g ^ ^ P R O F E S S O R I N C H A R G E O F T H E S IS

bad of

S chool or D epartm ent

T O T H E LIBRARIAN:-THIS THESIS IS N O T T O B E R E G A R D E D A S CONFIDENTIAL.

'K O r e e s O H D J C H A R G E

G R A D . S C H O O L F O R M 9 —3 - 4 9 —1M

IODINE REQUIREMENTS OF RATS DURING GROWTH, REPRODUCTION, AND LACTATION

A Thesis

Submitted to the Faculty of Purdue University

by Herbert Edmund Parker In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

August, 1950

ProQuest Number: 27714182

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uest ProQuest 27714182 Published by ProQuest LLC (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

ACKNOWLEDGMENT The author wishes to express his appreciation to Dr. F. W. Quackenbush for his suggestions and guidance throughout the course of this investigation. He also wishes to thank Dr. S. M. Hauge for his council in the conduct of the animal experiments and Dr. F. N. Andrews for his assistance in the preparation and interpreta­ tion of the slides showing the histology of the thyroid glands. The cooperation and assistance of Mr. C. E. Peterson and Dr. J. E. Christian in determining the amount of radioactive iodine absorbed by the thyroid glands of rats was greatly appreciated.

TABLE OF CONTENTS Page ABSTRACT .................................................

i

INTRODUCTION ..............................................

1

ANALYTICAL ................................................

9

Reagents and Special Apparatus ..........................

9

Procedure ...................................... *......

10

Results and Discussion .................................

12

EXPERIMENTAL................................................

17

Rations ..................................................

17

Design of Experiments

...............

Experiments with Rats during Growth ...................

21 22

Reproduction Experiments ..................................

22

Studies with Increased Levels of Other Dietary Components..

23

Absorption of Radioactive Iodine ........................

24

RESULTS......................................................

26

Experiments with Rats during Growth.......................

26

Reproduction Experiments ..........................

39

Studies with Increased Levels of Other DietaryComponents..

49

Absorption of Radioactive Iodine ........................

52

DISCUSSION...............................................

54

SUMMARY.....................................................

60

BIBLIOGRAPHY ................................................

61

FIGURES Figure 1.

Page Fresh Thyroid Weights of 9 Week Old Rats of the First Generation on Experimental D i e t s .......

29

Dry Matter Content of Thyroid Glands from 9 Week Old Rats of the First Generation on Experimental Diets ....

30

Fresh Thyroid Weights of 9 Week Old Rats of the Second Generation on Experimental Diets .....................

33

Dry Matter Content of Thyroid Glands from 9 Week Old Rats of the Second Generation on Experimental Diets

34

The Neck Area Showing the Thyroid Glands of a 9 Week Old Rat Which Was Grown on a Diet Containing 25 fig. of Iodine per kg.....................................

35

The Neck Area Showing the Thyroid Glands of a 9 Week Old Rat Which Was Grown on a Diet Containing 100 jig. of Iodine per kg.....................................

36

The Neck Area Showing the Thyroid Glands of a 9 Week Old Rat Which Was Grown on a Diet Containing 225 ug. of Iodine per kg.....................................

37

The Neck Area Showing the Thyroid Glands of a 9 Week Old Rat Which Was Grown on a Diet Containing 525 Ug* of Iodine per kg.....................................

38

Iodine Content of Thyroid Glands from 9 Week Old R a t s

40

10.

Fresh Thyroid Weights of Rats at B i r t h ........

45

11.

Fresh Thyroid Weights of Weanling Rats ........

46

12.

Iodine Content of Thyroid Glands from Weanling Rats and Their Dams ......................................

47

Sections of the Thyroid Glands from Adult Female Rats Which Were Autopsied at the Time the Young Were We a n e d ..............................................

50

2. 3. 4.

5.

6.

7•

8.

9.

13*

TABLES Table

Page

1.

Iodine Content of Possible Dietary Components ...........

13

2.

Recovery of Iodine Added to Samples ....................

15

3•

Composition of Basal Diets .............................

19

4*

Analysis of Variance of Fresh Thyroid Weights per 100 Grams Body Weight of Rats in Experiment 1 ............

27

Iodine Requirements of Growing Rats of the First Generation on the Experimental Diets .................

28

Iodine Requirements of Growing Rats of the Second Generation on the Experimental Diets .................

32

7•

Effect of Iodine on Reproduction ....................

42

8.

Weight and Iodine Content of Thyroid Glands of Young Rats ............

43

Weight and Iodine Content of Thyroid Glands of Adult Female Rats ...................................

44

Effects of Sodium chloride. Calcium carbonate, and Wheat Gluten on the Thyroid Glands of Rats on a Diet Deficient in Iodine ..................

51

Effect of Iodine Content of the Diet on the Absorption of Radioactive Iodine by the Thyroid Glands of Rats ....

53

5•

6.

9• 10.

11.

ABSTRACT The iodine requirement of rats during growth was studied using diets containing 25, 50, 100, 225, and 525 ug. of iodine per kg.

The

minimum amount of iodine needed in the diet of rats during growth to prevent significant thyroid enlargement was found to be 100 - 225 ug./kg. Although sane hypertrophy was observed in the thyroids of rats on the high­ est level of iodine, the general histological appearance of the thyroids of rats on the different levels of iodine was closely associated with the degree of thyroid enlargement. The thyroids of rats on the low levels of iodine contained only small amounts of colloid, had tall columnar epithelial cells, and showed considerable hypertrophy and hyperplasia. The growth rates of rats fed the different levels of iodine did not differ significantly; however, rats on the lowest level did grow slightly faster than rats on higher levels.

The amount of iodine needed to prevent

thyroid enlargement of rats of the second generation on the experimental diets was found to be the same as that required by rats of the first generation. Reproduction experiments with rats showed that the level of iodine required in the diets of pregnant and lactating rats to prevent thyroid enlargement in the young or in the dams was 100 - 225 ug./kg.

Adult

female rats on the lowest level of iodine produced larger litters than rats grown on higher levels of iodine.

The thyroid glands of newborn

rats from dams at the lowest level of iodine had the histological appear­ ance of embryonic glands.

However, these rats grew just as fast as rats

from dams on higher levels of iodine.

ii

Increasing the level of NaCl or CaCO^ in iodine-deficient diets did not significantly affect the weight, dry matter content, iodine content, or histological appearance of the thyroids from growing rats. Raising the protein level in the iodine-deficient diet by the substitu­ tion of

20 per cent wheat gluten for 20 per cent of the ground corn did

cause a

slight increase in thyroid size even though the iodine content

of the diet was increased. Studies of rats

using radioactive iodine showed thatthe thyroid glands

on the lowest level of iodine absorbed approximately ten times

as much radioactive iodine in one hour as the glands of rats on the highest level of iodine. The thyroid glands of male rats on the iodinedeficient diet were more efficient in absorbing radioactive iodine than the glands of female rats. Rats were grown for three successive generations on diets contain­ ing different levels of iodine.

Rats on the lowest level of iodine did

not show any inhibition of growth or reproduction.

It was concluded that

lowering the iodine level in the diet to 25 ug./kg. did not noticeably interfere with the metabolic functions of the rat.

IODINE REQUIREMENTS OF RATS DURING GROWTH, REPRODUCTION, AND LACTATION

INTRODUCTION Increased thyroid size, abnormal thyroid histology, and low iodine content of the thyroids have been observed in large numbers of newborn lambs and pigs grown in Indiana.

Since the effects of iodine deficiency

were most marked in newborn and young animals, the question was raised whether or not the iodine requirement was greater during gestation and lactation* Andrews et al.(1)

studied the gross and microscopic anatomy and

the iodine content of thyroid glands from 338 newborn lambs and 407 newborn pigs in three central Indiana sheep flocks and two swine herds which were fed rations commonly used under mid-west farm conditions. Enlarged hyperplastic thyroid glands having low iodine contents were observed in a high percentage of lambs frcm a flock fed a commonly used ration not supplemented with iodine.

In one swine herd fed a ration

not supplemented with iodine, of 111 glands studied 39 per cent contained only a trace of iodine, 45 per cent showed only a trace of stored colloid, and over 30 per cent were hyperplastic. When stabilized iodized salt was used only five of 106 glands weighed more than 0.47 gnu, only 14 contained less than 0.20 per cent iodine and none was hyperplastic* Some of the commonly accepted evidences of iodine deficiency such as hairlessness in pigs and scanty wool in lambs have been observed throughout Indiana.

However, the severity of the deficiency does not

appear to be nearly as great as that observed in the Northwest where

2

losses in newborn lambs and pigs were so great that farmers ceased sheep and swine breeding until it was learned that the us e of potassium iodide (35) would prevent these losses. The lack of information regarding the iodine requirements of farm animals makes it impossible to clearly evaluate the iodine situation in Indiana.

Although some rough estimates have been made of the iodine re­

quirements of farm animals based on the amount of iodine needed to prevent the development of goiter in rats during growth, no comprehensive study has been made of the iodine requirements of animals during reproduction and lactation* Studies of iodine requirements of animals are complicated by several factors.

In the first place, the iodine requirements of animals

are apparently very low, and therefore, the problem of preparing a diet which is deficient in iodine yet adequate in all other nutritional factors is difficult *

The levels of other nutritional components may affect the

iodine requirements.

In view of the complications listed above, it was

felt that a study should be made of the iodine requirements of rats and of the factors affecting the iodine requirements before more costly experiments are undertaken to establish the iodine requirements of farm animals.

The information obtained by rat experiments including the entire

life cycle should be invaluable in estimating the iodine requirements of other animals and in establishing technics which may be used in experiments with larger animals. Several investigators have shown that a deficiency of iodine in the diets of animals causes hypertrophy and hyperplasia of the thyroid gland.

Krauss and Monroe (15) discovered that the rachitic ration of

Steenbock and Black (32) produced, simultaneously with the rickets.

3

enlarged thyroid glands of the rat. tion:

This diet has the following composi­

yellow corn, 76 parts; wheat gluten, 20 parts; calcium carbonate,

3.0 parts; and sodium chloride, 1,0 part. this diet in goiter studies with rats.

In 1932, Thompson (34) used

Hyperplasia of the thyroid was

observed when this ration was fed either with or without vitamin D.

The

factors underlying the development of hyperplasia were reported to be a deficiency of iodine associated with excessive amounts of calcium carbon­ ate.

Addition of small amounts of potassium iodide prevented hyperplasia. In 1933, Levine, Remington, and von Kolnitz (17) published a

dietary technic for the study of goiter in the rat, using the rachitic ration of Steenbock: and Black with 0.2 gm. of irradiated yeast added per 100 gm. of ration.

These investigators (18) used this ration which con­

tained 15 ug. of iodine per kg. in a study of the iodine requirements of rats.

Using the smallest amount of iodine necessary to prevent any

significant thyroid enlargement as a criterion, these investigators re­ ported the TTnnimmn iodine requirement of rats to be one to two micrograms per rat per day.

It was suggested that an iodine intake of 20 to 40

micrograms per 1,000 calories of ration be considered as the minimum iodine requirement for farm animals until precise data are obtained for different species. Remington (25) has pointed out that the diet of Levine et al. was not ideal for the study of iodine deficiencies because the growth of young rats fed the diet was subnormal.

This diet was too deficient in

nutritional factors other than iodine to be used in reproduction studies. Remington reported that improved growth could be obtained if part of the wheat gluten was replaced with 1 ) purified casein, 20 dried pig liver, or 3) dried brewers1 yeast.

The high iodine content of casein prohibits

its use in iodine requirement studies.

Remington reported that on a

diet containing two per cent dried pig liver, rats reached maturity and produced normal numbers of living young despite almost complete ab­ sence of iodine and colloid from the thyroid gland.

No data were pre­

sented on the iodine requirements of rats during pregnancy and lactation. Sharpiess (29) failed in an attempt to produce goiter in rats using the diet of Levine et al. (18), but was successful in obtaining thyroid enlargements of 3 - 5 times normal when rats were grown on a diet con­ taining unprocessed soybean flour. Several investigators have reported that raw soybean flour has goitrogenic properties which cannot be accounted for by its low iodine content.

In 1933, McCarrison (24) reported a high incidence of thyroid

enlargement in rats fed a diet containing raw soybeans.

His results

indicated that raw soybeans produce goiter even when iodine is fed in large amounts.

Sharpless efc al. (31) reported that soybean flour con­

tains a positive goitrogenic property that is partially removed or destroyed by fat solvents (ether or acetone) or by steam.

These investi­

gators found the minimum iodine requirement of rats fed a diet containing 75 per cent unprocessed soybean flour to be two times that reported by Levine et al. (18).

Wilgus et al. (36) confirmed the observations of

Sharpless et al. (31) that soybeans are goitrogenic and that this goitrogenicity is partially inactivated by Heat.

Halverson et al. (8) found

that the iodine requirements of rats on diets containing raw or heated soybeans was slightly greater than that of rats on diets similar to those used by Levine et al. (18). Enlargements of thyroid glands of animals fed diets containing liver have been reported by Hunt (13), Marine (19), Burget (3), and Hou (12).

5

The results reported by Hou (12) indicated that the factor in liver which caused an enlargement of the thyroid glands of rats was present in the alcohol-soluble portion of the dried liver. The substances present in soybeans and liver which cause excessive thyroid enlargement have not been identified.

However, Greer et al.(7)

were successful in the isolation and identification of a natural occur­ ring goitrogen of the Ithiocarbàmidée series, 1 -5 -vinyl-2-thiooxazolidone, from turnips and rutabaga roots. The possibility of the presence of natural goitrogens in diets used for iodins requirement studies must be considered.

In these studies

an attempt was made to find a ration suitable for iodine requirement studies without using any of the feeds which have been reported to con­ tain goitrogens. The levels of other dietary components may affect the iodine re­ quirements of animals.

A review of the literature on this subject revealed

the need for further work to clear up the confusion due to the conflicting results which have been reported.

Thompson (34) reported that the addition

of calcium to an iodine-deficient diet exaggerated the clinical picture, caused enlargement of the thyroid gland, and degeneration of the para­ thyroid gland.

Hellwig ,10) found that the addition of two per cent cal­

cium chloride to a diet of corn meal and rolled oats caused the development of goiter in rats whether or not iodine was added to the diet.

However,

Remington and Levine (2?) reported that varying the calcium content of the rations, its calcium-phosphorus ratio, and the presence or absence of vitamin D did not significantly affect the degree of goiter produced. Sodium chloride as well as calcium chloride was reported to cause thyroid hyperplasia by Hibbard (11).

However, Sharpless and Anthony (30)

6

found no significant increase in thyroid size of rats fed increased amounts of chloride.

There was a decrease in iodine concentration in the thyroids

of rats receiving chloride. The presence of fluorine in sheep rations was reported by Hatfield et al. (9) to increase the percentage of iodine in the thyroid gland, both when there was adequate and inadequate iodine intake. The relationship between vitamin A and thyroid function has been studied by several investigators.

According to Remington et al. (26)

vitamin A deficiency was not an etiological factor in development of simple goiter, but there was increased need for vitamin A in thyroid hyper­ function and decreased need in thyroidectomy.

However, Coplan and Samp­

son (5) reported that vitamin A deficiency produced definite hypertrophy of the thyroid glands of female rats during a limited period of experi­ mentation, but caused consistent atrophy of the thyroid glands of male rats.

Deficiencies of iodine and vitamin A in the diet produced an ini­

tial hypertrophy in both males and females, but in the males the initial hypertrophy was followed by atrophy of the thyroid glands.

Lepsett and

Wingler (16) used radioactive iodine to study the effects of vitamin A deficiency on thyroid function.

These workers found that in vitamin A

deficiency the thyroids of rats were heavier than controls.

Degenerative

changes and distended follicles were found to be present within the same gland.

The uptake of radioactive iodine by the thyroid was found to be

the same as that of the controls but the rate of formation of thyroxine was decreased when vitamin A was deficient. Excessive feeding of

ck -tocopherol has been reported to cause the

development of abnormal changes in the thyroids of male dogs. fed from 5.5 to 12.5 mg. of

Huter (14)

o< -tocopherol acetate per kilogram body

weight daily for 22 to 96 days to 10 young dogs.

The following changes

7

were noted in the thyroids of the male dogs:

abnormal increase in size

and volume, deficiency of iodine and histological changes, especially increased epithelium*

The effects were much less marked in the females.

A survey of the iodine in water, soils, and crops of Indiana was made by the author (23 and 24) for his M. S. thesis.

The iodine content

of water samples varied greatly, ranging from 1.4 to 37«0 parts per billion and averaging 7*3 parts per billion.

A large variation was found

in the iodine content of water samples taken from the same counties.

In

spite of the large variation within counties, some conclusions were drawn regarding the general distribution of iodine in water. The water samples / taken from the northern part of the state were found generally to contain less iodine than those from the southern part.

Twenty water samples from

the region within 100 miles of Lake Michigan had an average iodine content of 3*5 parts per billion, while 33 samples from other parts of the state had an average iodine content of 9.6 parts per billion.

The "t" value

for these data was found to be 3*02 which showed that the observed differ­ ence was statistically significant at the one per cent level. The iodine content of Indiana soils was found to be dependent upon the organic matter content, texture, drainage characteristics, and age of the soil.

The iodine content of soil was found to vary directly with the

organic matter content of the soil. iodine than light textured soils.

Heavy textured soils contained more Poorly drained soils which did not have

an accumulation of organic matter contained less iodine than soils with better natural drainage.

Samples of soil of the Clermont and Guthrie

series were particularly low in iodine. The residual soils, millions of years old, in the southern part of the state, generally contained more

8

iodine than soils from, the Illinoian, Early Wisconsin,and Late Wisconsin glaciers.

The iodine content of soils derived from till of more recent

glaciers was slightly lower than that derived from till of older glaciers. The iodine content of crop samples varied greatly with the type of crop and the part of the plant analyzed.

Cereal grains were generally found

to be low in iodine; however, oats and speltz contained considerably more iodine than c o m and wheat.

All of the hay samples analyzed contained

considerably more iodine than corn and wheat.

A H of the hay samples

analyzed contained several times more iodine than any of the seed samples. The data obtained from the crop analysis indicated that the iodine in plants was concentrated more in the leaves and stems than in the seeds* The iodine content of crops was not found to be proportional to the iodine content of the soils on which the crops were grown. The results of the survey confirmed the contention that Indiana is on the border line of an iodine-deficient area.

Samples of soils, crops,

and water from Indiana were found generally to contain less iodine than samples from states farther south and more iodine than that reported from states farther north.

9

ANALYTICAL Determinations of the iodine contents of dietary components, rations, thyroids, and blood samples were made using modifications of the catalytic procedures described by Chaney (4), Taurog and Chaikoff (33) and Barker (2) for the analysis of blood iodine. in all of the procedures.

The fundamental reactions are the same

The only modifications made consisted of adapt­

ing the procedures to the equipment available at this laboratory.

Combus­

tion and solution of the feed samples were accomplished by the procedure described by Godfrey, Parker, and Quackenbush (6). Reagents and Special Apparatus Double-distilled water.

Distilled water was redistilled from 1 per

cent potassium hydroxide solution in an all glass distill. Oxygen (linde VSP).

Iodine was removed by passing the oxygen through

a gas absorption tower containing 1 per cent potassium hydroxide solution. Sulfuric acid (Baker’s analyzed C. P. special).

Iodine was removed

by adding 2 ml. of hydrochloric acid to 400 ml. of the sulfuric acid aid boiling for one to two hours. 10 M Chromium Trioxide Solution.

2000.2 gms. of chromium trioxide

(du Pont, flake, technical) were dissolved in redistilled water and diluted to 2 liters. 5 M phosphorous Acid.

102.5 gms. of crystalline phosphorous acid

(General Chemical Co., Reagent grade) were dissolved in redistilled water and diluted to 250 ml. 0.01 N Arsenic Trioxide Solution in 0.1 N Sodium Hydroxide.

0.4947

gms. of arsenic trioxide (Kallinckrodt Analytical reagent) and 4 gms. of sodium hydroxide (Mallinckrodt USP pellets) were dissolved in redistilled

10

water and. diluted to 1 liter. 0.15 N Arsenous Acid in 3.25 N Sulfuric Acid.

3.71 gms. of arsenic

trioxide (Mallinckrodt analytical reagent) were dissolved in an aqueous solution containing 2.5 gms. of sodium hydroxide (Mallinckrodt USP pellets). The solution was diluted to 250- 300 ml., neutralized with repurified sulfuric acid, 45 ml. of repurified concentrated sulfuric acid added, and the solution diluted to a volume of 500 ml. 0.1 N eerie ammonium sulfate solution in 3.5 N sulfuric acid. 31.62 gms. of eerie ammonium sulfate (G. Fredric Smith Chemical Company reagent grade) were dissolved in redistilled water, 48.7 ml. of repurified concentrated sulfuric acid added, and the solution diluted to Standard iodine solutions.

500 ml.

Potassium iodide (Mallinckrodt analytical

reagent) was dissolved in redistilled water.

A solution containing 10

micrograms of iodine per ml. was prepared fresh every few months.

Solu­

tions containing 0 .1 , 0 .075, 0 .050, and 0.025 micrograms per ml. were prepared fresh every two weeks from the solution containing 10 micrograms of iodine per ml. Combustion apparatus.

The combustion apparatus was the same as that

described by Godfrey, Parker, and Quackenbush (6). Coleman Junior Spectrophotometer Model 6 A. Distillation apparatus.

A modification of the distillation apparatus

described by Mathews, Curtis, and Brode (21) was used. consisted of the omission of the concentric trap

The modification

(Some iodine was found

to be retained in this trap).

Procedure The combustion procedure developed by Godfrey, Parker, and Quacken­ bush (6) was used to destroy the organic matter in the feed samples.

11

The fundamental steps in this procedure consisted of burning the samples in a vicor glass combustion tube and passing the gases produced by the combustion through a gas absorption tower containing glass beads, 5 ml. of 10 K CrO^ solution and 50 ml. of concentrated The ash in the combustion tube was then combined with the solution from the absorption tower in an 800 ml, digestive flask.

The resulting

solution was boiled down until its temperature reached 220°C., and the solution was digested at this temperature for five minutes.

After allowing

the solution to cool to 100°C., the sides of the flask were washed down with 25 ml. of redistilled water.

The solution was then reheated to 220°C.,

cooled to 100°C., 75 ml. of redistilled water added, and the flask con­ nected to the distillation apparatus. As soon as distillation commenced, a 50 ml. graduated cylinder con­ taining 5 ml. of 0.01 N arsenous acid in 0.1 N sodium hydroxide was placed under the condenser so that the tip of the condenser extended down into the solution.

Four ml. of 5 M phosphorous acid were added to the distillation

flask and distillation continued until 50 ml. of distillate collected in the graduate cylinder.

The distillate was transferred to a 100 ml. beaker

and evaporated to approximately 35 ml., over a hot plate.

The distillate

was then transferred to a volumetric flask of such size that when diluted to volume the resulting solution contained 0.002 to 0.2 micrograms of iodine per kg.

Five ml. aliquots of this solution were placed in 19 x

150 selected Covette colorimeter tubes.

One ml. of redistilled water and

0.5 ml. of 0.15 N arsenous acid in 3.25 N sulfuric acid were added to each tube.

The tubes were then placed in a water bath at 30oC.

to c ome to constant temperature.

0.1° and allowed

0.375 ml. of 0.1 N eerie aivmonium sulfate

was added to each tube and the per cent light transmission at 440 mu. read

12

with a Coleman Junior Spectrophotometer at exactly fifteen and thirty minutes after the addition of the eerie ammonium sulfate ♦

The iodine content of the

sample was then determined by comparing the readings with those obtained with tubes in which known quantities of iodine were added,

A series of standards

containing 0.000, 0,025, 0.050, 0.075 and 0.100 micrograms of iodine per tube were run with each group of samples.

By staggering the addition of

eerie ammonium sulfate solution to the tubes at one-minute intervals, fifteen tubes could be run at one time. The iodine contents of the thyroids were determined by wet oxidation of the thyroids in a solution containing 5 ml. of 10 M CrO^ and 50 ml. of repurified concentrated sulfuric acid.

The iodine content of the resulting

solution was determined as described for the analysis of feed samples. The blood samples were analyzed for iodine by oxidizing 2 ml. samples of the blood in a solution containing 10 ml. of 10 M CrOg and 50 ml. of repurified concentrated HgSO^.

The iodine content of the resulting solution

was determined as described above.

In all analyses, a reagent blank was run

with each group of samples using the same quantity of reagents as was used in the analyses.

Results and Discussion The iodine contents of the possible dietary components which were analyzed for iodine are shown in Table 1.

Many of the substances analyzed

contained too much iodine to be used in rations for iodine requirement studies. Using feeds which were found to be relatively low in iodine a ration suitable for iodine requirement studies was prepared.

The results

of analysis of the mineral compounds was particularly useful in the prepara­ tion of a mineral mixture having a low iodine content.

13

TABLE 1 IODINE CONTENT OF POSSIBLE DIETARY COMPONENTS

Sample analyzed

Iodine content _

Corn (Lafayette Cooperative Elevator)

_

_

14

8

Corn (George Dilts Farm) Wheat

20

Oats

25

Wheat gluten

26

Wheat germ

26

Wheat germ (defatted, dehydrated,and pulverized)

34

Sodium proteinate (soybeans)

94

Corn germ

29

Soybean meal

34

Fresh lean beef

45

Dried lean beef (Rund

PackingCompany)

100

Dried beef liver

255

Liver extract

175

1 :20 Liver extract (Wilson)

282

Blood fibrin

1300

Dried eggs

1000

Dried egg yolk

24^

Wesson oil containing vitamin A acetate and calciferol

134

CaCO^ (Mallinckrodt,

AR)

10,000

CaCO^ (prepared

from CaO,

lot

1

36

CaCOg (prepared

from CaO,

lot

2

24

14

TABLE 1 (continued) Iodine Content of Possible Dietary Components

Sample analyzed

Iodine content Pg./kg.

NaCl (Baker* s Analyzed, CP)

35

MgSO^ (Mallinckrodt, USP)

60

Na^ 1P04 12 h2° (Mallinckrodt, AR)

43

K 2HPO4 (Mallinckrodt, AR)

340

K ^ F O ^ (Baker's Analyzed CP)

896

KH2P04 (Mallinckrodt, AR, lot 7100)

22

RB^PO^ (Mallinckrodt, lot 7096)

24

K 2CO3 (Baker USP)

18

KOH

32

(Mallinckrodt, AR)

CaH^f(P0/f)2 (Mallinckrodt, lot 4256)

25

Ferrous lactate (Mallinckrodt, NFV)

7200

Ferrous ammonium sulfate (Baker, purified)

34

Calcium lactate (Mallinckrodt, lot 4208)

73

Mineral mixture (McCullum No. 185) Mineral mixture used in diets

330 39

15

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CV ON

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O

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NO

NO

ON

CV

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-4

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8

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t?

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8

2

I

U"\

33

820

-e

0

100

200

300

MICROGRAMS OF IODINE

400

500

600

PER KILOGRAM OF DIET

Fig. 3 Fresh Thyroid Weights of 9-Week Old Rats of the Second Generation on Experimental Diets

34

40

PER CENT

DRY

M ATTER

30

20

/00

200

300

MICROGRAMS OF IODINE

400

500

600

PER KILOGRAM OF DIET

Fig. 4 Dry Matter Content of Thyroid Glands from 9-Week Old Rats of the Second Generation on Experimental Diets

Fig. 5 The :3ck Area Showing the Thyroid Glands of a 9-Week Old Rat whi i: was Grown on a Diet Containing 25 ug. of Iodine per kg.

Fig. 6 The Neck Area Showing the Thyroid Glands of a 9-Week Old Rat Which was Grown on a Diet Containing 100 ug. of Iodine per kg.

37

Fig. 7 The Neck Area Showing the Thyroid Glands of a 9-Week Old Rat Which was Grown on a Diet Containing 225 ug. of Iodine per kg.

Fig. 8 The Neck Area Showing the Thyroid Glands of a 9-Week Old Rat Which was Grown on a Diet Containing 525 Ug. of Iodine per kg.

39

of the thyroid glands of rats fed different levels of iodine.

With the

exception of the rats on the highest level of iodine, rats of the first generation on the experimental diets had thyroids with higher iodine con­ tents than rats of the second generation (Figure 9).

The histological

appearance of thyroids from rats of the second generation (experiment 3) was somewhat similar to that of rats at corresponding levels of iodine in experiment 1.

Thyroids from rats on the basal diet in experiment 3

were larger and showed slightly more hypertrophy and hyperplasia than thyroids from rats on the basal diet in experiment 1.

Rats on the 30 pg.

per kg. level of iodine had thyroids which contained slightly more colloid than rats at the lowest level; however, evidence of hypertrophy and hyper­ plasia were still apparent in some thyroids of this group.

Thyroids from

rats at the 223 pg. per kg. level could not be distinguished from those at the 525 pg* per kg. level; however, hypertrophy was observed at both levels in spite of the fact that abundant colloid was present. Some difficulty was encountered in determining the iodine content of the blood samples*

For some unknown reason, the reagent blanks and the

blood samples from rats in experiment 1 were contaminated with iodine when the analyses were made.

The blood samples from rats on diets containing

25, 50, 100, 225, and 523 pg. of iodine per kg., in experiment 3, were found to contain 0.26, 0.52, 1.69, 2.07, and 4.41 pg. of iodine per 100 ml. , respectively

Reproduction Experiments A tabulation of some of the data obtained in the reproduction ex­ periments is shown in Table 7.

Female rats grown on the basal diet produced

more live healthy young than female rats grown on diets containing added

PCR CENT

IODINE

IN

DRY

THYROIDS

0 .4

o FIRST GENERATION x SECOND GENERATION

0.1

100 MICROGRAMS

200

300

OF IODINE

400

500

600

PER K IL O G R A M OF DIET

Fig. 9 Iodine Content of Thyroid Glands from 9-Week Old Rats

41

iodine.

An analysis of variance of number of live young per litter in

experiment 2 showed that the treatment means varied significantly (Pig./kg. level (P < 0.01).

The average birth weight of the individual rats was approxi­

mately the same in all groups.

No differences were observed in the weights

of the weanling rats from dams fed different levels of iodine. The weight and iodine content of thyroid glands from rats at birth and weanling rats from dams grown on diets containing different levels of iodine are shown in Table Ô.

It should be noted that the thyroid weights

per 100 g. of body weight of rats at birth were approximately twice as large as those of weanling rats.

As shown in Figures 10 and 11, the effect

of the iodine content of the diet on the thyroid weight of rats at birth and weanling rats is very similar to the effect observed in growing rats.

The

thyroid weights of weanling rats of the second litters from dams grown on the experimental diets were slightly larger than those of the first litter. However, the thyroid weights of weanling rats of the third generation on the basal diet were no larger than those of the second generation. The differences in thyroid weights of the adult female rats used in the reproduction experiments (Table 9) were similar to those found in the experiments with growing rats.

The thyroid weights of adult female rats

were not significantly changed by increasing the iodine content of the ration above 225 micro grams per kg.

The per cent iodine in dry thyroids

of the adult female rats and weanling rats is shown in Figure 12.

42

I

Q LTxITv » O

E

co

t-l

Q

feS.

Ox

o

o

( —1

O i—1

XO CN

Ox

cd

CV CV

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CN

to

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>

s §

0

0)

• xf a •H bû O

XI to CD

k 8

a

-ti H

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O

5

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^-1

.5

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1 •r l

O

to

ÿ

I

p

ro

ë S'S

s

ÏH

s

i

t £

u •H (i*

01 U •H

8

§

-p *H

s t #"ë

^ 58*3

cQ

a

CV (N

Lf\

LT\

45

Q

0

100

200

300

MICROGRAMS OF IODINE

400

500

PER KILO G RAM

600 OF DIET

Fig. 10 Fresh Thyroid Weights of Rats at Birth Second Generation First Litter

GRAMS BODY WEIGETT

* FIRST LITTER

THYROID

WEIGHT IN MILLIGRAMS PER 100

SECOND L I TTER

0

100

200

300

MICROGRAMS OF IODINE

400

500

600

PER K ILO G RA M OF DIET

Fig. 11 Fresh Thyroid Weights of Weanling Rats Second Generation

47

PER CENT IODINE IN DRY THYROIDS

0.30

0.25

0.15

0.10

•W E A N L IN G *

0.05

0

100

200

300

IO D IN E

FIRST LITTE R

WEANLING RATS SECOND LITTER

o AD U LT

M IC R O G R A M S O F

RATS

F E M A LE

400

PER K IL O G R A M

500 OF

Fig. 12 Iodine Content of Thyroid Glands from Weanling Rats and Their Dams

RATS

600 D IE T

48

The typical follicular arrangement of the more mature thyroid was was not present in the glands of newborn rats from dams on the lowest level of iodine.

The thyroids had the general appearance of embryonic glands.

The colloid was extremely thin and scant.

The epithelial cells were of

the tall columnar type and had the appearance of masses of disorderly ar­ ranged cells showing considerable activity and exhibiting considerable hyperplasia.

The histological appearance of thyroids from newborn rats on

the 100 pg. per kg. level of iodine was more typical of the mature gland. However, the colloid was still extremely scant and hypertrophy and hyper­ plasia were apparent.

The epithelium ranged from cuboidal to tall columnar.

The thyroids of newborn rats on the 225 pg. per kg. level of iodine con­ tained only traces of colloid which had a thin granular appearance. epithelium was cuboidal in type and showed slight hypertrophy.

The

Only moderate

amounts of colloid were present in the thyroid glands of newborn rats on the highest level of iodine. trophic.

The epithelial cells were cuboidal but not hyper­

The thyroids of newborn rats of the second litters from dams on

the different levels of iodine had the same general histological appearance as those of the first litters.

Thyroids of weanling rats on the lowest level

of iodine contained no more than a trace of colloid and the epithelium was predominantly of the tall columnar type exhibiting both hypertrophy and hyperplasia.

All of the thyroids from weanling rats on the 100 pg. per kg.

level of iodine contained moderate amounts of colloid and most of the glands had cuboidal epithelial cells.

Four of twelve glands examined contained

low columnar cells and showed slight hypertrophy.

Thyroids from weanling

rats on the 225 pg. per kg. level of iodine contained moderate to large amourts of colloid, and the epithelium varied from cuboidal to low columnar but

49

evidences of hypertrophy were limited.

The histological appearance of thy­

roids from weanling rats on the 525 pg. per kg. level of iodine was almost identical to that of thyroids from rats on the 225 pg. per kg. level of iodine group. All of the adult female rats examined were autopsied at the time the young were weaned and were therefore in a lactating condition. had some effect on thyroid function

This may have

since it is well known that certain

relationships exist between the thyroid gland and milk production.

Both

hypertrophy and varying degrees of hyperplasia were found in the thyroids of adult female rats on all levels of iodine intake.

With the exception of

differences in gross size, the thyroids of adult female rats on diets con­ taining 25 and 100 pg. of iodine per kg. were similar.

Only a trace of

colloid was present in these thyroids, the epithelium was of the tall columnar type, and both hypertrophy and

hyperplasia were evident.

Although the iodine

content of thyroids from adult female rats on the $25 pg. per kg. level of iodine was almost twice that of rats on the 225 pg. per kg. level, the histo­ logical appearance of the thyroids was very similar.

Colloid was present in

moderate to large amounts with a tendency for more to be present in the 525 pg. per kg. level group.

In both groups epithelial hypertrophy and slight

hyperplasia were evident in at least some areas of nearly all of the glands.

The histological appearance of sections of the thyroids from adult female rats is shown in Figure 13. Studies with Increased Levels of Other Dietary Components The effects of increased levels of sodium chloride, calcium carbonate, and wheat gluten on the thyroid glands of rats grown for 6 weeks on iodinedeficient diets are shown in Table 10.

The degree of thyroid enlargement of

rats in the high NaCl and high CaCO^ groups was not quite as great as that

d. Figs 13 Sections of the Thyroid Glands from Adult Female Rats Which Were Autopsied at the Time the Young Were Weaned. Hematoxylin and eosin x 300. a. b. c. d.

Section of Section of Section of Section of

a thyroid a thyroid a thyroid a thyroid

from a rat from a rat from a rat from a rat

on the 25 ug./kg. level of iodine. on the 100 Ug./kg. level of iodine. on the 225 ug./kg. level of iodine. on the 525 pg./kg. level of iodine.

51

v\

8

Effects of Sodium

Chloride, Calcium

Carbonate, and Wheat

Gluten

O

•5

i

to

nO

i

8

O