• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 Introduction
 Review of literature
 General experimental procedure
 Experiment 1- The effect upon rats...
 Experiment 2- The effect upon rats...
 Experiment 3- The effect of short-term...
 Experiment 4- The effect of short-term...
 Experiment 5- The effect of short-term...
 Experiment 6- Preliminary study...
 Experiment 7- The effects of varying...
 General discussion and conclus...
 Summary
 Bibliography
 Biographical items
 Copyright














Title: Interrelationships of molybdenum, copper and zinc in the simple stomached animal
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Title: Interrelationships of molybdenum, copper and zinc in the simple stomached animal
Series Title: Interrelationships of molybdenum, copper and zinc in the simple stomached animal
Physical Description: Book
Creator: Kulwich, Roman,
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
        Page v
        Page vi
        Page vii
    Introduction
        Page 1
        Page 2
    Review of literature
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
    General experimental procedure
        Page 23
        Page 24
        Page 25
        Page 26
    Experiment 1- The effect upon rats of varying levels of copper, molybdenum and zinc in a natural ration
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
    Experiment 2- The effect upon rats of varying levels of copper, molybdenum and zinc in a purified ration
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
    Experiment 3- The effect of short-term supplementation with molybdenum, zinc, and calcium pantothenate upon growth and radiocopper distribution in the rat
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
    Experiment 4- The effect of short-term supplementation with molybdenum upon growth and radiocopper distribution in the weanling rat
        Page 60
        Page 61
        Page 62
        Page 63
    Experiment 5- The effect of short-term supplementation with molybdenum upon growth and radiocopper distribution in the mature rat
        Page 64
        Page 65
        Page 66
        Page 67
    Experiment 6- Preliminary study of the effects upon swine of high dietary levels of molybdenum
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
    Experiment 7- The effects of varying dietary levels of molybdenum, copper, and zinc upon swine
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
        Page 86
        Page 87
        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
    General discussion and conclusion
        Page 95
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
    Summary
        Page 101
        Page 102
    Bibliography
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
        Page 108
        Page 109
        Page 110
        Page 111
        Page 112
    Biographical items
        Page 113
        Page 114
    Copyright
        Copyright
Full Text
Interrelationships of Molybdenum, Copper Zinc in the Simple-Stomached Animal
By
ROMAN KULWICH
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA August, 1951


X would like to express 137 gratitude to the Staffs of the Animal Nutrition 2k*fcor^tory of the University of Florida and the University of Tenneseee-Atcaaic Energy Comsiseion Agricultural Eeeearch Program at Oak fftdge, Tennessee, which Bade possible the research for this dissertation. I em particularly indebted to 3)r. George E. Davie, Br, Ray L. Shirley, t*r. Tony Cunba of the University of Florida, and to Br# C# 1. Cesar, Professor S. I#. lansard, Pr. S, L. Hood, and Professor Ira B* JMtney of the IJalirersity of feaaessee for their aid in planning and conducting this investigation.
I am grateful to the Oak Bidge Institute cf Buelear Studies and the Atomic Energy Commission, which granted the fellowship which enabled me to conduct my research at the splendid facilities of the University of SennesseeAtomlo Eaergy Commission Agricultural Eesearch Program laboratories at Oak Bidge.
I also wish to thank Mrs. Philip J* Doherty for her aid in correcting and typing this dissertation.
n


Page
Introduction ,*> 1
Eeview:'Of literature .* > * <.<*> :.# .# .# 3
Copper 3
Holyb&enua -* ? 12
Zinc * ? # 3.8
General Experimental Procedure 23:
Experiment 1 The Effect upon Eats of Varying Levels' f Copper^
Molybdenum arid Zinc in a Natural Ration ? 27
Experimental Procedure'and Results # , 27
Discussion and Conclusions ? * 32
Experiment 2 fhe Effect upon Eats of Tarying levels of Copper*
Jfalybdenum and Zinc in a Purified Eation # # ? 39
Experimental Procedure and Results 39
Discussion and Conclusions ? i
Experiment 3 Effect of Shert~terBi Supplementation with Molybdenum, Zinc and Calcium Pantothenate upen Growth
and Radiocopper Oictribution in the Rat ..... $$
Experimental Procedure and Results ? ...# 53
Discussionand Conclusions^' .# ~ .* * $>$
Experiment k Effect of Short-term Supplenientation with -Molybdenum-upon Growth and Radiocopper Distribution'in
the Weanling Eat *# '*"?* *-> 60
Esperimental Procedure and Results *. 60
Discussion and Conclusions * 63
Experiment 3? Effect of Short-torra Supplementation with ^lybdenum upon Growth and Radiocopper Distribution in
"the Mature Bat = # # Esperimental Procedure find Results ..,. 6b
Discussion and Conclusions ......... 6?
Ill


Experiment 6 Preliminary Study of the Effects upon Swine
of High Dietary Levels of Molybdenum 68
Experimental Procedure and Results * 66 Discussion and Conclusions 7k
Experiment 7 the Effects of Varying Dietary Levels of
Molybdenum, Copper and Zinc upon Swine 76
Experimental Prcwe ?6
Discussion and Conclusions *** 86
General Discussion and Conclusions ** 9$
Summary 101
Bibliography .# 103
TABLESt
1* Properties of Radioisotopes of Copper Molybdenum and
Calcium #** # .*..*... 23
2* Composition of S- Rat and Swine Basal Ration 27
3. Growth of Albino Rats Maintained on Natural Rations with Varying Levels of Molybdenum* Copper and Zinc from Weaning until Twelve Weeks of Age 28
U* Serum Phosphorus and Alkaline Phosphatase Levels of Albino Rats Maintained Fourteen Weeks oh Natural Rations with Varying Levels of Copper, Molybdenum and Zinc 29
%m Accunulation of Orally Admiriisired Radioactive Calcium in Tissues of Albino Rats Maintat ned on Natural Rations with Varying Levels of Copper*' Molybdenum and Zinc * 30
6m Excretion of Orally Administered Labeled Calcium by Albino Rats Maintained on Natural Rations with Varying Lvels of Copper* Molybdenum and Zinc *'# 32
7* Calcium Turnover In Tissues of Albino Rats Maintained on Natural Rations with Varying Levels of Copper* Molybdenum and Zinc 33


8* Accumulation of Copper, Molybdenum and Zinc in Tissues of Albino Rats Maintained on Natural Rations with Varying Levels f Copper, Molybdenum and Zinc from Time of Meaning until Eighteen Weeks of Age ** 35
9* Ca-3 Rat Ration with Modifications UO
10* Growth of Male Albino Rats Maintained on Synthetic
Rations with Varying Levels of Molybdenum* Copper
and Zinc from Weaning until Twenty-nine Weeks
of Age ......*.* Ul
>
11* Serum Phosphorus and Alkaline Phosphatase Levels of
Male Albino Rats Maintained on Synthetic Rations with
Varying Levels of Molybdenum, Copper and Zinc from
Time of Weaning until Seven Months of Age 1*2
12 # Accumulation of Orally Administered Radioactive Copper, in Tissues of Male Albino Rats Maintained on Synthetic Rations with Varying Levels of Molybdenum* Copper and Zinc U3
13 Alimentary Tract Content and Excretion of Orally
Administered Labeled Copper in Male Albino Rats
Sacrificed Eighteen Hours after Dosage 16
lit* Accumulation of Copper and Molybdenum Tissues of Male Albino Rats Maintained on Purified Rations with Varying Levels of Copper, Molybdenum and Zinc from Time of Weaning until Seven Months Old it6
15* Copper Turnover in Select Tissues of Male Albino Rats Maintained on Synthetic Rations with Varying Levels of Molybdenum* Copper and Zinc *.*.. h9
16* Effect of Molybdenum* Zinc* Molybdenum plus Zinc* and Molybdenum plus Calcium Pantothenate Drenching upon Growth of Weanling Albino Rats $k
17* Effect of Prior Drenching with Molybdenum* Zinc*
Molybdenum plus Zinc* and Molybdenum plus Calcium Pantothenate on Tissue Accumulation of Intramuscularly Administered Labeled Copper in Wean* ling Albino Rats * * 56


18. Alimentary Tract Distribution and Excretion of Labeled Copper in Weanling Albino Eats Sacrificed twenty* Four Hours after Intramuscular Dosage 8
19m Effect of Prior Drenching with Molybdenum upon tissue Distribution of Orally Administered Labeled Copper in Weanling Female Albino Eats Sacrificed Twenty-Two Hours after Dosage 61
20 Effect of Prior Drenching with Molybdenum upon Gastrointestinal Tract Content and Excretion of Orally Administered Labeled Copper in Weanling Female Albino Rats Sacrificed TwentywTwo Hours after Dosage 62
21 Effect of Drenching with Molybdenum upon Growth of
Female Weanling Albino Rats . . 62
22* Effect of Prior Drenching with Molybdenum upon Tissue Distribution of Intramuscularly Administered Labeled Copper in Twenty-Four Week Old Male Albino Rats Sacrificed Twenty-Two Hours after Dosage * 65
23* Effect of Prior Drenching with Molybdenum upon Gastrointestinal Tract Content and Excretion of intramuscularly Administered Labeled Copper in Twenty-Four Week Old Male Albino Rats Sacrificed Twenty-Two Hours after Dosage 66
2iu Effect of Drenching with Molybdenum upon Growth
of Twenty-Four Week Old Male Albino Rats 66
25* Composition of Swine Basal Ration Used in Preliminary
Study * ' *"'''" 68
26* Accumulation of Copper and Molybdenum in Various
Tissues of Spotted Poland China Swine Maintained on Natural Rations with Varying Levels of Copper and Molybdenum * ?1
27* Accumulation of Copper and Molybdenum in Various Tissues of Duroc Boars Maintained on Natural Rations with Varying Levels of Copper and Molybdenum ' ' " ' y 72
28. Excretion of Labeled Molybdenum Administered
Orally to Swine 73


FIGURESt
1 Pig 3 of Lot I after Four Months on Basal Ration ...... 78
2* Pig 3 of Lot II after Four Months on Basal Ration
plus 1000 p.p.n, of Zinc ....... * 78
3* Pig 1 of Lot III after Four Months on Basal Ration
plus 200 p.pum. of Capper *..* 79
U* Pig 1 of Lot 3V after Four Months on Basal Ration
plus 1000 p.p.m* of Molybdenum > 79
5* Fig 2 of Lot V after Four Months en Basal Ration plus 1000 p*pm Molybdenum and 200 p.p#m# of Copper * * 80
6, Pig 2 ef Lot VI after Four Months on Basal Ration
plus 1000 p.p.m. Zinc and 200 p.p.m. of Copper 80
vn
29* Growth Rate and Feed Consumption of Hampshire Swine on Natural Rations with Varying Levels of Molybdenum, Copper and Zinc .............. 81
30. tissue Distribution of Orally Administered Labeled Copper in Hampshire Swine Maintained on Natural Rations with Varying Levels of Copper* Molybdenum and Zinc *..*#*... 83
/pf
31* Alimentary Tract Distribution end Excretion 'Orally Administered Labeled Copper in Swine Twenty-Four Hours after Dosage 8?
32 Accumulation of Copper and Molybdenum in Tissues Of Hampshire Swine Maintained on Natural Rations with Varying Levels of Copper* Molybdenum and Zinc from Time of Weaning until Nine Months of Age *** 88
33* Copper Turnover in Select Tissues of Hampshire Swine Maintained on Natural Rations with Varying Levels of Molybdenum, Copper and Zinc * 50


INTRODUCTION
It has been known since 1938 that molybdenum has a certain antagonistic effect upon copper metabolism (39) When cattle and sheep were grassed on pastures high in irolybdenum content.:/_4iatottaj loss 0 condi-tion and even deaths resulted* Supplemental copper proved to have a therapeutic effect* indicating a relatlenship the nature of which is not yet entirely clear*
In certain areas of the United Kingdom stock grassing upon pastures with normal copper content which should supply adequate copper develop characteristic copper deficiency symptoms (1* 7# $$)* this is thought to be due to the interference with copper metabolism of a ^conditioning factor"* In some Instances, molybdenum has been ruled out as a factor (7).
Smith and Larson (101) demonstrated an antagonism between zinc and copper* When high levels of sine were fed to young rats subnormal growth and a microcytic hypochromic anemia resulted. When,, supplemental copper was supplied, hemoglobin levels were significantly higher.
The purpose of this investigation was to study toe effects of varying dietary levels of molybdenum* copper and sine upon rats and swine. Special emphasis was placed upon determining any possible effects of molybdenum and zinc upon copper metabolism* Chemical analyses were performed to determine copper, molybdenum and sine content in rat tissues and copper and moiybdenum content in swine tissues after the animals had been on experimental rations* Serum alkaline phosphatase and inorganic


phosphorus determinations were also made on rats maintained on high molybdenum and sine dietary levels* Radioactive copper was used to determine the effect of both long and short term supplementation with high levels of molybdenum and alnc upon copper metabolism* The eaccretion of radioactive molybdenum in swine was investigated* Radioactive cal cium was adminlsterid to rats to determine if high dietary levels of sine or molybdenum Influenced its absorption* tissue distribution and excretion*


REVIEW OP LITERATURE
Copper
Although copper Is universally present in all plants and animals (37)* the fact that tt is essential for life has only been established during the last three decades* Early investigators did not associate the presence of copper in various biological materials with any definite function* The historical development of knowledge conceraing the role of copper in plant and animal life has been reviewed by several authors (15* 23.1 37, k9$ 72* 78# 68* 89* 99).
Oecttrrenee in plant materialst as early as 1817 Seissner (76) re* ported the presence of copper in plant tissues* HcHargue (7U) noted the widespread occurrence of copper in sea water* rocks* soils* plants and animals and inferred that copper had a biological function* Lindow* Elvehjem and Peterson (65) reported the copper analyses of about 160 samples of common plant and animal foods* Copper was detected in all foods examined* the lowest level being 0*1 milligram per kilo of fresh celery* Porty**two livestock feeds examined by Hveh^em and Hart (36) contained an average of 13*5 milligrams of copper per kilo of dry matter1* These authors found that fertilization with copper sulfate could increase the copper content of plants as much as litS percent* Cunningham (21) found more copper in the aerial portion of plants than in the roots* with a lower copper content associated with fibrous portions of plants*
Copper la known to play a role in certain plant enzyme systems (23,


62) It has been established that copper is necessary for the growth and development of numerous plant species* Stiles (102) and Gilbert (U2) have reviewed the literature concerning copper deficiency diseases in plants*
Occurrence in animal tissues t Early studies concerning the role of copper in animal life were concerned with marine forms* In 1920 Rose and Bodansky (90) reported the occurrence of copper in the common ^eHyflsh* toe Portugese man-of-war* oysters* and numerous species of common fish, and expressed the belief that it might be an essential constituent* McHargue (Ik) reported in 1925 that newborn calves have considerably higher liver copper concentrations than mature animals* which he considered to be evidence that copper plays an important part In embryonic development and early growth stages* Quern and Hellwig (86) presented data concerning the copper content of milk in 1928. the values reported were 0*26 0*52 milligrams per liter for raw cow** milk, 0*1*5 0*50 milligrams per liter for raw sheep* s milk* and 0*19 0*25 milligrams per liter for Toggenburg goat milk* Cunningham (21) in 1931 reported the copper content In various organs of toe following t|>eeiest human* bovine* sheep* horse* pig* dog, cat, guinea pig, rabbit, rat, badger and domestic fowl* In general the liver, kidneys, heart, brain and hair contained higher levels of copper than did the skin, lungs, pancreas, spleen and flesh* Bruckmann and Zondek (12) reported the Iron, copper and manganese in human organs in 1939* liver copper concentration was shown to be at a maximum at birth, falling rapidly after two months


with & mean value for adults of about 3^*6 milligrams per kilo* Kehoe, Cholak and Story 08) using a ojaantitative spectrograph! method, fouad that copper was present in all biological materials studied* Blood copper was reported to be almost evenly divided between the plasma and the formed elements* "
Copper metabolism in animals t FUnn and Inouye (1&) carried out an investigation concerning the elimination and distribution of'copper in rats and guinea pigs. Most of the supplemental copper which was administered was recovered In the feces, and the liver was shown to have the greatest tissue accumulation* Using dogs and guinea pigs as experimental mlmale, an increase in the cxygen-carrying capacity of the blood was dersonstratcd when'copper was fed*
Perla, Sandberg, and Holly (83) in an experiment with rats, demonstrated a drop in copper retention when the manganese intake was increased 50 percent above the basal''level of 100 micrograms daily. Thompseit (106) demonstrated with mice that copper absorption was increased when the calcium content of the ration was increased and also when acid was added to the ration* Sftsa (35) found that when rabbits were in copper equilibrium* more than 96 percent of the copper intake was excreted in the feces* The infection of $ milligrams of copper as the sulfate or 100 milligrams given in solution by stomach tube proved fatal while 200 milligrams given in a dry form with the feed had no harmful effect*
Window* Peterson and Steenbock (66) found that with rats maintained on a ration containing 3*29 milligrams of copper per kilo the urinary if ecal


ratio of copper was lt2, bat 96 percent was excreted in the feces when five milligrams of copper were added daily per rat* Rats receiving this supplemental copper had five times as much copper in their spleens and 20 times as much copper in their livers as did toe controls at seven to eight months of age*
In a study using radioactive copper* Comer, Davis.and Singer (18) determined the fate of rally administered and intravenously injected doses of labeled copper in the bovine* Seventy*flVe percent of the oral dose was excreted in the feces* and three percent in the urine during five days* the copper which was absorbed was distributed generally with the highest concentration found in the liver and kidney* the use of radioactive copper in nutritional studies has been reviewed by Comar OS. 16%
Lorenaen and Smith (67) studied the liver copper content of rats* rabbits and guinea pigs at various ages* It was found that all three species have a reserve store at birth* There, was a decrease in total copper and copper concentration of the liver during toe suckling period for the rabbit and the guinea pig* while rats showed only a decrease in copper concentration*
Sehultse (93) has reviewed the literature dealing with the role of copper in hemoglobin and red blood cell formation* In 1928 HcHargue* Healy and Hill (?5) demonstrated that giving copper which had been extracted from calf liver ash raised the hemoglobin levels of rats which had become anemic on a milk diet* During toe same year Hart* Steenbock*


Waddell and ELveh^em (1*0) reported that copper in amounts'as small as 0*01 milligram were capable of curing the anemia produced in rats main* tained on a whole milk diet supplemented with ferric chloride* T.'addell, Steeribock and Blyeh^ea (Xlh) gave further proof that this anemia produced on a whole milk diet was caused solely by a copper deficiency* These re* suits have been confirmed in later investigations (57# $9$ 85* 113)* Teague and Carpenter (105) produced a copper deficiency in young pigs. Accompanying the dietary anemia which resulted was an unusual leg condi tion resembling rickets* The forelegs became crooked and the hocks extremely flexed, while the joints lacked rigidity* The administration of copper stepped the development of this abnormal condition* leil and Helson ($9) observed a graying produced by nutritional anemia in dark coated rats which the addition of 0*05 milligrams of copper daily restored to the original color* Hontwyler and Hanzal (60) reported that when copper was administered to anemic rats it caused a mobilisation of the iron stores* Liver iron concentration was reduced and blood hemoglobin and laythreeytes increased* Scbultse* Elvehjem and Hart 05) found that copper from copper easeinate* glycine amide biuret, hemocyanin and whole wheat was in a form readily available for the treatment of nutritional anemia of rats, while copper hematoporphyrin was not utilised* These authors reported that 0*005 milligrams of copper daily produced a response in anemic rats* Schultae (9k) investigated the relation of copper to cytochrome oxidase and hematopoietic activity of the bone marrow of rats* A considerable decrease of cytochrome oxidase activity of the bone marrow


was fand. to accompany copper deficiency. 0pon the administration of copper there was an immediate increase in cytochrome oxidase activity, approaching a maximum within 2h hours, thus demonstrating a relation between cytochrome oxidase activity of the bone marrow and hematopoiesis.
Schultze and Kiuken (96) investigated the effect of copper en cata-lase activity in rats, finding that there was a considerable decrease in catalase activity of liver, kidney and blood in copper deficient rats, while there was an increase of catalase activity of the heart. Davis and Hannon (29)* in a study of copper deficiency with Devon cattle, observed Increases in alkaline blood phosphatase activity as blood copper levels decreased while at the same time blood inorganic phosphorus content rose from below 6*0 milligrams percent to over 12.0 milligrams percent* The bones of these animals became brittle* Administration of copper brought about a quick return to normal blood phosphatase and inorganic phosphorus values* Lemer, Fitapatrick, Calkins and Summerson (63) demonstrated that mammalian tyrosinase required copper for ensymatic activity* Activity was inhibited by substances combining with copper, and then activity was restored by the addition of cupric ions* The role of copper in ensyme systems was reviewed in 1950 by Lehninger (62)*
Flesch (liO)investlgated the role of copper in mammalian pigmentation* It was found that black or gray hair generally contained more copper than white hair in the same individual, although this was not always the case* The epidermis of human skin contained more copper than the cerium* Cupric ions bad a stronger catalytic action in autoxidation of dopa in vitro


than any other heavy metal salts investigated* This author hypothesised that copper acts as a local catalyst in mammalian pigmentation* Singer and Davis (100) produced graying of the haircoat in a group of black and piebald rats by feeding a simplified diet containing less than one part per minion copper. Another group which was fed the same basal ration with the copper level increased to 20 parts per million showed no change in pigmentation. A daily dose of 20 micrograms of caloium pantothenate showed some effect in restoring the hair color of the rats which had grayed* while 30 and bO micrograms had a considerable effect* Hundley (53) reported that Black Long Evans weanling male rats developed achromotrichia after five to six weeks on a purified ration conatining 1U*6 parts per million of copper* The addition of 2*5 micrograms of copper per gram of ration prevented change in haircoat* In a later paper (5U)* Hundley and Ing demonstrated about a fourfold increase of skin copper content of pantothenic add deficient rats as compared to litter mates receiving sufficient pantothenic acid* The pantothenic acid deficient rats devel* oped gray hair in about seven weeks and were retarded in growth* The authors suggested that pantothenic acid deficiency may produce achromo-trichia by blocking copper utilization in melanin formation and in hair growth*
Copper teaicltyt Boyden* Potter and ELvehjem (10) in a copper toxicity study with rats* reported slight toxicity at a level of 500 parts per million* After four weeks on a ration containing 2000 parts per mil* lion of copper* liver copper content was about 300 times normal* All rats


on ItOOO parts per million of copper died daring the first week on rations. Boughten and Hardy (9) reviewed the early literature concerning copper poisoning and described an outbreak of chronic copper poisoning of range t&sep in West Texas caused by ingestion of salt mixtures containing copper sulfate. The symptoms described were yellow discoloration of the tissue, brown to black urine, weakness and loss of appetite* The disease proved fatal in most cases after the symptoms had appeared. The disease was produced experimentally in healthy sheep. Marston and tee (71) found that impaired kidney function was invariably the cause of death in experimentally produced chronic copper toxicity of sheep. Cunningham (2U) experimentally produced acute copper poisoning in the bovine* The lethal dose was between 200 and 1,00 grams of copper sulfate. The administration of 0.8 to $ grams of copper sulfate daily failed to produce chronic poisoning.
Naturally occurring copper deficiency diseases in animalst There are several excellent reviews of the literature concerning copper deficiency diseases in animals (1$, 1*2* 91, 102). In 1931 leal, Becker and Shealy (81) reported a natural iron and copper deficiency in the vegetation growing on white or gray sand, Everglades muck or Homestead marl soils in Florida. Cattle, sheep, goats and swine were affected in the deficient areas. Animals affected by this condition, known as "Salt Sick" were emaciated and weak, and showed very low hemoglobin levels. A dally drench of ferric ammonium citrate and copper sulfate was effective in treating affected animals. A copper deficiency disease of cattle


ectt*ffLag la the southwest portion of Western Australia has been (Ascribed by Bennetts* Beck* Harley and Evans (5)* This disease* known as "Falling Disease" is characterised by macrocytic, hypochromic anemia* malnutrition* depraved appetite* temporary sterility in cows* and abj^rmal bone developmeat* The range of pasture copper content in affected areas Was 1*1 to 3*2 parts per million* The mean liver copper concentration of affected animals studies was 3*7 parts per million of copper compared to 122 parts per million for cows in areas not affected* Merston* Lee and IfcBoaald (70) in an experiment with steep on copper and cobalt defi* cieht pastures of the South Australian coast* found that from 1 7 milligrams of supplementary copper daily were needed to meet the re* quirements* When sheep on these deficient pastures received cobalt but no copper* low liver copper levels ranging from 2*3 *"*>? parts per mil* lion and high liver iron concentrations of 7*700 35*700 micrograms iron per gram..of dry weight were observed*' The first/indication of ..copper deficiency was a characteristic lesion of the wool* Demyelination of the central nervous system of lambs occurs when the copper stores of the eves have been seriously depicted. The growth rate was retarded and anemia occurred in copper-deficient sheep* (Mnningham (22* 25* 26) described the occurrence of copper deficiency in Hew Zealand* In deficient areas, the pasture plants are deficient in copper* and stock maintained in these areas have below normal liver and blood copper levels* Davis* Kidder and Comar (28) reported that copper deficient cattle main** talned on Saint Augustine grass in the Everglades section of Florida lost
n


-weight* became anemic* showed bleaching of the haircoat and 3&eketf*like swelling were observed in the long bones* Bone fractures occurred with older cattle* Copper deficiency diseases have been reported (1, 7, $$) to occur in areas of the United Kingdom where pastures contained normal amounts of copper* indicating that interference in copper metabolism is perhaps a factor*
Molybdenum
Mstrlbutlon of molybdenum. In 1932 ter Meulen (77) reported on the distribution of molybdenum in nature* Molybdenum was found in small concentrations in a large variety of plants and vegetable products* fhe greatest concentration (3-9 milligrams per kilo) was found in peas* with cereals having 0*2 0*6 milligrams per kilo and wood* leaves* various fruits and vegetables containing lesser amounts* Soils* coal ash and mineral oils also were found to contain this element* Human and animal tissues were eaamined for molybdenum* The greatest concentration was found in the liver (1*5 milligrams per kilo in pig or ox liver) and the spleen. Molybdenum was also found in smaller concentrations in the blood* bile* milk, eggs and various tissues (0*llj, 0*03 milligrams per kilo)* Dingwall* McKibbin and Beans (33) reported the presence of molybdenum in vetch* com stalks* red clover, Canada thistle* timothy, ragweed, alfalfa* celery* alsifce, beets* goldenrod, carrots and reed canary grass grown in the Province of Quebec* Jfolybdenaa is needed by plants in small amounts, but because of its wide distribution in soils*


naturally occurring molybdenum in plants is not likely (k%$ 102) Vanselow and Batta (112) described an experimentally produced molybdenum deficiency of the citrus plant which was cured by levels as low as 0.0001 part per million of molybdenum in toe culture solution. There are several reviews of the literature concerning molybdenum deficiency in plants (1*2. 78. 102* 112).
The role of molybdenum in animal nutrition! In 1938 Ferguson* Lewis and Watson (39) reported that high levels of molybdenum in the pastures in Somerset* England were responsible for a disease of cattle characterised diarrhea* loss of condition and even death. The disease was produced experimentally by feeding soluble molybdates. Supplemental copper was found to prevent or cure the disease. Pastures on which molybdenum toxicity occurred contained from 20 100 parts per million of molybdenum on the dry basis. Potentially toxic levels of molybdenum have been reported in Florida (18) and California (3* 31) Cunningham (2?) has suggested that a small excess of molybdenum together with a deficiency of copper is responsible for peat scours" of cattle in New Zealand. Britton and (Joss (11) found that in an area of California where toxic levels of molybdenum occurred young cattle were more susceptible than older ones* dairy cattle were affected more than beef cattle* while swine and horses were not affected at all*
Teresi, Hvehjem and Hart (105a) found that no growth improvement resulted when additional molybdenum was given to rats fed a goat's milk diet which supplied 0*5 micrograms of molybdenum daily* and


concluded that If molybdenum ie needed by the growing rat* 0*5 micrograms daily fulfils the requirement* Falrhall at al. (38) reviewed the literature on molybdenum and reported the results of a study of molybdenum toxicity with particular reference to industrial toxicology* Doses of from 1*200 to 6*000 milligrams of molybdenum per kilogram in the form of molybdenum triextde* calcium molybdate and ammonium molybdate proved fatal when fed to rats and guinea pigs* Intraperitoneal injection of from ItOO to 800 milligrams of soluble molybdenum compounds produced a high mortality rate with guinea pigs* the kidneys and bones had the greatest accumulation of molybdenum* It was shown that both absorption and excretion of molybdenum occurred rapidly*
McQowan and Brian (73) suggested that the diarrhea which prevailed with cattle suffering from molybdenum toxicity may be due to a reduction of the bacteriostatic activity of catechols as a result of complex formation with molybdates*
Hollands* strong and Elvehjem (82) studied the toxicity of molybdenum for rata when added to a purified ration in the form of sodium molybdate* A level of 500 milligrams percent of molybdenum produced death after one week* while at levels of 100 and $0 milligrams percent a greatly reduced growth rate resulted* with no gross pathology or change in the blood picture* The addition of two milligrams percent of copper to a ration containing 7*73 milligrams percent of copper was found to largely counteract the toxicity of 1*0 milligrams percent of molybdenum. A tracer study with radlomolybdenum indicated that much of the ingested
m <


molybdenum was rapidly absorbed and then excreted. The kidneys and bone had a greater accumulation of labeled molybdenum than other tissues two days after oral dosage* Simultaneous administration of copper did not affect the distribution of radiomolybdenum, while a simultaneous dose of catechol increased the retention of molybdenum* These authors suggested that ittolybdenum toxicity may be neutralised to some extent by the forma* tion of combinations with copper and perhaps other substances in the diet*.
Comar, Singer and Davis (10) demonstrated that the toxicity of a dietary molybdenum level of 80 parts per million depended upon copper in** take* When rats received this level of molybdenum and two parts per mil* lion of copper* retarded skeletal growth* rough haircoat and diarrhea were observed, while rats on the same molybdenum level and % parts per million of copper were normal in appearance* Data for tissue distribution of radiomolybdenum in the bovine was presented Which indicated a similarity in the behavior of phosphorus and molybdenum* Interrelationships of molybdenum* copper and phosphorus in the rat were investigated, using radioisotopes* The authors suggested that molybdenum toxicity may be due to interference in enzyme systems involved in skeletal metabolism caused by a lowering of liver copper, direct inhibition of these ensyme systems by molybdenum, and competition between phosphorus and molybdenum for deposition in bone*
Shirley, Owens and Davis (98) demonstrated an effect of high molybdenum and copper diets upon excretion of intramuscularly injected radio-phosphorus by rats* The control group excreted the most labeled phosphorus
- 15


la feces and urine, while the high molybdenum, high copper, and high molybdenum plus copper groups excreted decreasing amounts, respectively.
Jeter and Davis ($6) reported the effects of varying levels of molyb* denura upon fertility, gestation and lactation of rats. levels of 0, 20, 80, and IhQ parts per million of molybdenum were added to a purified ration. Growth retardation was observed with all groups receiving added
molybdenum. Histological studies revealed some degeneration of the
my
epithelium of the seinlferous tubules in rats on high levels of molybdenum. TOiile all male rats receiving 0 or 20 parts per million of molybdenum sired litters, T$ percent of the males receiving 80 or Uj0 parts per million of molybdenum failed to sire litters when bred to females on the Same ration*
Gray and Ellis (hh) confirmed the growth retarding effect of molybdenum and found that 0*01 percent added copper did not counteract the toxicity of molybdenum at the 0*08 percent level*
In 19h$ Dick and Bull (32) reported that feeding ammonium molybdate over a three-year period to cattle resulted in lowered liver copper storage* this was also found to be the case when sheep were given10 or 100 milligrams of supplemental molybdenum daily for six months*
Davis and Hen son (30) reported that levels of molybdenum equivalent to 200 parts per million of the diet fed in the form of sodium molybdate to cattle resulted in somewhat lower liver copper values and a rise in liver molybdenum level* Davis (27) found that giving high levels of molybdenum to cattle for seven months resulted in a change in the pathway of excretion of radiophosphorus* Considerably more labeled phosphorus


was excreted in the feces and less in the urine of the steer receiving a high level of molybdenum than was the case for the control animal*
Cunningham (26) observed that in some regions of New Zealand sheep and cattle diseases resulted from a simple copper deficiency* while in other regions they resulted from a copper deficiency complicated* and partially induced by excess molybdenum in the feed* In cattle, both simple and complicated copper deficiency resulted in lack of thrift, anemia and brittle bones in young animals* while a severe seasonal diarrhea occurred only with complicated copper deficiency* Both types of deficiency in sheep were manifested by the development of osteoporosis or ataxia in lambs*
Marston (69) found that when sheep were grafted in areas normally providing sufficient copper, the addition of molybdenum reduced liver copper concentrations more rapidly when seasonal conditions decreased the pasture copper content below that necessary for the sheep to be in positive copper balance* When pasture conditions Improved* the rate of copper storage was lower than that of an untreated animal* However* when sheep on deficient pastures of the calcareous littoral of South Australia were given $0 milligrams of molybdenum dallyi the rate of depletion of liver copper was decreased, blood copper concentration was maintained, and the symptoms of severe copper deficiency were manifested sooner* A supplement of 10 milligrams of copper dally, which Insured a positive copper balance in sheep on these pastures, prevented the j^mptoms of copper deficiency from occurring In sheep treated with molybdenum and lead to the recovery of affected sheep* Blood copper levelu of sheep receiving


supplemental copper and molybdenum were very significantly higher than those of sheep receiving only supplemental copper* Marston hypothesized that under these conditions molybdenum either fixed the copper in a nonutilizable form or antagonised copper-containing enzymes, this phenomenon was also demonstrated with rats maintained on rations supplying just sufficient copper for maximum growth when given one milligram of molybdenum daily* Copper deficiency symptoms resulted, while tissue copper concentration increased materially*
Russell (93.) has reviewed the literature to 1916 on the occurrence of high levels of molybdenum in pastures and its relation to animal health*
Cesser (1, 16) has reviewed the use of radioactive molybdenum in nutritional studies*
Zinc
Occurrence of slnot In 1077 Iie^hartler and Bellamy (61) reported the presence of sine in wheat, corn, barley, other grains, and in the liver and muscle of an ox* In the same year BAoult and Breton (87) analysed four human livers, finding a range in sine content from 10-76 milligrams per kilo* In 1920 Bodansky (8) found that sine was a normal constituent of the twenty species of marine animals which were studied* Zinc is present in aH foods and tissues (99) Jait (68) reviewed the early literature on the occurrence of sine in biological materials and reported on sine distribution in the rat, cat and man* Vallee and Gibson (108, 309) reported on the sine content of the blood components of normal and anemic humans*


The role of zinc in ratritlont There are several reviews of the early literature on sine (3h$ 88, 8?)* The literature concerning the role of sine in plant growth has been extensively reviewed by Stiles (102) and Gilbert (1*2) The literature on sine was reviewed to 1936 by Hart and Elvehjem (1*9). In 19^9 Vallee and Altschule (111) published an exhaustive review of the literature concerning the role of sine in the mammal3an organism*
Bertrand and Benson (6), using a ration lacking in vitamins, observed that mice receiving sine lived 25 to 50 percent longer than those on a very lew sine diet* Todd, Elvehjem and Hart (10?) found that rat growth rate and maximum weight on a synthetic ration containing all known minerals and vitamins, but with only 1*6 milligrams of sine per kilo, was considerably inferior when compared to the growth rate and maximum weight attained when zinc was fed at the normal level, thus demonstrating that Sine is an essential element in the nutrition of the rat* Stirn, Elvehjem and Hart (100a) presented additional evidence of growth retardation in rats en a synthetic diet low in sine* By lowering the zinc level in the low-ainc diet to 2*2 milligrams per kilo and by the addition of water soluble factors from milk serum, greater differences in growth rate were observed between rats on the low sine and the ainc-containing ration* There was interference with development of normal haircoat on the low zinc ration, characterized by softening of the fur and graying of black portions in six or seven weeks* Further studies with these zinc-deficient rats (52) indicated that there was a delayed absorption of glucose from the gastrointestinal tract and a disturbance in protein metabolism*


Montgomery. Shelino and Chaikoff (79) administered labeled Kino intravenously in dogs end found that as such as 11 percent was excreted in the pancreatic juice in lit days. Cemar (15) has reviewed the literature on radioisotope studies with sine.
In 191)0 Kellin and Mann (6) showed that sine forms the prosthetic group of the enzyme carbonic aafcydrase* They found 0.33 percent zinc in a purified preparation of the enzyme obtained from ox or sheep erythrocytes. Vallee et al. (110) found that there was a good correlation between zinc content and carbonic anhydrase activity of erythrocytes in a study with humans. Patients with pernicious anemia showed increased zinc content and carbonic anhydrase activity per unit of blood, while both were lowered in most patients with other anemias. The role of line in enzyme systems has been extensively reviewed by lehninger (62) and Vallee (111)*
Zinc toxlcltyt Heller and Burke (51) found no harmful effect in rats when 0*25 percent zinc in the form of the oxide* chloride* carbonate or Sulfate was added to a normal ration in a study carried through three generations. Only a slight increase in the zinc content of livers and kidneys was observed in toe rats receiving supplemental zinc* The chief pathway for excretion of zinc was the feces* Bartow and Weigle (U) observed that only a slightly slower growth rate resulted from giving rats 1000 parts per million of zinc in their drinking water* In 1936 Orimmett and Hclntesh (%$} reported the death of 1*0 or 50 young pigs that had been fed Skim milk which had taken up 1.90 parts per million


of zinc from galvanized iron pipes* Zinc lactate was added to whole milk so as to supply 1000 parts per million of zinc* This was fed together with a little meal to three weanling pigs (1*6)* The pigs became unthrifty and lame. Analysis showed accumulation of zinc in kidneys* liver and bones* Sutton and Nelson (10U) found that rats fed 0*1 and 0*5 percent zinc
-.
deaths occurred within four weeks and most of the rats failed to grow* There was a failure in reproduction after the first pregnancy on the 0*5 percent level* and no reproduction when the 1*0 percent level of zinc was fed* There was a decrease in hemoglobin and red blood cell count after 12 to 16 weeks on the 1*0 percent level* Sutton (103) reported that the lew hemoglobin values observed in rats on the 0.5 percent level of zinc were improved to 90 percent of normal by the addition of 0*50 percent iron as the chloride to the ration* Graham* Sampson and Hester (1.3) observed no toxic symptoms when 0*8 grams or 8*0 grams of zinc as the lactate were fed to breeding marcs for two and a half years* Smith and Larson (101) found that liver extract helped overcome the growth depressing effect of one percent zinc as the carbonate and increased longevity, although hemoglobin values were low* When 0*? percent zinc was fed, copper or liver extract produced higher hemoglobin values than those of rats receiving high zinc levels without added copper or liver extract* Iron or cobalt salts alone had no effect on hemoglobin* In 1950 Gray and Ellis (hk) confirmed the anemia producing effect of zinc by


feeding 0*5 percent zinc to rats and correction of the anemia was effected by the addition of 0.01 percent copper* The therapeutic effect of copper.in correcting "zinc anemia* was less effective when there was a high level cf molybdenum.


GENERAL EXPERIMENTAL PROCEDURES
Certain of the procedures were used in many of the experiments and the details thereof are presented in this section to avoid repetition*
Whenever radioactive materials were used, monitoring devices, shielding and other appropriate health precautions were employed (Hi)*
Radioisotopes of copper* molybdenum and calcium were used to trace toe metabolism of these elements in the animal body under the varying experimental conditions* Table 1 presents the properties of these isotopes (115)* The irradiation unit of copper consisted of 0*32 grams of
TABLE 1* Properties of Radioisotopes of Copper Molybdenum and Calcium*
Specific
Activity
Radiation Energy Projectile Initially
(MKT) and Form Available
Isotope Half-Life Beta Gamma Target Available mc./g.
0u61* 12.8 hrs* 0*li8 1.2 (weak) Neutrons Cu 300
0*06p4>,K Cu
67 hrs* 1.3 0*77* Neutrons M&O3 7
0*815*
CakS 0*8it0
152 days 0*26 none Neutrons CaClg 5-20
CaC03 solution
pure copper wire with an initial activity of about 100 millicuries (2)* After removal from toe shipping container the copper was dissolved in
three milliliters of concentrated nitric acid* evaporated nearly to dryness with an infra red heat lamp* treated with concentrated hydrochloric


acid to convert to the chloride and then evaporated to dryness, the resulting cupric chloride was then taken up in distilled water to give a pH of about three*
The irradiation unit of molybdenum consisted of 10 grams molybdenum tri03d.de with an initial activity of about 1*6 mini curies. This material was dissolved in an excess of sodium hydroxide using a mechanical stirrer, the solution neutralised to litmus with hydrochloric acid, and then made to a volume of 200 ml 111 liters with distilled water*
The ealcismU was received as a solution of calcium chloride which was diluted to an appropriate volume with distilled water* and the pB then adjusted to 6*5 using dilute sodium hydroxide*
Methods of Analysis
Detailed descriptions of methods suitable for the preparation of biological tissues for radiocopper assay have been reported by Coraar (16). In general* the samples were wet-ashed In concentrated nitric add and made to a volume of 10 milliliters* Samples with a sufficient amount of activity were counted by placing 10 milliliters of the solution In a 60 millimeter diameter Petri dish directly beneath a thin mica end-window counter* With samples containing small amounts of activity* a one milliliter aliquot was pipetted into a tared, one inch diameter stainless steel cup and evaporated to dryness. The dry sample was counted in a restricted atmosphere proportional counter manufactured by the Nuclear Measurements Corporation* Indianapolis, Indiana* The measurements made


In this way were about Ik times as sensitive as compared with solution counting* It was necessary to correct for self-absorption in the dry samples, and a self^bsorptiOEi ^Irfeetlon curve was determined by adding known amounts of sodium chloride to a constant amount? of copper^* activity contained in the steel cups* Typical values for self -absorption were as follows*
Fraction of Cu^ Mass in
Activity Counted mg/cm2
1.00 0
0.9U 3*0
0*88 8*2
0.82 13.3
0.75 18.ST
The radiomolybdenum was measured in essentially the same manner as described for wet-ashing and solution counting of the copper6!**
The analyses for radiocalcittm and total calcium were carried out as described in detail (19). In general, the samples were dry-ashed in a muffle furnace at about 550C*, the ash taken into solution, the calcium precipitated as the oxalate, and the precipitate collected for measurement of activity with a thin mica window or internal Geiger counter* Total calcium was determined either graveraetrically or by titration of the oxalate with permanganate* The usual corrections for self-absorption were applied. ,
When samples were to be analyzed chemically for copper, molybdenum or zinc in addition to radioactivity measurements, the wet-ashing of soft tissues was continued by the method described by Lindner and Harley (61*) For wet-ashing bone samples concentrated nitric acid and repeated


additions of hydrogen peroxide were used, the carbamate method (92) was used for copper determinations, modified by the use Of isoarayl alcohol as the solvent for the extraction of the copper dlethyldithiocarbamsto (13) The thiocyanate-stannous chloride method (8U) using separator/ lunnola, was used for molybdenum determinations. Zinc was determined by the dithisone method ($>?)#'using a pH meter for the adjustment of pH* Machine shaking* by means of a rack with a capacity of 12 separatory funnels was used in all three determinations*' The Jajmetron photoelectric colorimeter was used for the molybdenum and copper determinations* A sample holder with a 10 millimeter light path was used for copper determinations, while one with a 3J> millimeter light path was used for molybdenum determinations. The Fisher colorimeter was used for sine determinations*
Serum inorganic phosphorus and alkaline phosphatase activity were determined by a modification of the method described by Hawk* Oser and Summerson (0), modified by the use of 0*1 milliliter of serum and proportionately less reagents*


EXPEPJMEHT 1 The Effect upon Rats of Varying levels of Copper, Molybdenum and Zinc In a Natural Ration*
Experimental Procedure and Results
All rats used in these studies were from a highly inbred Wistar strain. Oral administration of radioactive solutions was accomplished by use of a specially designed stomach needle manufactured by Phlpps and Bird, Incorporated, Richmond, Virginia. After administration of the radioisotopes, the rats were kept in metabolism cages which permitted the separate collection of feces and urine.
Twenty-four weanling* four-week old rats were lotted into eight groups and started upon the S-5> natural ration (see Table 2 for composition), and modifications of it derived by varying the molybdenum, copper and sine
TABLE 2* Composition of S- Rat and Swine Basal Ration
Ground yellow corn 61.3
Soybean oil meal, solvent process, hh% protein 30*0
Alfalfa meal, 17% protein 7*0
Sodium Chloride 1.0
Calcium Carbonate 0*7
content. They were given feed and water ad libitum. The copper, molybdenum and sine content of the basal ration, as well as the amounts of copper*, molybdenum and sine which were added for the various groups of rats, are given in detail in Table J>. Groups VII and fill were females, while groups I through 1 were mies,


TABLE 3
Growth of Albino Rats1 Maintained on Natural Rations wim Varying Levels of Molybdenum* Copper and Zinc from Weaning until Twelve Weeks of Age*
Group No.
Treatment
'ma
Rat Weight NO. (1* Weeks)
Weight Weight Gain Daily
at 12 Weeks
During 8 Weeks
Gain (8 Weeks)
1 Basal Ration
Average H2 High Mo Average
HI High Zn
Average IV High Cu
Average V High Mo, Cu
Average VI High Zn, Cu
Average VII2 High Mo, Zn Average VIII High Mo, Zn, Cu
Average
1 2 3
1 2
1 2 3
1 2
3
1 2 3
1 2 3
1 2
1 2 3
35 1*0
51
6? 51
SO
57
*2 53
58 1*8
W
38 50 52
3h 52
50 0
1*1 US
13U IBS
9U 99 "Jf
152 155 167
w
171* 171 359 TO
>87 93 79
~H5
119 173 l6o
106
113 SB
117 91*
27 1*8 "15*
102 98 111*
w
116
123 117 XE9
1*9 1*3 27
"to
85 151
115 157
56 66
77 %
2.17 1.7U 2*2*8
0.50 0,89
o".7o
1.89 1*81 2*11
2*15 2*28 2*17
0.91
0.80 0.1
1.57 2.21* 2.13
1*02* 1*22
1.1*3
IaH rats were males except those in groups VII and VIII which were females*
2In groups H and VII one rat died after 8 and 5 weeks respectively on experimental ration, and data are presented for the two remaining rats.


These animals were maintained on the rations specified for a fourteen-week period and were weighed weekly. A summary of weight changes during the first eight weeks is presented in Table 3* After eight weeks the rats* tails were clipped in order to obtain blood samples. Several rats Jest excessive blood and since this appeared to have an effect on growth the gains from the eighth to the fourteenth week on experimental rations were not considered in comparing the various groups*
After fourteen weeks on experimental rations* each rat was given orally a tracer dose of h$0 micrograms of labeled calcium. Each rat was sacrificed after four days in the metabolism cage* Serum inorganic phosphorus and alkaline phosphatase activity were determined (Table h) The tissue accumulation of labeled calcium in rats on the various rations was determined
TABLE U* Serum Phosphorus and Alkaline Phosphatase Levels of Albino
Rats Maintained Fourteen Weeks on Natural Rations with Varying Levels of Copper, Molybdenum and Zinc*
Group No* Treatment Serum P (mg* P/100 ml*)* Phosphatase Activity (Ecda^sky Units)1
3- Basal Ration 7*2 19*8
n High Mo 9.7 20*7
m High Zn 10*0 17 .h
IV High Cu 9*0 20.0
V High Me, Cu 8.2 19.9
VI High'Zn, Cu 7*9 17 *5>
VII2 High Mo* Zn 6*9 15*2
VIII2 High Mo, Zn, Cu 7.U 19.lt
%alues given represent averages of duplicate determinations on serum which was pooled for the 3 rats in each group*
2Groups VII and VIII consisted of 2 and 3 females respectively* All other groups consisted of 3 males except group II which had only 2*


TABLE 5 Accumulation of Orally Adminftefcerad Radioactive Calcium in Tissues of Albino Hats1 Maintained on Natural Rations with Varying Levels of Copper, Molybdenum and Zinc. (Expressed as Xerograms of Labeled Calcium per Oram of Fresh Tissue Based on 10
Micrograms Dose per 100 Grams of Body Weight}2
Group No* Ration Rat Rat Wt. No* (Gms) Tibia Shaft Tibia Epiphysis Incisors Jawbone Liver x 103 Fooled Blood x 103 Pooled Kidneys x 103
I Basal* 9.2 p.p*m* Cu, 2*7 p.p.m. Mo, !|1.6 p.p.m. Zn Average 2 3 ISO 158 210 0.305 0.737 0*293 b'.l*l*5 0.892 0*9li8 1*10* TW 0.96% 1.88 1.11 r;ir 0.3H 0.965 0.500 0*1*21* 0*7l48 O.T38 0.637 1.11* 1*06
n Basal plus 1000 2 p.p.m. Mo as 3 NagMoOj^^oQ Average 0.53? 0*595 '07565 2.25 1.28 1.77 1*39 1.13 ras 1.23 0.87I* 1.05"' 0.828 0.698 0".?63 1*00 1.52
hi Basal plus 1000 p.p.m. Zn as ZnSO^.THgO Average 1 2 3 121 177 0.267 0*61? 0*228 6'.37l" l.uo 1.88 ** 0.619 1.25 0.953 oM 0.363 0*900 o.iioa 0.557 0*71*2 0.790 0*871 0.801 1*13 1.25
IV Basal plus 200 p.p.m. Cu as GuSOij Average 1 2 3 206 213 203 Wf> 0*359 0*516 0.377 'o".i*I7 0.881* 2*25 1.8? 1.31 1.1*? 0.931* TtST 0.811 o.l*5o 0.771 W 0*811 0*93? 1*52 TlW 0.931 1.38
Basal plus 1000 p.p.m. Mo and 200 p.p.m. Cu 1 2 3 112 160 109 0.276 0*605 0.520 0.6?? 3.91 2.55 0.600 1.19 X.29 0*291 1.01 ****' 0.1*88 1.16 0*61*1* 0.900 1.26
Average w 0.1*67 2.38 1.03 0.651 O.76I*


TABLE 5 (ConH*) Accumulation of Orally Administered Radioactive Calcium in Tissues of Albino Ratal Maintained on Natural Rations with Varying Levels of Copper, Molybdenum and Zinc* {Expressed as Micrograms of Labeled Calcium per Gram of Fresh Tissue Based on 10 Micrograms Dose per 100 Grams of Body Weight)2
Group No* Ration Rat Rat Wt. No. (Cms) Tibia Shaft Tibia Epiphysis Incisors Jawbone Liver x 103 Pooled Blood x 103 Pooled Kidneys x 103
VI Basal plus 1000 p.p.m. Zn Average 1 3 U*2 197 T?o* 0*31*8 0.501* 5*1*26 1*38 2.69 1.30 0.86? 0.283 0.333 5*3oH 0.60? 0.91*6 6,m 0.679 1.21*
VII Basal plus 1000 p*p.m* MO plus 1000 p*p.m* Zn Average 2 3 120 Il*Q 1^ 0*255 0*1*63 0"*3f 0.81*1* 2.17 T~5T i.ol* 1*?6 Xm 0.371* 0.1*07 0.721 1*01* o.tjai 0*731* 1.12
vm Basal plus 1000 p.p.n* Mo plus 1000 p*p.m* Zn plus 200 p*p*m* Cu Average 1 2 3 11*3 litO 97 HT 0*1*23 0.1*1*2 0*206 0*3*7 1.16 1,80 1.02 1*35 1.35 0.986 0.555 0.961* 0.598 1.15 0.H8 %m 0.61*9 0.1*26 0.533 tr* 0.829 0.911
^Groups I through VI were males* groups VII and VIII were females* 2A11 rats were dosed with 1*50 micrograms of labeled calcium*


(Table $) and the four*day collection of feces and urine was pooled for the three rats in each group for determination of the percent of dose excreted as reported in fable 6* fable 7 presents calcium turnover data in these rats expressed in terras of micrograms of labeled calcium (cor* reeted on the basis of 10 micrograms dose per 100 grams body weight) per Milligram of calcium* Samples of liver, kidney, and femur were obtained and copper5 molybdenum and zinc were determined (fable S)*
TABLE 6. Excretion of Orally Administered Labeled Calcium by Albino Eats Maintained on Natural Rations with Varying Levels of Copper, Molybdenum and Zinc*
(Percent Dose Excreted During Four Days Following Dosage)*
Idroup l'n,TI' *" "' '"' .' ''"" "'' ''"' '' '
No* Treatment Feces2 Urine2
2 Basal Ration 55.6 o.Li
II High Me bO.h 1*12
hi High Zn $2.0 0,12
i? High Cu U9*9 0*08 0.3U
V High Mo, Cu !*2*9
vi High Zn, Cu 52*5 0*96
?n High Mo, Zn U8*l 0*11
vm High Mo,.Zn, Cu 50.5 0*22
*4?he dose was J|0 micrograms of labeled calcium*
2peces and urine determinations were for pooled samples of three rats from each group except groups II and VII which had two rats each*
Discussion and Conclusions
The growth study data indicated that all animals vfaieh received an added 1000 parts per million of molybdenum (groups II, V, VII and VIII) were retarded in growth (see Table 3)* The deaths which occurred in groups II and VII may be indicative of the toxicity of these rations* Zinc at the


TABLE 7 Calcium Turnover in Tissues of Albino Rats1 Maintained on Natural Rations with Varying Levels of Copper, Molybdenum and Zinc.
(Expressed as Microgram of Labeled Calcium x 103 per Milligram of Calcium)2
Group Mo. Ration Rat No. Tibia Shaft Tibia Epiphysis Incisors Jawbone Liver Pooled Blood Pooled Kidneys
I Basalt 9.2 p.p.m. Cuy 2.7 p.pvm. Mo, 1*1.6 pp.m. 2n Average 1 2 3 1.8? 3*5* 1.38 SttT 6.62 6.92 8.6? TOO lt.76 6.19 7.21 oToT 2.52 7.26 3.19 3.62 7.33 9.66 8.35
H Basal plus 1000 p.p.m. MO as la^foO{(2H^Q Average 2 3 3.1|8 2.86 jin 10.6 7.33 5.33 7.55 5.22 5.15 5.79 6.65 11.0 12.3
ni Basal plus 1000 p.p.m. Zn as ZnS0^.7H20 Average 1 2 3 1.32 2.90 2.71* 1^,6 .... TO* 3.89 2.31* 6.31 5.53 5.75 1..58 5.1*9 7.85 13.0 12.6
IV Basal plus 200 p.p.m. Cu as CuSOj* Average 1 2 3 2.60 3-11 2.31 OTf 10.8 15.2 13.9 TO 6.10 6.99 9.69 735 5.1*5 lt.88 5.16 5135 5.92 8.1*1* 8.00 6,29 11.0
V Basal plus 1000 p.p.m. Mo and 200 p.p^i. Cu Average 1 2 3 1.39 3.16 2.66 f3jS 7.25 23.3 11*.2 H*7T 2.# 9.62 8.86 7.U* 2.63 6.72 ... Tuff 5.03 til 9.68 8.75


TABLE 7 CeoaH.) Calcium Turner in Tissues of Albino Eats* Maintained on Natural Rations with Varying Levels of Copper, Molybdenum and Sine*
(Expressed as Micrograms of Labeled Calcium x 10 per Milligram of Calcim)2
Group No. Ration Eat No. Tibia Shaft Tibia Epiphysis Incisors Jawbone Liver Pooled Blood tooled Kidneys
VI Basal plus 1000 p.p.m. Zn Average JL 3 1.66 2.52 9*73 26.2 mr 6.5& 10.6 "TOST 2*81* 1**27 J3S 5*23 9*59 TM 9*10 10*6
vn Basal plus 1000 pp.m Mo plus 1000 p.p.m..2a Average 2 3 1.19 2.12 Y35 6*9? 16.0 6.20 "TO? 2*68 6*27 11*1 "8~63f 12.2 6M
vin Basal plus 1000 p.ji*. Mo plus 1000 p.p.m. Zn plus 200 p*p*m. Cu Average 1 2 3 2.6? 2.11 0.932 13.3 13.5 J.U3 lo.r 7.33 U.20 3.3? .....MM 3.5i 6.71* 2.68 rat 6*90 5.26 3.70 8.73 9.69
^Groups I through VI were males, groups VII and VIII were females. All rats were dosed orally with U50 .micrograms of labeled calcium.
^The figure for micrograms of labeled calcium was corrected on the basis of 20 micrograms dose per 100 grants of body weight.


TABLE 8 ? Accumulation of Copper, Molybdenum and Zinc in tissues of Albino Rats1 Maintained on
Natural Rations with Varying levels of Copper, Molybdenum and Zinc from Time of Weaning until Eighteen Weeks of Age*
(Expressed as Parts per Million on a Dry Matter Basis)2
Pooled Pooled Pooled
Croup Rat Liver Liver Liver Kidneys Kidneys Kidneys Femur Semur
Ifo* Ration Ho. Cu Mo Zn Cu Mo Zn Mo2 Zn
I Basal: 9*2 p.p.* 1 11.8 2,$ 62*1* 0* 259*
Cu, 2*7 p*p*m. Mo, 2 21*1 2.U 55.2 20*0 1*0 97.2 11*1*.
1*1.6 p.p*m* Zn 3 17*9 JU5 77*1 0.5 lj*6*
ATERAge HX 1507
H Basal plus 1000 l3 1*3.0 22.0 139. 13*5 93.9
p.p.m* Mo as 2 63*0 38*2 91*9 252. 41*6. 99.1 20.2 129*
Na^o0jt*2H20 3 38.6 1*0*0 71*8 22*9 138*
Average 1*B.I B2* WT I5&T
HI Basal plus 1000 1 18.0 i*.2 157. 1.3 300*
p.p.m. Znas 2 9.9 1.8 65.2 21.1 1.V 95.5 1.7 258.
3t^O.*7%0 3 11.6 1.9 76.7 2.1 273.
Average IX? 99.6 TTf I??.*
IV Basal plus 200 1 13.3 2*1* 90*3 0*1* 166.
p*p*m. Cu as 2 9.3 0.9 72.3 20.7 0 99*6 *.* 106*
CuSOj, 3 28*7 2.5 67.7 0 161.
Average TflT "1.5 7OT IS*.
V Basal plus 1000 1 30.1* 6*8 79*3 35.1* 88*5
p.p.m. Mo and 200 2 181*. 5.8 157* 131. 56.6 102. 11**7 169*
p*p.a. Cu 3 1030* 91**1 11*5. 27*1 129
Average 127. 19.X 129.


TABLE 8 (Con*t) Accumulation of Copper, Molybdenum and Zinc in Tissues of Albino Rats1 Maintained on Natural Rations with Varying Levels of Copper, Molybdenum and Zinc from Time of leaning until Eighteen Weeks of Age*
(Expressed as Parts per Million on a Cry Matter Basis)2
Croup No. ... Ration Rat No, Liver Liver Cu Mo Liver Zn Pooled Pooled Pooled Kidneys Kidneys Kidneys Su Mo Zn Femur Mo2 Femur Zn
VI Basal plus 1000 p.p.m. Zn and 200 p.p.ru Cu Average 1 2 3 20.3 10.1* 11.6 2.2 1,8 2.? 68.0 78.2 67.0 TTTI 2U.3 0 90.9 3.6 1.7 0 V it X.Q 3h0. 166. 181.
VII Basal plus 1000 p.pra. Mo and 1000 p.p.m. Zn Average 13 2 3 31.5 1*5.5 6.3 W.U 31.2 6.9 190. 8U.1 120. mr 8U.6 U6.5 75.it 30.0 28.lt 13.5 WS 129. 155. 132.
VHI Basal plus 1000 1 p.p.m. Mo, 1000 2 p.p.m. Zn and 200 3 ppm Cu Average mt 19.5 6.0 18.2 12.6 75.7 105. 77.U 31.8 17.6 86.6 13.S 2l*.6 15*0 188. 198. 162. W7
^Groups I through VI were males, groups VII and VIII were females. ^Femur values represent micrograms per gram of fresh weight.
%ats which died during experimental period. Rat 1 of group II died after 8 weeks; rat 1 of group VII died after 5 weeks.


level of 1000 parts per million added in rations of groups 321 and VI "did not seem to have an adverse effect upon growth when gains were compared to those of groups I and 3V, which had the same copper contents respectively* as the rations of groups 111 and VI* Group V* which was a ration with the high level of molybdenum (1000 parts per million added) and copper (200 parts per million added), did not gain appreciably better than group II* on a high molybdenum level with 9*2 parts per million of copper* This Indicated that with this basal ration there was little* if any, therapeutic effect caused by the addition of 200 parts per million copper when a level of 1000 parts per million of molybdenum was present* However, a greater number of experimental animals are needed to prove the statistical significance of the growth effects indicated here*
The data shown in Table k indicated that the various rations produced very little effect upon either the serum inorganic phosphorus or the serum alkaline phosphatase*
The varying dietary levels of copper, molybdenum and sine did not appear to cause an appreciable difference in the accumulation of orally administered labeled calcium in the various tissues analyzed (see Table ) Calcium turnover did not seem to be affected in the tissues which were sampled, and the excretion data did not indicate any modifying influence of the various rations (Tables 6 and 7)*
Analyses of livers, kidneys and femurs for sine indicated that the addition of 1000 parts per million of zinc to the basal ration containing hi.6 parts per million of zinc caused very little increase in the sine


content of these tissues after fourteen weeks on the ration (see fable 8). Individual rats varied considerably in tissue sine concentrations. The molybdenum analyses shewed that molybdenum was definitely cumulative at the 0.1 percent dietary level. All rats receiving added dietary molybdenum showed appreciably higher liver, kidney and femur molybdenum concentrations. The tissue copper analyses of group IV indicated that little additional liver or kidney copper accumulation was caused by the addition of 200 parts per million of copper to the basal ration. The addition of 1000 parts per million of sine did not alter appreciably the copper concentration in the livers and kidneys of group HI, as compared to group I which received the same dietary copper level (9.2 parts per million). In comparing the high copper group (IV) with group VI which received the same level of copper plus the high zinc level, no appreciably modifying effect of the added zinc was seen upon liver and kidney copper accumulation. However, this was not the case when the effect of added dietary molybdenum upon liver and kidney copper accumulation was considered at the basal copper intake level (groups I and H) and the high copper level (groups IV and V). The average liver copper value in group H (high molybdenum) was more than three times as high as in the basal group. The pooled kidney copper value for group II showed a seven-fold increase above that of the basal group. The livers of group V (high molybdenum and copper) varied considerably, but there was a trend for much greater copper accumulation than was the case for group I?, with the same dietary copper intake,.


EXPERIMENT 2 The Effect upon Rets of Varying Levels of Copper* Molybdenum and Zinc in a Purified Ration*
Experimental Procedure and Results
In order to study the effects of varying dietary levels of molybdenum* copper and sine in purified rations, six groups of three weanling, four-week old male albino rats each were fed the rations whose composition is given in Table 9* The Ca-3 basal ration has been used In numerous studies at the University of Tennessee-Atomic Energy Commission Agricultural Research Program and contains 0*5 percent calcium, 0*1* percent phosphorus, 22*5 parts per million copper, less than one part per million molybdenum, and 33*6 parts per million sine*
These six groups of rats were given feed and water ad libitum and were maintained on experimental rations for twenty-five weeks* Weekly weights were taken, and a summary of weight changes during this period is presented in Table 10*
After twenty-five weeks on experimental rations each rat was dosed orally with 320 micrograms of labeled copper* This was a physiological dose, since normal daily copper intake on the basal ration was around 225 micrograms* The initial activity of the dose was about 100 micro-curies* After eighteen hours these rats were sacrificed* Serum phosphorus and alkaline phosphatase levels were determined on serum samples which were pooled for each lot and these data are presented in T&le H


TABLE 9m Ca*3 Rat Ration Kith Modifications*
Group No*s II in w t
Rationt Ca*3 Ca3 Ca*3 c**3 Ca*-3 Ca-3
Basal Mo Zn Cu MoCu Zn*Cu
Ingredients* Grams
per 1000 gm. ration
Corn Starch 61*2.6 61*0.0 638*0 3*2*0 639.5 637.7
Casein 280*0 180.0 180*0 280*0 180*0 180*0
Irradiated feast 10*0 10*0 10.0 10*0 20*0 10*0
KH2P0i| 13.2 13*2 13.2 33*2 13*2 33*2
CaC03 12.2 12*2 12*2 25.2 12*2 12*2
u"T*AEC Min* Mix I1 20.0 20.0 20.0 20.0 20*0 20*0
Wesson Oil 1*0.0 Uo.o 1*0*0 1*0.0 1*0*0 1*0*0
Vitab Uo.o 1*0.0 ItO.Q 1*0*0 140*0 1jQ0
Cod Liver Oil 2.0 2*0 2*0 2*0 2*0 2*0
Cellulose 1*0.0 liO.O IjO.O 1*0*0 1*0.0 1*0*0
Ha2MoO^*2HgO 0 2*52 0 0 Wt 0
0 0 1**1*0 0 0 0*50 1**1*0
CuSOj 0 0 0 o*5o 0*50
%JTAEC Mineral Mix I Ingredientsf (Grams per 2000 Grams)
Nad 398*1*
KC1 182*0
KHC03 262*0
t^%Ahhydrous)220.0
CuS0.5H2O l**0
MnSOjJbHjjO 2*0
Zn$<%*7H20 6.0
FeCl3.6H20 25.0 ^12(801^)14*2^ 0.2 l;:iOt 0*3
NaF 0*1
Vitab fortified with riboflavin (from Nope Chemical Co*, Harrison* N.J*)*
* I4O *


TABLE 10# Growth of Halo Albino Rats Maintained on Synthetic Rations with Varying Levels of Molybdenum, Copper and Sine from Meaning until Twenty-Kine Weeks of Age#
( Expressed in Grams)
Initial Weight Wt. Gain Daily Weight V;t* Gain Dally
Weight at During Gain at During Gain
(1* Weeks) 12 Weeks 8 Weeks (8 Weeks) 29 Weeks 25 Weeks (25 Weeks)
Group Rat
No* Treatment Mo*
I Basal Ration
Average
1 2 3
1*0 65 52
186 229
31*6 161*
279 336 32J
239 271 271 155
n High Mo 1 U5 75 30. 0*56 160 115 0.66
2 1*5 103 63 1*17 211 166 0*96
3 & 160 102 1.89 261* 206 1.19
Average if mr ~o]? IIS Wi T5"? Q~M
in High Zn I 1*9 199 150 2.78 315 266 1*51*
2 59 21*0 Ifll 3*35 399 3U0 1*97
3 h5 210 i6$ : 3.06 3U3 298 1*72
Average 5* 1XB I55" 3335 "352" 36i: TM
IV High Cu I 58 217 159 2*91* 31*8 290 1*68
2 35 151* 119 2.20 21*9 211* 1*21*
3 39 171 132 2.10* 310 271 1*57
Average Tar IHT 137 2"3? ^2" I*fo
V High Mo, Cu 1 56 109 53 0.98 196 11*0 0*81
2 35 91 56 1.01* 217 182 1.05
3 Jj5 0 0 5U 9 o*o5
Average (of I, 2 and 3) ^2 "35 TOT Ho olol
Average (of 1 and 2) U6 100 55 1*01 207 161 0.93
VI High Zn, Cu 1 37 171* 137 2.51* 301 261* 1.53
2 59 191* 135 2*50 339 280 1.62
3 1*3 193 150 2.78 322 279 1.61
Average US w U*T 231 351 271*


TABLE 11* Serum Phosphorus and Alkaline Phosphatase Levels of Hale Albino Eats Maintained on Synthetic Ration? with Varying Levels of MolyMenum, Copper and Zinc from Time f Weaning until Seven Months of Age*
Group Ho. Treatment .r..:. Serum J? (mg* P/3UD0 ml.)* Phosphatase Activity (Bedansky Units)
I Basal Ration 7*2 17*1
High Mo 7.6 20.U
hi High Zn $.9 16.3
IV High Cu 8*1 11**1
V High Mo* Cu 7.6 20.3
. VI High Zn, Cu 7.8 13.?
^Values given represent averages of duplicate determinations (except for group 3V, & single determination) on sera : which was pooled for the three rats in each group*
Samples of liver, blood, kidney, spleen, gastrocnemius muscle, hide and red bone marrow (from the femur) were taken and the concentration of labeled copper was determined (Table 12). For estimating blood volume in order te calculate total percent of dose in the blood of the rat* the value of 6.7 cubic centimeters per 100 grams body weight was used (20). The percent of dose present in the urine, feces and segments of the alimentary tract were also determined (Table 13)* Femur shaft samples were analysed f-'-r molybdenum} liver, kidney and hide samples were analyzed for copper and molybdenum (fable 1U). Because of the appearance of an interfering yellow color in some samples which was probably doe to organic matter present during the analytical procedure, accurate values were not obtained for kidney molybdenum content for rats in groups I, ill, IV and VI, and therefore no kidney molybdenum values were presented for these groups* There was ample evidence that these values would not exceed
* 1*2


TABLE 12 Accumulation of Orally Administered Radioactive Copper in Tissues of Male Albino Ratsl Maintained on Synthetic Rations with Varying Levels of Molybdenum, Copper and Zinc* (Expressed as Percent of Dose x lo3 per Gram of Fresh Weight)2
Group Ho.
Ration

Rat Wt. Ho. (Gms)
Liver
Blood2
Kidney Spleen
Red* Bone
Muscle Hide Marrow
I Basalt 22.5 p.p.m. 1 Cu. below 1 p.p.m. 2 332
Mo* 33.6 p.p.ra. Zn 3 322
Average
II Basal plus 2000 p*p.m. Mo
Average
HI Basal plus
2O0C p.p.m. Zn
Average
IV Basal plus 200 p.p.m. Cu
'"Average.
V Basal plus 1000 p.p.m. Mo and 2CC ppm. Cu Average
283 57*2 (0*u3)3 32.9 (0.61) 388* (.50) 2U*7 (.013) 2.31* 8*36 1*79* 71*1 (0*58) 27.2 (0.50) 196* (.27) 2U*0 (.021) 2.58
TO
2B.0 (O.U) 26.9 (0.50) 3B7* (.30) _7.09C.pl8) TOTOTJ 29.0 (6.$h) (.36) 1B.6 (*oii*)
1.1*2 2*11
1 161 70*8
2 217 358*
3 256 63.1
(0.66) (2.75) (0.53 TOT
(3.97) (3.1*5)
217* 288.
352;. (2.80) ffi6. (3-U15 397
312* 630*
(.1*9) (.81.)
2I48* (.37)
60*2 (.050) 23*7 75a (**039) 19*2 63*1 (*036) 6*59 66*1 (*0U2) I5.J-
1 305 58*2 (0*140
2 388 6.71(0*05)
3 33i 118* (0,92) -IfojTt
1**12(O*08) 216. (.31) 12*1* (*005) 0*52
13*0 (0.21*) 118*. (.17) 6.88(*003) 0*31*
13*9 (0.26) 561. (.80) 16*7 (.007) 2*70
lc*3 M ml (.1*3) .12*6 J.ou5) w
1 31*1 93*7 (0.71)
2 21*5 120. (0.91)
3 306 23*0 *2W *^9"
(0.17) 1Ko07
1.60(0.03) 293. (*U3) 126* (.01*9) 2.15
2.63(0.05) 177. (.21*) 12*2 (.006) 1*1*7
9*90(0.18) 230. (.36? 7.66(^303) ....
u.?I(0.69) 233. (.31*)-- OI
1 206 62.3 (0*52)
2 220 360* (2*83)
3 69 69,5
16*8 (0*31) 191*. (.28) 55*1 (1*01) 173* (*2lt) 39*8(*09) (.26)
36*0 (0*66) 135
50.3 (*030)
30*8,(*oih 33.8 (*028:
5.83 1*97. 90 208.
.05 *3*S7
19*5 25.8 23.1*
767* 529. 651**
6.71* 122.
7.87 1*9*1*
9*99 85.8
3*"2o" *~557Y
7*01 ....
3.97 205.
t .16 1*5*1*
.... 21*5*
7*71* 71.9
19.6 ....
T3TT -


TABLE 12. (Con'T.) Accumulation of Orally Administered Radioactive Copper in Tissues of Male Albino Hatsr Maintained on SjnthefcLc Ratioi^ with Varying Levels of Molybdenum, Copper and Zinc. (Expressed as Percent of Dose x 103 per Gram of Fresh Weight)*
Group No*
Ration
Rat Rat Wt. No* (Gma)
Liver
Blood2
Kidney
Spleen
Red Bone
Mqscle Hide Marrow
VI Basal plus 1000 p*p.m* Zn and 200 p*pm* Cu Average
171. (1.26J
1 293
2 327
W "*lii.i(b.7'2
85.8(0.671 307 26*2(0.210
6.05(0.11) 6.33(0.12) 9*02(0.17)
21*3. (0.39) 108. (0.17) 1*67. (0.751
13.8 (.006) 0.91* 9.9li(.O05) 1.12 lul9(.0Q2) 3.18
5.89 109*
2.05 101.
2.99 2l0t.
JM 151*
^These rats were maintained on the experimental rations from time of weaning until seven months old.
^The percent dose figure was corrected to a body weight of 275 grams. Each rat received 320 micrograms of labeled copper. Blood values are based on one ml. of whole blood.
%alues in parentheses represent percentage of dose in the whole organs*


TABLE 13 Alimentary Tract Content and Excretion of Orally Administered Labeled Copper in Male Albino Rats Sacrificed Eighteen Hours after Dosage.
(Expressed as Percent of Dose)!
Rat Group Group Group Group Group Group
No. i II in i? 7 71
Stomach plus 1 10.9 19.8 21.lt 3.76 6.21 5.92
Contents 2 7.1 10,6 6.79 0.70 3.73 6.26
3 2it.lt 1B.3 17.8 2.21 0.85 u.57
Average tot ittt 2.22 its? J38
Small Intes- 1 2.9 2.51 3.10 3.76 lt.70 1.98
tine plus 2 0.1tl 3.08 23.2 2.50 2.97 6.57
Contents 3 1.23 2.65 5.5? 5.26 8.87 5.68
Average 131 1 2.7? JM $3Z
Large Intest- 1 i9.it 21.6 13.1 12.it 27.3 23.5
ine plus 2 8.27 12.1. 30.1 5u.2 29.u 25.6
Contents 3 9.87 19.8 21.1 23.1 28.lt 25.2
Average Tsar Wk*' 29.9 28 .it ffc8
Feces'* 1 18.1 16.8 20.2 15*2 22.5 11.7
2 18.1 16.8 20.1 20.1 u7.8 1S.9
3 it.7it 15.0 20.2 15.2 28.7 11.7
Average ror ZO'.i i6.r B-6 lit'.!
Urine2 1 0.16 0.06 0.16 O.lii 0.08 o.iit
2 0.16 0.06 0.08 0.07 0.07 0.11
3 0.13 0.01. 0.16 0.11t 0.09 O.ut
Average .....6.15 0.05 ^m "O.'db ......
Total of Averages ltl.9 53.1 67.6 52.9 70.6 lt9.lt
iDose was 320 micrograms of labeled copper per rat.
2peces and urine were pooled for 2 rats of each group except group V*


TABLE lit. Accumlation of Copper and Molybdenum in Various Tissues of Male Albino Rats Maintained on
Purified Rations with Varying Levels of Copper* Molybdenum and Zinc from Time of Weaning until Seven Months Old.
(Expressed as Parts per Million on a Dry Matter Basis)!
Group Rat Liver JUiver Kidney Kidney Hide Bide Spleen Spleen Femur Shaft
Ho. Ration No. Cu Mo Cu Mo2 Cu Mo cu2 Mo2_Mo
I Basalt 22.5 p.p.m. 1 13.? 1.5 51.8 1**3 1.1 > 1.6
Cu, 33*6 p.p.m. Zn, 2 8*5 1.3 52.lt 1D.3 0.1 2.3
below 1 p.p.m. Mo 3 15.5 1.9 39.0 it.l 0.1 1.1
Average 1535 TO U7.T ~0 "suit IT?
II Basal plus 1000 1 8tt.lt 107* 1690. 917. 15*3 66.8 it36. 568* 85.3
p.p.m. Mo as 2 1*02. 177* HOG. 663. hu5 39*9 519* u62. 101.
Na-MoOh^oO 3 310.. lilt* 1020. 623. IB.? 5u.1 lt77.
Average4 TWT WT i2j0t 73tu iks 5370* #7
III Basal plus 1000 1 12*8 1*5 u8*5 u*2 0 2.7
p.p.m. Zn as 2 H4.i1 1.2 50.8 3*6 0 0*6
ZnS0^.7H20 3 15*6 U3 1*5.3 Jjh _0_ 1.8
Average io ^3 ^*T i.f
IV Basal plus 200 1 28.lt 2*0 110. 3.6 0.1* 2.0
p*p.m. Cu as 2 61t*3 2.1 68.8 7*8 0.2 2.5
CuSO^ 3 35j3 1.3 57*2 5*0 0 3*7
Average 37*3* "75*7 "TS "^2 **7
Basal plus 1000 1 2lt00* !6!u 131*0. 360* 26.0 37.5 787. 109.
p*p.m. Mo, 2 2280. 83.1 lfcOO. 209. 15.2 23.7 503. 322. 80.2
200 p.p.m. Cu 3 550* 336. 2J|30. 965* 71.5 8u.5 107.
Average TOT Tf2^ OTT 37.S Wit "W*T


TABLE 2k* (CenH#) Accumulation of Copper and Molybdenum in Various Tissues of Male Albino Bats Maintained on Purified Rations with Varying Levels of Copper, Molybdenum and Zinc from Time of Weaning until Seventh Months Old*
(Expressed as Parts per Million on a Dry Matter Basis)'1-
Group Rat Liver Liver Kidney Kidney Hide Hide Spleen Spleen Femur Shaft
Ration Ho. Cu Ms Cu Mo Cu Mo Cu2 Mo2 M6
VI Basal plus 1000 1 12..6 .* $3.3 3.9 0*1 1.1
p.p.m* Zn and 2 13.6 3.0 70*0 6.7 0 ia
200 p.p.m. Cu 3 13.0 0*6 88*ii g.8 0 2.1
Average u.7 W tots "5*0*3
values for femur shaft represent parts per million on a fresh weight basis*
Due to uncertainties in the method, accurate values are not available for kidney molybdenum in groups I, 111* 17 and VI. However, there was ample evidence that these values would not exceed 6%.? p.p.m. The analytic results for the spleens of these groups indicated that there was less than 10 p.p.m. copper, and no molybdenum determinations were made since the .hole sample was used for copper determination*


6u-7 parts per million, the analytical results for the spleens of groups I* Hit IV, and VI indicated that there was less than 10 parts per million of copper in any spleen, and no molybdenum determinations were made since the whole spleen was used for the copper determination. Copper turnover in terms of percent of labeled copper dose {corrected for body weight) per milligram of copper in the tissue was computed using the data which had been obtained for percent of labeled copper dose and total copper for livers, kidneys and hides (fable 15).
Discussion and Conclusions
Groups in and VI which received an added 1000 parts per million of zinc In their feed grew at about the same rate as the controls (group I). The addition of 200 parts per million of copper also had no effect upon grswth rate. However, both group II (high molybdenum) and group V (high molybdenum and high copper) were retarded in growth and thus the additional 200 parts per million of copper which group V received did not seem to have a therapeutic effect. One rat (number 3) of group V gained only nine grams during the 25 week experimental period. Even if this rat is not Included in the average, group V still did not have a faster growth rate than group H, which received no additional copper.
The data obtained for serum phosphorus and alkaline phosphatase activity (Table 10) did not indicate any appreciable effect due to the varying levels of copper, molybdenum and sine.
l>6


TABLE 15 Copper Turnover in Seleot Tissues of Male Albino Bats Maintained on Synthetic Rations vlth Varying Levels of Molybdenum, Copper and Zinc.
(Expressed as Percent of Labeled Copper Dose per Milligram of Copper in the Tissue)2
drouF
SO* :
Ration
Rat Wt.
IfQt ,fercs,),
''J&V&J,;>,......Kidney,,
'X* Basal: 22.5 p,p.. Cu below 1 p.p.su Mo, 33*6 p.p.m. Zn Average
II* Basal plus 1000 p.p.m. Mo
Average
III* Basel plus 1000 p#pm.*. Zn
Average
IV. Basal plus 200 p.p.m. Cu
Average
V. Basel plus IOO0 p.p.m. Mo end SCO p.p.m. Cu
Average
VI. Basal plus 2000 pp.m. Zn end SCO p.p.m. Cu Average
1
2
3
1 2 3
1 2
3
305
388.
3&1
33
2 20(5
2 220 3
327 30?, 309
12.7
25.7
2.79 2.96
1.87
13*3 1.5&
20*9
6.28 3.88
6.09m
:o..':
38.8 21.8
ri2 -
26.l1 17.2
0.601 2,20 0.Q22 It20"
26*7 8.87
2i .2
9.93 9*27 2|.0
0S2k
0.j 0.3"
16,1 6.32 20.< 53d
1.18 2.23
2.90 3.61 2.62
37oT
3.**0 k.B6 6.05
^77
lj.30 1.23
g.26
O.98 0.66
3*17
0*^6
%iese rats were jsaintaiaed on e^rimental rations: from time of weajflng until 7 months old*
2Labe!ed copper dosage was 320 adcrograms administered orally* The percent dose figure was corrected to a body weight of 275 grams*
1.9 *


The data for tissue accumulation and excretion of orally admin* istered labeled copper (Tables U and 12) did show differences between groups which may be attributed to the nutritional regime. Group U (high molybdenum) had a higher average accumulation of labeled copper in the liver, blood, kidney, spleen, gastrocnemius muscle, hide and red bone marrow than did the controls of group X. Group V (high molybdenum and high copper) had a greater average accumulation of labeled copper in the liver, blood, gastrocnemius muscle and hide than did group X? which received the same dietary copper level*
Molybdenum analyses (Table 1U) indicated a continuous accumulation of this element* In Experiment 1, after rats had been on 1000 parts per million of molybdenum or 1000 parts per million molybdenum plus 200 parts per million copper (groups H and V) for lit weeks, liver values rangel from 5*8 to 9U*1, femur values from 13*5 to 27*1, and pooled kidney values from 6.6 to Ut6 parts per million of molybdenum* Liver and kidney values are based on dry matter, femurs on fresh weight* In Experiment 2, after 25 weeks on experimental rations, the corresponding groups II and V (1000 parts per million of molybdenum and 1000 parts per molybdenum plus 200 parts per million copper) showed a range of liver molybdenum values of 83*1 to 336 parts per million} kidney values from 209 to 917} hide values from 23*7 to 8U.5f and spleen values from 332 to 568 parts per million on the dry matter basis* Femur shaft molybdenum values ranged from 80*2 to 109 parts per million on the fresh basis*
A comparison of tissue copper accumulation of group III (basal plus 1000 parts per million sine) with group I (basal) indicated that liver
5o


and kidney -copper seeiattilation were not affected by the addition of glne 'to the ration containing '22*5 parts per million copper.
Group I?, receiving the basal plus 200 parts per million copper* showed evidence of approximately a threefold increase in liver copper level and almost a twofold increase in kidney copper level when compared with the controls (group I)* Accumulation of copper in the hide was not increased by the added dietary copper*
There appeared to be considerable Individual variation in response among the groups receiving an additional 1000 parts per million of molybdenum insofar as its effect upon tissue copper accumulation was concerned* In all cases* however* there was a dramatic increase in the copper content of liver* kidney, spleen and hide as compared with the copper eon-tent of these tissues in the groups receiving the same dietary copper 'level without 'added molybdenum*
' At the-'22*5 parti- per 'million copper lavel, while'the'CMtrol'liv* ers, kidneys and hides ranged from 8*5 to 15*5# 19*0 to 51*8 and it*! to 10*3 parts per million on the dry matter basis respectively* the cor* responding;' ranges for .group'XX,- receiving an'additional'1000 -parts per .'million molybdenoa'were 01**1* to l*02,' 1020 to 1690 'and''11**5''to: 3U&>7* The spleen copper .content for grcups'-II and-?-''ranged from '1*36 t# 519 'parts per riUHta'6f copper, and $03 to 787 parts per million respectively*: ;-whlX@'-aXl- groups- had spleen copper values':of less than 10 parts'-per "' ''million*:'-- '--''- /
, At -the- -high copper level- (222*5 pwi* per 'million) the livers|' Iddneys and hides of group If (high copper) ranged from 19*3 to '61**3*


57*2 to 310 wd 3.6 to 7*8 parts per million of copper respectively, the corresponding values for group 7 (high molybdenum plus copper) were 550 to 21*00, 131*0 to 21*30 and 15*2 to 71*5*
The copper turnover data (Table 13) indicated that there was a con-siderably lower copper turnover rate in the kidneys and livers of the animals receiving high levels of molybdenum when compared to those on tee same dietary copper level without supplemental molybdenum* This is a reflection of the fact that the groups receiving supplemental molybdenum had accumulated large amounts of copper during the 25 week experimental period* The data for tissue accumulation of the labeled copper dose indicated that this tendency for the greater accumulation of copper still existed after 25 weeks*


EXPERIMENT 3 Effect of Short-term Supplementation with Molybdenum, Zinc and Calcium Pantothenate upon Growth and Radio-copper Distribution in the Rat.
Experimental Procedure and Results
In order to study the effects of molybdenum and zinc administered as a drench, 30 weanling, four-week old albino rats were selected from the stock colony. They were divided into five groups, each containing three males and three females* All groups were fed the Ca-3 basal ration (Table 9), modified by the mission of copper sulfate and zinc sulfate from the mineral mix* The resulting ration, designated as the Ca-3-A ration, contained 3.3 parts per million of copper and less than one part per million of molybdenum* Pyrex distilled water and the Ca-3-A ration were given ad libitum to the five groups*
The rats of each group were drenched daily for eight days* Group 1, the controls, received 0*5 milliliters of pyrex distilled water* The rats of group H each received 5.0 milligrams of molybdenum as NagMoOj^*2Hj^> in 0*5 milliliters of pyrex distilled water daily. The drench for group III was 5.0 milligrams of zinc as zinc sulfate in 0*5 milliliters of pyrex distilled water* Group IT received 5*0 milligrams of sine as the sulfate and 5*0 milligrams of molybdenum as sodium molybdate in two separate solutions of 0*25 milliliters each* Group 1 rats were drenched with 5*0 milligrams of molybdenum and 2*5 milligrams of calcium pantothenate in 0*5 milliliters of distilled water* The effect of the various drenches upon growth as measured by weight change is presented in Table 16*


TABLE 16. Effect of MelyMenum* Zinc* Molybdenum plus Zinc* and
Molybdenum plus Calcium Pantothenate Drenching upon Growth of Weanling Alblne Ratsl
(Weight Expressed in Grams)
Group No.
Treatment
"'Rat''' ''' '' ,r"|m^in'iiaW^,''""' "nW'weight No. Sex....... 29,' 3b" .. Change.
32
HI
IT
Daily RgO drench
Average
Dally drench of 5 mg Mo as Na2M0i4.2H20
Average
-Hit
IJaily. drh$h of
5 mg.yz8;'as
sine sulfate
Average
Daily drench of 5 mg. Mo and 5 mg* Zn
Average .
Daily drench of 5 mg. Mo and 2.5 mg, of Calcium Panto-thenate
Average
2 3 I.
i
2 3 h 5 6
1 2 3 It 5 6
1 2 3 U
I
1 2 3 I* 5 6
Female
Female
Female
Male
Male
Male
Female
Female
Female
Male
Male
Male
Female Female Female
Male Male Male
F Female
Female
Male
Male
Male
Female
Female
Female
Male
Male
Male
5o 55 5? 52 51 M
1*7 38
1*9 38
33 U7 53 52
I7 60
~W
33 50 52 58
"TJET
52 It? It! 1*5 58 1*8
6$ 81 79 81 76 61
**75
35 32 25
29 1*0 IF
1*7 52 59 70 53
-J
35. 1*9 55 50 50 ||2
1*9 31 30 3U 57 1*1
19 26 22 29 25 20
*13*
*. 12
35 ..** 9 ** 5 9
' 13
*n8
II*
I
18 16 IS
5* 3 1 3
.** 8 0
;t -'"'1
* 3 16
* 11
.* 1 * 7
Ifhes. rat. .MdtUinml on sjrthrtle U editing 3.3 P.M.
of copper and less than 1 p*p.m. of molybdenum*


On the eighth day of drenching each rat was injected intramuscularly in the hind leg with %&& micrograms of labeled copper as a solution of the chloride in 0*$ milliliters* The initial activity of the dose was about $0 mlcroeuries. The three males and the three females of each group were put into separate metabolism cages*
After 2k hours all rats were sacrificed* Radioactivity was determined on blood* liver* spleen, kidney, hide, alimentary tract plus con* tents, urine and feces* These data are presented in Tables 17 and 18*
Discussion and Conclusions
The sine drench which group IH received had a significant depressing effect upon the weight gains of these rats as Shown by comparison with the control group* The rats of group XI {molybdenum) and group f (molybdenum plus calcium pantothenate) actually lost weight in distinction to the weight gains shown by the groups net receiving molybdenum* The rats of group If (zinc plus molybdenum) did not lose as much weight as those of group II (molybdenum) or group V* (molybdenum plus calcium pantothenate)* possibly in the case of group IV Insoluble sine molybdate was formed in the alimentary tract, decreasing sine and molybdenum absorption* The differences in weight changes mentioned above were significant at the five percent level (P 0Q*>)# The weight changes in grams during the one week drenching period for groups I to v, respectively, expressed as a % per* cent confidence interval (31), were as fellowsi 2h It, -10+U* 12 * *lrjjl* and'*>*|-6*


flSLI If* Effect of -.Prior Breaching with fkS&afimm* 2is% iBlyhdeaaa pins Zinc* and Wolyodeamm
Has Calcium Panthstfaanats on -tissue.- Accumulation of Intramuscularly Aomiiiistered tiabeled Copper in Weanling AXMne Sats*3,
(Expressed as Percent fiose per Cram of fresh lsight)2
Croup
10*.. .
Prior"; 'fratent-
Eat No*
Bat Wt* in Grams
Blood
Idver
Spleen
Hide
IX
HI
Bally H| drench for- 8 -days- pre*- ceding, dosing.
Average
Bally drench of 5 mg. Ho for 8
days preceding
Average
Daily drench of 5 mg* Zn for 8
- days- '.preceding dosing
Average
1 69
2 81
3 79
1* 81
76
6 61
W
1 35
2 32
3 29
it ItU
$ 29
6 ttO

lt7
2 52
3 59
it 70
$ 53
6
0*i.2CUitl}3 0*33(1.10) 0*52(1.73) 0*29(0*98)
0.51(1.72} 0*6^(2*0^) 0*h5uh9i
0*81t(2*8l) 0*i.3(l*52) 0*10(0.33) 0*54(1.81) 0*62(2.06) 0,53(1*77)
G.5K1.7D
0*31.(1.12) O.6l(2.0U) 0.67(2.2lt) 0.53(1*78) 0*37(1*23) 0*85(2*83) 0*56(1.875
%*68(l4.8) 5.30(12.8) 5*53(12*1) 5*01.(12.6) 6*51(13.2)
|*mS*51
5.3^(15M
lt*60 (7*35) 6*25 (9.65) 3*57 (6*95) 1..18 (10.lt) 7*19(11.9) lt.23 (7*61) 5*03 (8.98)
lt*25 (9.52)
lt.3lt(ll.l)
5*51(12*8)
it.82(12*6)
3.05 (8.35)
7*llt(15*8)
It .B5 11.7
0*65(0.10)
0.^(0.053)
0.7lt(0*10)
o.a(o.io)
0,73(0*076)
Q.35(O.Oltl
4.61(5.678
1*55(0*10)
1*27(0.091*)
0.1^(0*026)
0.63(0.082)
0.86(0*063)
0.82(0.063
o*9i(o.o8a
0*53(0*060) 0.89(0*151) 0*63(0*059) 0.63(0.070) 0*1.8(0*059) 0.60(0.056) 0*63(0*076)
2*1*2(X*29> 1*77(0*79) 2*07(l*01t) 2*1*6(1*21*) 1*79(0*76) f.l7(l*28) CT^.0fJ
1.01(0.61t)
1.20(0*75)
0.91(0.53)
1*83(1*15)
1.51(0.97)
1.26(0.80
1.29(0.81
lt.83(2.78
5.72(3.08.
1**98(2*76)
5*88(3*010
5*1*5(3.2$)
1**63.(2,19)
$.2*<2.flS>
0*21* 0,20 0*20 0*25 0*18 0*18 0*12!
0.16
0*18
0*091
0*30
0*23
0,18
0*W
0*15 0*20 0*17 0*22
0*17 0*28 0*20


TAE 17. {Gen*t.) Effect of Prior Drenching with Molybdenum, Zinc, Molybdenum plus Zinc, and Molybdenum plus Calcium Pantothenate on Tissue Accumulation of Intramuscularly Administered labeled Copper in Weanling Albino Ratsl
(Expressed as Percent Dose per Cram of fresh Weight)*
Group
Prior Treatment
Rat Bat wi.
No. in Grams Blood
liver
Spleen
Kidney;
Hide
If Daily drench of 5 mg* Mo and 5
mg. Zn for 8 days preceding dosing
Average
V Daily drench of 5 mg* Mo and 2*5 mg* calcium Pantothenate for 8 days preceding dosing
Average
1 35 0*21.(0*818)3
2 U9 0.23(0*772)
3 55 0*72(2*1*1) k 50 0.90(3*00)
5 50 0*1*9(1*63)
6 I|2 0*27(0.91)
1 h9 0*76(2.51*]
2 31 0*50(1.67
3 30 0*55(1.86) It 3l* 0*28(0.931*)
5 57 1.37CU.58;
6 lil 0.25(0*1*9] 2*0* 5*60(2.01
1*1{6 (2.30) 2*78 (8*35) 3*27 (8*80) 2.28 (6*27) 1**93(10.6)
km "
m?
0*37 (7*25) 2.0k <3.7i*) 3.11 (7.07) 3.21* (5.11) 1*80 (i*8it) lt.86
1.27(0*052) Q.ii5 0*1*6(0*029) 0.69(5*067)
0.1*6(0.050)
0.72(0*Oli5)
0.91(0.051)
0.83(0.031)
l.l6(0*18)
0.57(0*035)
1.0l.{0.62) 3.09(1*95) 3.70(2.13) 3.00(1.67) 2*98(2*02) 2.31(1*30) 2,69(1*62)
1*83(1*21) 1*25(0,85) 0.98(0.66) 0.57(0.36) 2.20(1.30) 1.21(0.63) 1.32(5*82)
0.15
0.25 0.25 0.30 0.17 0.19
0.25
0*12
0.16
0.093
0*30
0*063
each lot, rats one through three are females and rats four through six are males.
%a2aes for percent does per gram of fresh weight are corrected to a body weight of 50 grams. Values for blood are for percent dose per ml. of blood.
%alues in parentheses represent percent dose in whole tissues or organs.


TABLE js Alimentary Tract Distribution and Excretion of Labeled Copper in Weanling Albino Rats1 Sacrificed Twenty-four Hours after Intramuscular Dosage.
(Expressed as Percent of Dose)2
Group Eat I
$ (H0 Drench)
Group Group Group Group
H III IV V
(Ho Drench) (Zn Drench) (Ho*Zn Drench) (Ho.P.a Drench)
Alimentary Tract plus Contents
Average Feces-*
Average
Brine3
Average
1 2 3 U
5 6
1 -3 h 6
1-3
l*-6
31.9
9.1*1* 10.1
8*10 20.3
1.U3 5.33
0.72* 0*55
6.68 5*95 1*.27 8*21 1**21
0*78 1*20
0.22 0*53 ^.3t
12*5 6.73 7.22
12*1 8.33 1**1*9
9.91 23.3 5.21
9.91 5*1*5 8.1*1*
15.9 8*21* 6.83
10.0 7.05 8*59
11.7 8.17 TM
1.30 0.8o 0*1*6
1.91* 1.5u 0.20
TM
0*69 0*90 0.21
0.7k 0.76 0.30
6.72 6*fe 072S
lln each group, rate 1 * 3 are females and 1* 6 males.
^Dosage was 160 micrograms of labeled copper per rat.
^Feces and urine were pooled for the 3 females and 3 males in each group,


The data for tissue accumulation of the intramuscularly administered labeled copper did not show any great differences for the blood* Group V (molybdenum plus calcium pantothenate) had a higher percent of the dose in the blood than did the other groups, but the differences between groups were not great* The control group had the greatest accumulation in the liver, about twice as much as group V, which had the lowest liver accumulation of the labeled copper* Spleen and hide values for the various groups varied little* Kidney values varied considerably according to the drenching treatment* Group II (molybdenum) and group V (molybdenum plus calcium Pantothenate), had somewhat lover kidney accumulation of the labeled copper dose than did the control group* Group III, receiving the sine drench, had more than twice as much of the labeled copper dose in the kidneys* Group 17, receiving mol^fcden'j'A plus sine, had a kidney accumulation of labeled copper which was intermediate between that of the control group and the zinc drench group, perhaps indicating a modifying effect of the molybdenum upon the tendency of sine administered as a drench to Increase kidney accumulation of intramuscularly administered labeled copper*
The excretion data in Table 18 indicated that -considerably less labeled copper was excreted by group II (molybdenum drench) than by the control group* The percent dose in the alimentary tract plus contents was almost twice as high with the controls as it was with group IX* About three times as much was excreted in the feces and twice as much in the urine-by the control group 2U hours after intramuscular administration of the labeled copper* This indicated that molybdenum drenching tended to increase copper retention*


EXPERIMENT i* Effect of Short-term Supplementation with Molybdenum upon Growth and Radiocopper Distribution in the Weanling Rat
Experimental Procedure and Results
Ten weanling, four-week old female albino rats were selected from the stock colony and divided into two groups of five each* These rats were weighed at the start of the experiment and again after one week* For eight days the rats of group I were each given a dally drench of 0*5 milliliters of pyrex distilled water, while those of group II received 5*0 milligrams of molybdenum as sodium molybdate in 0*5 milliliters of pyrex distilled water* Both groups received the Ca*3A purified ration, containing 3*3 parts per million copper and less than one part per million molybdenum and pyrex distilled water ad libitum*
On the eighth day of the experiment each rat was dosed orally with 160 micrograms of labeled copper as the chloride in 0*5 milliliters of solution after which the daily drench was administered* The initial activity of the dose was about 50 microcuries* Each rat was placed in an individual metabolism cage and after twenty-two hours all rats were sacrificed and the accumulation of labeled copper in blood, liver, kidney, Spleen, hide, gastrointestinal tract plus contents, feces and urine was determined* These results appear in Tables 19 and 20* Data concerning the weight changes of these rats after one week of drenching have also been tabulated (Table 21)* Rat $ of group 21 died after 8 days of molybdenum drenching*


TABLE 19 Effect of Prior Drenching with Molybdenum upon Tissue Distribution of Orally Administered Labeled Copper in Weanling Female Albino Rats Sacrificed Twenty-two Hours after Dosage* (Expressed as Percent of Dose per Gram of Fresh Tissue)!
Group So*
Prior Treatment
Rat No*
Blood?
Liver?
Spleen?
Hide
Daily H2O drench for 8 days
Average
5 mg* Mo drench daily for 8 days
Average
1 0.22*1* (0*521)
2 0*0967(0.3i*3)
3 0.21.7 (0*522) 1* 0.132 (0.1*67) 5 0.061*6(0*220)
Q.m (6*1*114)
1 0.251* (0.900)
2 O.I*59 (1*63)
3 0.732 (2.60) k 0.201
0.313(0*537)
0.302(0.51.6)
0.1*9l*(l.l*0)
0.231(0*337)
0.309(0*537)
***. ...*
0*312(0*852) 0.1.70(1*10) 0*l*Ql*(O*686) M<%(S*8^)
1.25 (0.65?) 1.35 (0.621*) 0.73U(0,398) 0.71*3(0*397)
0*71*2(1*31*) 0.9c(o.6a)
0*589(0*1*06)
0,725(0*559) .....(0.273)
0.665(0*525) 6.6&(6.3S>
0*209(0.0303) 0.085(0*0159) 0*197(0.0378) 0*173(0.0287) 0,207(0.031*9) 6.17f*(6l0i^i
0.177(0.0131*) 0.153(0.0372) 0.208(0.0139) 0.253(0*011*8) 5.1k8(5.01983
0.132
0*0995
0.205
O.077I*
0*0555
oiow
0*108 0.122 0.139 0*0691
values for percmt dose per gram of fresh weight were corrected to a body weight of 50 grams, values in parentheses represent percent of dose in whole tissues or organs.


TABLE 20. Effect of Prior Drenching with Molybdenum upon Gastroin-
testinal Tract Content and Excretion of Orally Administered Labeled Copper in Weanling Female Albino Rats Sacrificed Twenty-two Hours after Dosage*
(Expressed as Percent of Dose)
Group'Prior Ho. Treatment
1
n
Rat No.
Gastrointestinal Tract and Contents
Feces Urine
70.0 0.17
62.8 0.18
9.69 0.1*7
72.lt 0.3u
1.8.3 0.31
o10
58.2 0.038
58.3 0.30
u9#5 0.091
12. u 0.30
TOT 6.18
Daily HgO 1 0.683
drench for it .51
8 days 3 2.62
it 3.71
5 11.6
Average lt.62
5 mg. Mo 1 1*1.6
drench dally 2 2.75
for 8 days 3 llul
Average it 7.76 *t.5t
TABLE 21 Effect of Drenching with Molybdenum upon Growth of Female Weanling Albino Ratsl
(Weight Expressed in Grams)
Group No. Treatment Rat No. Age in Weeks Net Weight Change
I Daily HgO 1 79 29
drench 2 55 79 21t
3 .55 80 25
it 59 85 26
h5 69 2lt
Average
II Daily drench :. 1 50 39 11
of 5 mg* Mo as 2 63 lt3 20
NagKoO^.^O 3 51 39 12
it 52 39 13
5 56 38 18
Average fit *' 15
*These rats were maintained on a purified ration containing 3*3 p*p*m* of copper and less than 1 p*p*m. of molybdenum.


Discussion and Conclusions
The rate which received the molybdenum drench accumulated about three times as much labeled copper in the blood 22 hours after dosage as did the controls* Liver and hide values were somewhat higher* while kidney and spleen values were lower than with the controls which received the pyrex distilled water drench* The gastrointestinal tract labeled copper content was slightly higher for the controls* However, the con* trole had a higher fecal and urinary excretion of labeled copper than did the animals drenched with molybdenum* In general the excretion data were erratic, however there seemed to be a somewhat greater excretion of labeled copper by the controls than by the group receiving the molybdenum drench. This trend was also seen for the weanling albino rats in the previous experiment (Table 18) which received the labeled copper intramuscularly. However, the inteamuscularly dosed rats of the previous experiment did not show increased blood accumulation in the case of the rats receiving the molybdenum drench to the extent which was observed In this experiment after oral dosage*. Ilolybdenum, when given as a dreaeh, seemed to increase the retention of orally or intramuscularly administered labeled copper*
The loss in weight due to molybdenum drenching (Table 21) was significant at the five percent level (P 0*05)* The 9$ percent con* fidenee intervals (31) for weight change in grams in the control group was 26 j- 3| while it was -15 5 for group H which received the daily molybdenum drench*


EXPERIMENT $ Effect of Short-terra Supplementation with Molybdenum upon Growth and Radiocopper Distribution in the Mature Rat*
Experimental Procedure arid Results
Male albino rats, twenty-four weeks old* which had been maintained on the Ca-3 ration (Table 9) since weaning were divided into two groups of five each* for the next eight days the rats of group I received a daily drench of 1*0 milliliters of pyrex distilled water while those of group H were given 10.0 milligrams of molybdenum as sodium molybdate in 1.0 milliliters of pyrex distilled water. Both groups were fed the Ca-3-A purified basal ration* containing 3.3 parts per million copper and less than one part per million molybdenum* and were given pyrex distilled water ad libitum. The rats were weighed at the start of the drenching period and again after one week.
On the eighth day of the experiment each rat received a dose of 320 micrograms of labeled copper as the chloride in 1*0 milliliter of pyrex distilled water. The dose was injected intramuscularly into the hind leg and each rat was then placed in an individual metabolism cage.
These rats were sacrificed twenty-two hours after dosage and the accumulation of labeled copper in the blood, liver, kidney, spleen, hide, gastrointestinal tract plus contents, feces and urine was determined (Tables 22, 23)* The effect of molybdenum drenching upon the weights of these rats is shown in Table 2k*
* 6k -


TABLE 22. Effect of Prior Drenching with Molybdenum upon Tissue Distribution of Intramuscularly Administered Labeled Copper in Twenty-four Week Old Male Albino Rats Sacrificed Twenty-two Hours after Dosage*
(Expressed as Percent of Dose per Gram of Fresh Weight)1
'"Prior" Treatment
Rat No.
Group Ho.
Blood2
Liver
Kidney2
Spleen2
Hide
II
Daily H2O drench for 8 days
Average
10 mg. Mo drench daily for 8 days
Average
1 2
3 1* 5
1 2 3 h 5
0.0363c 0.70?) 0.07qm 1.38)
0.362 ( 5.1*10
0.180 ( 3.53) 0*51*2 (10*5)
U.31)
is.
0.811 0.802
0*551* 0.360 0.550
(15.8)
(15.7) (10.8)
7.05)
(10.8)
(12..0)
2.01 1.9? 2.03 1.66 2.1*?
1.1.9 1**1*2 1.17 2.01
"of
2.55 (3.1.6) 2.90 (3.87) 3.7u (5.36) 2.13 (3.33) 0*71*7(1*31*)
2.1.1 ilm
1*1*3 1.1*5 1.09 1.60 1.81
(2.23) (1*95) (2.15) (1*23) (2.58)
0*219(0*Hi*} 0.230(0.103) 0.51.8(0*226) 0.255(0.128) 0.1*37(0.182)
0.338(5*151)
0*1*99(0*181*) 1.13 (0*331) 0*1*29(0*270) 0*31*2(0*156) 1*35 (0.g03) 0*750(5.280
0.0601
0*12!*
0*292 0-136 0*132
0*215 0*297 0*0652
0.187 QlWT
%alue for percent dose per gram of fresh weight is corrected to 275 grams body weight* %alues in parentheses represent percent of dose in whole organs or tissues*


TABLE 23
Group No.
Effect of Prior Drenching with Molybdenum upon Gastroin-teestinal Tract Content and Excretion of Intramuscularly Administered Labeled Copper in Twenty-four Week old Male Albino Rats Sacrificed Twenty-two Hours after Dosage* (Expressed as Percent of Dose)
Prior '" Treatment
Rat Gastrointestinal """* No* Tract and Contents
Feces
Urine
II
HgO drench
daily for 8 days
Average
10 mg* Mo drench daily for 8 days
Average
1 2 3 1* 5
1 2 3 k
1**25 7.1*0
1.1*7 3*61
3*63
1**22 10*1* 3*07 8*51 1.16
T*Vf
$09
19*0 17*1* 30*8 9*86 TOT
1*87 1*82 2*39 2*26 2.80 "2723*
0,800
2*37 1*06
24*1 1.1*2
TABLE 2k* Effect of Drenching with Molybdenum upon Growth of Twenty-four Week Old Male Albino Rats,1
. (Weight Expressed in Grams)
Group No* Treatment Rat Ho* Age 21* in Weeks 25 m weight Change
I Daily H2O drench Average 1 2 3 k $ 273 308 369 287 331* 275 309 371 287 337 US 2 1 2 0 3
II Daily drench of 10 mg* Mo as NasMoQ^HgO Average 1 2 3 1* 5 330 312 261 318 321 313 309 21*1* 319 317 355 -17 3 17 1 m k O
During the week of drenching, these rats were maintained on a purified ration containing 3*3 p.p*m* copper and less than 1 p,p,m molybdenum*


Discussion and Conclusions
In comparison with the controls, the rats of group H, which received the daily molybdenum drench, had almost three times as much labeled copper in the blood twenty*two hours after dosage* The liver and hide concentrations of labeled copper were somewhat higher, and the spleen concentrations were about twice as high in group II as they were in the control group. The kidney accumulation of labeled copper was considerably higher in the control group, as was the urinary excretion of labeled copper* There was little difference in the percent of the labeled dose in the gastrointestinal tract or the feces of the two groups* The wide difference in the percent of labeled copper found in the blood of the two groups indicated that drenching with molybdenum Increased the retention of intramuscularly administered labeled copper in these twenty-four week old albino rats*
The adverse effect upon weight gains of drenching these twenty-four week old rats with 10*0 milligrams of molybdenum daily for eight days was much less severe than that of drenching weanling rats with 5*0 milligrams daily*
While -toe control group gained an average of two grams during a week of drenching, the high molybdenum group lost an average of eight grams* With the weanling rats of Experiment !., the control group gained an average of 26 grams while the group receiving a drench of 5*0 milligrams of molybdenum dally lost an average of 1$ grams in one week*


EXPEftXMBHT 6 Preliminary Study of the Effects upon Swine of High Dietary Levels of Molybdenum*
Experimental Procedure and Results
Pour Spotted Poland China litter mates and two Duroc litter mates were used in this preliminary study concerning the effects of high levels of molybdenum upon swine*
The Spotted Poland China pigs were four months old at the start of the experiment and bad been on pasture prior to the experimental period* They were placed in individual pens with concrete floors located within a shed* Feed was supplied in self-feeders* and water in individual wooden water troughs.
Pig one* a barrow, was fed the basal ration whose composition is given in Table 25* The copper content was 8*1 parts per million and the
TABLE 25* Composition of Swine Basal Ration Used in Preliminary Study*
li^redient _..........; \.'r [..'/.". '"'['!.'"'.",'"^n. '"".,,., '...Li'.al'-, Perc^"^n:Ration 11'
Ground yellow corn $%*$
Soybean oil meal, expeller, l&% protein 1.0*0
Alfalfa meal, 15^ protein 7*0
molybdenum content was 2*6 parts per million. Pig two* another barrow, received the basal ration with hPO parts per million of molybdenum added* Pig three, a gilt, received the basal ration with 800 parts per million of mrlybtift^jm added* while pig four, another gilt, was fed the basal ration
Bonemeal Sodium chloride Calcium carbonate
0*5 0*5 0.5


with 800 parte per million of molybdenum and 200 parts per million of copper added* the supplemental molybdenum was supplied in the form of NajMoOj^HgO and the copper as CoSO^*
After three and a half months on experimental rations* haemoglobin values were determined on blood obtained from the ear vein, using the acid-hematin method (50)* the values for pigs one to four were 10.0, 11*2, 12*1 and 12*1 grams per 100 milliliters of blood respectively*
Pig one, which was used for a radlomolybdenum study to be discussed later, was sacrificed after lk weeks and pig four after 20 weeks on ex* perimental ration* Pig two died during an extremely hot period after 16 weeks on experimental ration* this barrow evidently became overheated in attempting to get out of a metabolism unit* Pig three died after lU weeks on the experimental ration* This gilt had been placed in a metabolism unit for a three-day preliminary period, then had a urinary catheter inserted and kept in place for two days* At the end of three days, she was taken off the balance trial because of bleeding around the catheter. She apparently did not recover from this irritation, probably due to inflammation and perhaps septicemia* On opening this animal, the most striking symptoms were gross inflammation, thickening of the bladder and uterus, a total lack of urine in the bladder, emaciation and inflammation in the urethra, and apparent stoppage of urine flow in the ureters with enlargement and necrosis of the kidneys. The kidneys had large areas of necrosis and a nutmeg-like appearance. The fluid around the heart was cloudy, indicative of generalized uremic poisoning and septicemia*


The livers* spleens and kidneys of these four swine were analyzed for copper and molybdenum and the femur shafts for molybdenum* The results have been tabulated in Table 26*
Two Duroc boars were also used in this preliminary study* These pigs* which were litter mates, were extremely unthrifty* Pig one weighed 13 pounds and pig two weighed 10 pounds at the start of the experimental period at which time they were four months old* Pig one was fed the basal ration (Table 21.) plus 1.00 parts per million of molybdenum for two weeks, followed by the basal ration plus 1000 parts per million of molybdenum for five weeks, after which the basal plus 800 parts per million of molybdenum and 200 parts per million of copper were fed for the final 11 weeks of the experimental period* Pig two was fed the basal ration plus 600 parts per million of molybdenum for two weeks, followed by 16 weeks of the basal ration plus 1000 parts per million of molybdenum* At the end of the 18*week experimental period these swine were sacrificed and the copper content of liver, kidney and spleen as well as the molybdenum content of these tissues plus the femur shaft was determined (Table 27)*
These two Duroc bears and the control Spotted Poland China barrow were dosed with radioactive molybdenum, using a stomach tube* The data for fecal and urinary excretion of labeled molybdenum during the six days following administration of the dose are given in Table 28*


TABLE 26. Accumulation of Copper and Molybdenum in Various Tissues of Spotted Poland China Swine1 Maintained on Natural Rations with Varying Levels of Copper and Molybdenum.
(Expressed as Parts per Million on a Dry Matter Basis)
Pig No. Ration weeks '"-'-tai-k : Ration Final Weight (lbs.) Liver Cu liver Mo Spleen Cu Spleen Mo Kidney Cu Kidney Mo Femur Shaft Mo
1 Basal: 2.6 p.p.m. Mo 8.1 p.p.m. Cu Ik 132 51.5 7.7 3.6 1.9 33.0 k.7 0.5
2 Basal plus kOO p*p*iu Mo 1 171 108. 6k.9 5.5 18.9 k57. 223. I8.k
3 Basal plus 800 p.p.m. Mo lit 122 8k,5 73.6 18.6 20.7 .... ... 28.8
It Basal plus 800 p.p.m* Mo. 200 p.p.m. Cu 20 7k 1090. 17.5 6.9 16.8 58.2 61.3 k5.6
^These pigs were litter mates. They had been on pasture prior to being put on experimental rations at four months of age. Pigs 1 and 2 were barrows and 3 and k were gilts.


TJSLE 27 Accumulation of Copper and Molybdenum in Various Tissues of Duroc Boars Maintained on Natural nations with Varying Levels of Copper and Molybdenum*
(Expressed as Parts per Million on a Dry Matter Basis)
Femur
Liver Liver Spleen Spleen Kidney Kidney Shaft
No* : Cu Mo Cu Mo Cu Mo Mo
i* 81.6 22.9 16*0 7*6 82.1 37.8 18.6
22 125. 53.2 8*5 3.1* 137. 117. 29.1
*Pig one was maintained on the basal ration plus 1*00 p*p*m* Mo for two weeks* followed by basal ration plus 1000 pp*m. Mo for five weeks, after which the basal ration plus 200 p*p*m. Cu and 800 p.p.m. Me was fed for 11 weeks prior to dosing.
2Pig two was a litter mate of pig one* This pig had been maintained on the basal ration plus 800 p*p.m* MO for two weeks, followed by 16 weeks of the basal ration plus 1000 p*p*m* Mo*


TABLE 28 Excretion of Labeled Molybdenum Adainistered Orally to Swine.
(Expressed as Percent of Pose per Day)
Pig Number I1 22 33
Age in Months 7 8| 8|
Weight (in pounds) 132 99 l$h
Grams of K0O3 administered 0.25 0.55 0.70
Feces brine Feces Urine Feces Urine
% 0. 52.0 0. 57.0 0.53 52.U
2 0.01 1.51 0.30 1.16 0.06 2.03
3 39.8 0.1*5 7.83 0.1*1 18.2 0.1*7
a 2.32 0.28 1.25 0.12 3.91. 0.17
5 2.21. 0.20 2*70 0. 0.13 0.13
6 0.1$ 0.12. 0.97 0. 0.05 G.Oi,
Total for 6 days UU.52 5U.58 13.05 58.69 22.91 55.21*
^Pig 1 was a spotted Poland china barrow which had been on basal ration for lit weeks.
*Pig Z ** a Duroc bear. fhia pig had boon stained on tt. baoal ration plus too p.p.*. Ho for to
weeks, followed by basal ration plus 1000 p.p.m Mo for five weeks, after which the basal ration plus 200 p.p.m. Cu and 800 p.p.m. Mo was fed for 11 weeks prior to dosing.
3pig 3 was a boar litter mate of pig 2. This pig had been maintained on the basal ration plus 800 p.p.m. Mo for two weeks, followed by 16 weeks of the basal ration plus 1000 p.p.m. Mo.


Discussion end Conclusions
With this small number of pigs it was not possible to draw any definite conclusions concerning the effect of molybdenum upon growth* Of the four Spotted Poland China pigs* pig two (basal pins 1.00 parts per million molybdenum) grew somewhat faster than the control pig, pig three (basal plus 600 parts per million molybdenum) grew somewhat slower, while pig four (basal plus 800 parts per million molybdenum and 200 parts per million copper) was very unthrifty in appearance at the start of the experimental period and grew at a much slower rate than any of the other pigs*
Feeding these high dietary levels for three and a half months did not appear to have an appreciable effect upon haemoglobin level*
the continued feeding of hPO to 1000 parts per million of molybdenum to swine for 1U to 20 weeks resulted In considerable accumulation of molybdenum in the liver, kidney, spleen and femur shaft* Liver molybdenum values ranged from 17*5 to 73*6 parts per million on the high levels of molybdenum feeding* Spleen values ranged from 3*U to 18*6 parts per millionj kidney values from 37*8 to 223$ and femur shaft values ranged from 18.U to U5*6 parts per million of molybdenum* These values were con siderably higher than for the corresponding tissues of the control pig maintained on the basal ration* Liver, spleen, kidney and femur shaft values for this animal were 7*7, 1*9, U*7 and 0*5 parts per miUbn of molybdenum respectively. All values are reported on a dry matter basis*
<** 7lt


High dietary intake of raolybdenum bad a considerable effect upon liver, spleen and kidney accumulation of copper* All pigs which received supplemental molybdenum in the ration had higher concentrations of copper in liver, spleen and kidney* Pig four (basal ration plus high molybdenum and high copper) (Tfc le 25) had a liver copper value of 1090 parts per million* Although thera was no pig on this high copper level without added molybdenum, this level of copper alone would hot be expected to cause such a liter copper accumulation*


EXPERIMENT 7 the Effects of Varying Dietary Levels f Molybdenum. Copper and Zinc upon Swine.
Experimental Procedure and Results
Eighteen weanling Hasfwhire swine from tw> litters were 'divided into six lots* The litters were eight and ten weeks old respectively* In distributing these pigs among the six lots, they were selected for uniformity of weight and even distribution of barrows and gilts* The three pigs of each lot were placed in pens nine feet long and eight feet wide with concrete floors! each with a self-feeder, waterer and shelter* Straw was used for bedding. All pigs were weighed biweekly during the experimental period of seven months.
Lot I was fed the S-5 natural ration, whose composition is given in Table 2. This basal ration contained 8*9 parts per million of copper. 2*1 parts per million of molybdenum and 39*2 parts per million of sine* Lot II received this basal plus an additional 1000 parts per million of sine as ZHSOj^HgO* Lot III received the basal plus 200 parts per mil* lion of copper as CuSoh. Lot IV was fed the basal ration plus 100 parts per million of molybdenum as Naglf^.gRgO* Lot V was fed the basal ration with the same level of molybdenum added as for lot IV, and in addition, 200 parts per million of copper as the sulfate* Lot VI received the basal ration with the same level of sine added as for lot H, plus 200 parts per million of copper as the sulfate* The rations were mixed in a Twin Spiral mixer of one half ton capacity manufactured by the Prater Pulverizer Company, Chicago, Illinois.


After 11 days on experimental ration, one gilt from lot IV (high molybdenum) developed a prolapsus anl condition, and passed bloody feces. This pig was reaoved from the experiment and a barrow from the same lit* ter was substituted* One month after the start of the experiment a barrow from the same lot died during a period of freeaing weather.
The data for growth rate, feed consumption and efficiency of feed utilisation during 2k weeks on experimental ration are presented in fable 29*
Figures 1-6 show the appearance of a pig from each lot after four months on experimental rations.
After 27 weeks, swine from each group were oriented in swine metabolism units (u7) for several days without any change in ration. Feed was given four times daily and water was before them constantly* Urine conduits were fastened over the vulva in the case of gilts to effect quantitative separation of urine and feces while with the barrows, the urine was collected separately through a funnel and the feces fell into a col* lection pan*
Radioactive copper was administered orally to all swine* For the first group of pigs treated, the labeled copper chloride solution was placed in a gelatin capsule and administered by means of a balling gun* Use of a survey meter indicated that the capsule went into the stomach only in the case of two of the animals dosed* In the case of the other four, the capsule did not enter the stomach but apparently was lodged in the pharyngeal diverticulum^ these animals were not sacrificed but were




Fig. 3. Pig 1 of Lot III After Four Months on Basal Ration plus 200 p.p.m of copper.
Fig. U. Pig 1 of Lot 17 After Four Months on Basal Ration plus 1000 p.p.m. of Molybdenum.


Fig. 5. Pig 2 of Lot after Four Months on Basal Ration plus 1000 p.p.m. Molybdenum and 200 p.p.m. of Copper.
Fig. O. Pig 2 of Lot VI after Four Months on Basal Ration plus 1000 p.p.m. Zinc and 200 p.p.m. of Copper.


TABLE 29 m Growth Rate and Feed Consumption of Hampshire Swine on Natural Rations with Varying Levels of Molybdenum. Copper and Zinc*
Lot 1 (Basal Ration) Lot U (High Zn) Lot III (High Cu) Lot ivi (High Mo) Lot V (High Mo.Cu) Lot VI High ZnCa)
Hteber of pigs 3 3 3 2 3 3
Days on test 168 163 168 268 168 268
Av. initial wt*, lb* 31*2 31*8 31*7 21* *0 32.7 31*3
Av final wt** lb* 211*0 199*0 195*7 283*0 193*3 198.7
Air* daily gain, lb* 1*07 1.00 0*98 0*95 0.96 1.00
Av daily feed, lb* *t*53 3s*.15 3*89 U.39 1**00 1**1*1*
Feed per 100 lb* gain 1*23 1*15 397 L62 1*17 hhh'
*Of three pigs which were started in lot IV, one pig died after one month on experimental ration. In computing feed consumption for the remaining two pigs of this lot, allowance was made for feed consumed by this pig*


held ever until the following week and re-treated at which time the first dose of copper^, had decayed to a negligible amount* As a result of the experience with capsule administration it was decided that the use of the stomach tube might offer some advantages* A thin polyethylene tube was inserted into an ordinary human stomach tube and the assembly inserted into the esophagus of the animal. After indications that the tube was not in the lungs a large syringe containing water was attached to the end of the polyethylene tube* the radioactive solution, about eight milliliters, was measured into a syringe and the hypodermic needle inserted into the polyethylene tubing, and the dose was slowly forced out with positive pressure being maintained by the large syringe* The dose was 102.li milligrams of labeled copper for each animal except pig two, lot V, which received 7U*2 milligrams, and pig one, lot I, and pig X, lot HI, each of which received 98*5 milligrams of labeled copper. After administration of the activity, a survey meter was used to ascertain if the dose went into the stomach*
All swine were sacrificed 21* hours after administration of the labeled copper. Each animal was stunned, bled, and the alimentary tract removed to another room for sampling to preclude the possibility of contaminating tissue samples with the high activity present in the intestinal contents* The tissue distribution of labeled copper in the animals of the various lots is presented In Table 30* The percent dose figures for red blood cells were calculated, using the values determined for whole blood and plasma and the nematoerit determination. A barrow of lotUI which received the labeled copper dose in the lungs had uniformly higher tissue accumulation and was not included in Table 30*
The blood was estimated at 7*0 percent of body weight for computing the percent of dose present in the blood* The percent of dose in the


f ABLE 30, Tissue Distribution of Orally Administered Labeled Copper in Hampshire Swine1 Maintal ned on Watural Rations with Varying Levels of Copper* Molybdenum and Zinc*
(Expressed as Percent of Dose x 105 per Gram of Fresh Weight}2
Pig Lot I Lot H
Tissue No* Basal Ration : .0.1$Zn
Lot III Q.02# Cu
Lot I? 0.3g Mo
" LoT? 0*1$ Mo plus 0*02# Cu
0.1$ Zn plus 0*02$ Cu
Whole Blood
1
2 3
16.7 (l*ll.)3 12*3 (0*8lt)
Average ih.5 (0*99)
Blood Plasma
Average
Red Blood Cells Average
Liver
Average Kidney
Average
1 2 3
1 2 3
1 2 3
1 2 3
25*6 19.2
mi-"
3.36 lt.$0
333"-"
.7 (0.5?)
ai.a (i*oi)
102. (0.1u) 1.8.1 (0.0?)
?5*i (0,11)
7*31(o*5o) 13.1. (0*85) 11*0 (O.70 lo*6 (5*68
9*37 35.9 35.3
s.f
i..97 10*1.
6.57 "w
65.0 1.7.8
(0.71.) (0.70) 32.0 (0.1.6) 6.3 (5.65)
82.7
1.1..7 (0.06); 50*5 (0*07)
8.53(0.51.) 9.65(0.62)
32.3 11.7
H.o~
i..a
7.23
1.2.8 (0.51.) 39.9 (0.56)
lil.it (6.5^5
67.0 (o*h) 32.3 (0.05)
1.9*? (o.ob)
18.6 (1*27)
2l.*S
12.1
137. (1*68)
79.5 (0.11)
11*2 (0.71) 8.80(0.60) 11.1 (0.71) 10*1. $.677
19.9 8*57 17*9
35*3-
1.08 9.16 3*89
36.9 (0.50) 1.0*8 (0.1.8) lg*6
38.0 (0*01.) 37.2 (0*06)
37*1
11.6 (0.79) ?.00(O.u5) 9*90(0.63) 9.50(6.62)
17.9 9.79 h..0
TC?-
1..15 l.*17 5.33
1.2.0 (0*52 1.0*6 (0.1& 51..3 (0.78)
i.5.6 (0.58)'
127* (0.18) 39.8 (0.06) 35*0 (0*06) 67*3 (3 Joj


TABLE 30. (Cont.) Tissue Distribution of Orally Administered Labeled Copper in Hampshire Swine1 Maintained on Natural Rations with Varying Levels of Copper. Molybdenum and Zinc* (Expressed as Percent of Dose x 105 par Gram of Fresh Height)2
Tissue
Pig Lot I Lot n
No. Basal Ration 0.1% Zn
Lot HI O.Ogg Cu,
Lot iv
0.1$ Mo
0.1$ Mo plus 0.02% Cu
lot VI 0.1$ Zn plus 0.02S6 Cu
Spleen
Average Heart
Average
3 2 3
1 2 3
lt.oo(*Q05)
5*77(*01)
!.,!#(.01) 3.8a(*0l)
2.99 (.005)
3*62 (.01)
2*83 (*0C4)
3>ig (.006)
o.hW
O632(.O02)
3 90
1.66 (.062)
2.20(*0l) 2*35(*003)
i.28(*06t)
1*^7(*01) 1.68(.G05)
7*73(.02)
6.93(.02)
2*56 (.00U)
3*76 (.01)
2.37 (.003)
2.90 (.006)
6.85 (.02)
2.10 (.01)
2,39 (.01)
3.78 (.01)
2.82 (.003) 2.56 (.002) 3.37 (.01) 2.58 (.065)
2.83 (.01) 1.63 (.005) 0.670(.Q2)
141(*6ir
Gastroc- 1 2.06 1.52 2.2U 2.23 2.20 1.35
nemius 2 2.72 1.00 2,02 1.39 0.913
Muscle 3 1.30 0.970 1.38
Average 2.3$ 11 1.2? 03 '" 1.52 1.21
Hide 1 2.57 1.62 5.15 2.68 I*15 2.60
2 2.69 6.23 2.58 2*38 fc.71
3 6.88 1.53 1..90
Average 2.63 """ 11.91 ti.07
Femur 1 1.98 3*03 3.69 3.71 2.52 6.03
Epiphysis 2 2.08 2.25 1.90 0.287 2.37
3 2.6L 1.2k 1.18
Average i*o3- 2.6L 1 2*86 1*35 3.19


TABUS 30, (Con*t*) Tissue Distribution of Orally Administered labeled Copper in Hampshire Swine1 Maintained on Natural Rations with Varying .Levels of Copper, Molybdenum and Zinc* (Expressed as Percent of Dose x 10? per Gram of Fresh Weight)*
-^--^^ Lot V LoWI
Tissue Pig No. Lot I Basal Ration Lot XI O.lg Zn Lot III Q*Q2 Co, Lot I? 0*1% Mo 0.1* Mo plus 0.025S Cu 0*1% Zn plus 0.02$ Cu
Femur Shaft 1 2 3 3*20 1*63 0.5Iil 0.1.28 OTj> 0*289 0*1.29 1*16 0.1.66 0*781 0.770 1..22 0.597 1.25
Average 0*359 0*6?2 2.62"
Red Bone Marrow 1 2 3 1.11 1*36 THE 2*01 1.03 Q*7$h 0*51.0 0*303 3*1.2 3*02 0*1.10. 5*2? 1.91* 0,830
Average 1*26 d*l.& 2.91 1^9"
Rib Sternal End Average 1 2 3 11.4 6*7? 10*8 lt.21 *T3o* 1..53 3*12 9.75 6*05 2*13 5*19 W*W 5*73 5*80 0.77 135*
We swine were maintained on these experimental rations from time of weaning until nine months of age. All were barrows except lot I pig 2j lot H pig 3j lot V pig 1 and lot VI pigs 2 and 3 which were gilts*
^Values corrected for 200 pounds body weight* Blood, bile, urine and plasma values represent percent dose x lO? per milliliter* The dose was 102*1. mg* of labeled copper for each pig except pig 2 lot 7, which received 7l*.2 mg., and pig 1 lot I and pig 1 lot VI, both of which received 98.5 mg. of labeled copper.
^Values in parentheses represent percent dose in total organs or tissues.


various portions of the alimentary tract, feces and urine were determined (fable 31} liver, kidney and spleen samples vers analysed chemically for copper and molybdenum, blood plasma for copper, and femur shaft samples for molybdenum (Table 32)* Copper turnover data, expressed in terms of percent of labeled copper per milligram of copper in the tissue, are presented in Table 33* The percent dose figure used was corrected for body weight so that the data for all swine are on a comparable basis*
After about three months on experiment, varying degrees of weakness in the hindquarters was observed in some animals of all lets* Histological examinations were carried out after sacrifice and revealed the following.
In all but two animals examined, the digital pad of the foot examined showed various degrees ef neutrophilic exudate indicating some Irritating agent which elicited an inflammatory response* It is assumed, but is by no means certain, that this was due to standing on concrete*
Leg joints and muscles taken from the gluteal region were without change*
Sections taken from the cerebrum, cerebellum, cervical chord,
r
thoracic chord andlumbar chord showed no degenerative changes and are believed to represent normal tissues*
Discussion^ and .Conclusions
Although the swine in lot I (basal ration) had the highest average daily gains, the differences among the six lots wore not very great


TABLE 31* Alimentary Tract Retribution and Excretion Orally
Administered Labeled Copper in Swine Twenty-four Hours After Dosage*
(Expressed as Percent of Dose)1
Pig NO* Lot I 1111 Lot n Lot in Lot I? Lot V Lot VI
Stomach Average 1 2 ? 0*03 o.ok o.ol. 0*03 0*02* 0^03 6*63 0.03 0*09 o*66 0*10 0*06 0*02 0*12 6*0^ 0.0k 0.0k 0.06 ~ Stomach Contents Average 1 2 0.0k 0*03 -6761 0.0k 0*02 .....5*63' 0*06 0*03 0*k7 0*01 0.2E 0*62 0.01 0.16 0*06
Small'" Intestine Average 1 2 3 0.18 0*19 TOT 0.62 0.19 0*08 "TOT* 0*09 0.2k ~07Ff 0*51 0.12 0.05 0.09 o*ik 0.22
Small Intestine Contents Average 1 2 3 0*18 0*11 0*35 0*29 '"6".3S 0*2k 0*10 0.70 0.10 0.1k 0*30 0,36
Large Intestine Average 1 2 3 om 1*07 0*20 0.12 O.kO "OH 0.16 0*26 0*6k 0.19 0*37 0*k0 ~0*32 0*56 0*10 oJg TJ*38
Lg* Intest. 1 Cont* plus 2 Feces 3 Average 63.9 52.7 TO 36*2 k9*5 5k*9 50.9 55*8 k6.9 k8.0 51*3 31.3 50*9 13*9 32*1
Urine Average 1 2 3 0*33 0*51 'TO 0*27 0*07 0*26 0.31 6*kk "'63 0*k3 0*37 1.00 0.5? isiw 0*08 0*18 0.82
Total.^% Averages^ 60*0 Wi*8 5k*0 57*6 50.3 33.3
%he dose was 102.k mg. of labeled copper for each pig except pig 2* lot V* which received 7k.2 mg*. and pig 1* lot I, and pig 1, lot VI, both of which received 98.5 rag* of labeled copper*


TABLE 32. Accumulation of Copper and Molybdenum in Tissues of Hampshire Swine Maintained on Natural Rations with Varying Levels of Copper* Molybdenum and Zinc from Time of Weaning until Nine Months of Age*
(Expressed as Parte per Million on a Dry Matter Basis)!
Lot No*
Ration
Pig No*
Liver Cu
Liver Spleen Spleen Sidney Kidney Ride Hide Plasma Shaft
Mo
Cu
Mo Cu Mo Cu Mo CuA Mo
1*1* 0*8 1.0 1*1 $2*5 38*9 28.8 w 5.2 3*6 u*u 1..1 2.7 3.9 ~3.$ ? o*5 0* o.u "o".i 255 220 * *m 2*3 3.2 0*3
0.8 0.2 1.6 86*1* 20*1 35*7 "%m 5.3 2*5 2.1 3.1 2.5 "275 1*8 0*2 1.1 253 281. 2u2 0.1 0 0*1*
0 0*7 o.5 15.1. u5.5 33.0 99.2 3.9 2*u 3*2 11.8 6.3 0*8 0.1 1.7 222 217 255 2ll 0.5 0.6 3.3 r"t3t
16.3 26.1 7.0 1FZ 136* 151.: ** TO7 66*7 83.7 f3 k+6 ioa ..... 16*3 1.1.8 388 363 * 30*3 33.5 11*5
I Basalt 8.9 p.p.m. Cu* 2.1 p.p.m. Mo*, and 39.2 p.p.m. Zn
Average'
II Basal plus 1000 p.p.m. Zn as ZhS0ji.7H^) Average
III Basal plus 200
p.p.m. Cu as CuSOj^
Average
IT Basal plus 1000 p.p.m. Mo as
NakKbOjj^oO
1 2 3
1 2 3
1 2 3
1 2
32
Average (of pigs 1 and 2)
2lt*8 1.2.6 18*1
23.3 17*1.
"W 50.6
102. 28*1.
~w$
185. 113. 38.6 WT
5.5
li*o 5.6
6*3 6.1 l.*lt 3*5
2.5 5.5
5.9
69.1. 6u.G
if
7.8
34 8.9
2.3 1..8 2.1
6.5
3*2 3.6
~5.i
38.2 31.3 12.2
2pr


TABLE 32. (Cont*d) Aceumalation of Copper and Molybdenum in Tissues of Hampshire Swine Maintained on natural Rations with Varying Levels of Copper* Molybdenum and Zinc from Tim of Weaning until fens Months of Age* ,
(Expressed as Parts per Million on a Dry Matter Basis)1
Lot Ho* Ration Pig Liver Cu Liver Mo Spleen Spleen Cu Mo Kidney Kidney Cu Mo Hide Cu Femur Hide Plasma Shaft Mo Cu1 Ho
Basal plus 2000 pp*m* Mo and 200 p*p*m. Cu Average 1 2 3 253* 80.6 381*. W> 20.? 37*0 38.2 55.0* 1**6 7.2 9.0 6.7 5*7 lU*o 36*2 75*2 68*9 60*1 28.9 28.2 26*7 7*8 8.8 9.7 17*7 35.6 1*6.2 310 31*7 332 y 26*1 2l*.l 16*7 22*5
VI Basal plus 1000 p.p.m* Zn and 200 p.p.m* Cu Average 1 2 3 32.9 25*0 37.5 31.8 5*1 5.3 u.s 5.5 2a 6.8 "TO Qi8 0*6 0 72*3 30.7 27*0 ' w& 6*5 8*2 3*8 20.2 1**5 9.1 i*. 323" 2lt*0 17.6 26*2 255 238 2lt8 W 0.9 0*8 1*5 1.1
lvalues for plasma copper are for micrograms of copper per 100 milliliters plasma. %his pig died after one month on esperimental ration*


TABUS 33* Copper Turnover in Select Tissues of Hampshire Swine1 Maintained on Natural Rations with Varying Levels of Molybdenum. Copper and Zinc*
(Expressed as Percent of Labeled Copper Dose x 102 per Milligram of Copper in the Tissue)2
LoCr Pig Wt. Blood
No. Ration No. (lbs) Liver Kidney Spleen Plasma Hide
I Basalt 8.9 p.p.m. 1 178 6.65 9*21 2*20 10*0 1*31*
Cu, 2.1 p.p.m. Mo, 2 21*3 6.01 6*06 7.85 8.73 2.13
39.2 p.p.m. Zn ..... ....... _____ ,
Average "tci 6.33 *7.$7 T*Ol "%$J~T*%
II Basal plus 1000 1 19k k*92 i*.*8 5.1*7 3*?0 1.59
p.p.m. Zn as 2 220 6.69 8.71 3*29 5.60 I..33
ZnS0U*7Hg0 3 232 j.99 j6Jk 4& 4#-M
Average 215 5*b7 6.68 i*.8h 5*21 a.16
EI Basal plus 200 1 207 2.7k 7*05 1*51 5#* 0.?26
p.p.m. Cu as 2 211. 1.26 1**26 3*21 5*39 0*905
CuSOh__ _____ ________
Average "si ~Z&0 "JM "WB t3.7~ot5
IV Basal plus 1000 1 150 2.50 2.59 1.90 6.39 1.03 p.p.m. Mo as
NagMoOj^HgQ
V Basal plus 10C0 i 196 0.h53 U.82 2.1*2 6.1*2 1.15 p.p.m. Mo, 200 2 208 1.65 2.25 2.38 2.1*7 0.528 p.p.m. Cu 3 228 0*355 2.29 1*20 5.39 0.310 Average 211 0.819 3*12 2.00 h.?6 o.6b3
VI Basal plus 1000 1 236 3.1*8 7.02 1.91 5.88 0.522
p.p.m. Zn, 200 2 168 5.21 5*1*9 3.33 lull 0.502
p.p.m. Cu 3 236 1*.68 6.39 2.13 5*65 1*00
Average 213 U*uo 6.30 2.1*6 5.21 0.675
Irhese swine were maintained on these experimental rations from time of weaning until nine months of age. All were barrows except lot I pig 2$ lot H pig 3f let V pig If and lot VI pigs 2 and 3 which were gilts.
^The percent dose figure was corrected for 200 pounds body weight* The dose was 102.1* mg* of labeled copper for all pigs except lot V pig 2 which received 74*2 mg. and lot I pig 1 and lot VI pig 1, both of which received 96*5 mg. of labeled copper.


(Table 29). Let Hi (basal plus 200 parts per million of copper) shoved the lowest average daily feed consumption and the lowest amount of feed required to produce 100 pounds gain* Here again* there were no significant differences between the lots* The addition of 1000 parts per million of molybdenum to the ration apparently had a much smaller growth depressing effect for swine than for rats*
Although the animal in lot 17 (high molybdenum) had the greatest accumulation of labeled copper in whole blood* the differences between lots were not great (Table 30)* This was also the case when the accumulation of labeled copper in the blood plasma and red blood cells was considered* The liver accumulation for the barrow of lot 17 (1*68 percent of the dose) Was more than twice as high as the average liver accumulation of any other lot* The kidney accumulation of the lot IV barrow was about the same as that of lot I (controls) with 0,11 percent of the dose being found* The other lots averaged between 0*05 and 0*010 percent of the dose in the kidneys* The barrow on the high dietary molybdenum level (lot IV) had more than twice the accumulation of the labeled dose in the spleen (0*02 percent of the dose) that was found in the other groups, which ranged from 0*005 to 0*008 percent of the dose* The concentration of labeled copper in the heart varied considerably with the lot IV barrow showing the highest concentration, lot I (control) and lot V (high molybdenum plus high copper) having an intermediate value with the other three groups having a concentration about one half that of lots X and V* Lets X, 111 (high copper) and 3V had about twice the


concentration of labeled copper In the gastrocnemius muscle as lot XI (high sine) and lot VI (high sine plus high copper), while lot V had an Intermediate concentration. lots XX. XXX and VI had somewhat higher hide values than did the other three lots. Femur shaft and epiphysis and rib sternal end values varied considerably without showing a definite trend, the lot XV barrow and lot V had a considerably higher concentration of labeled copper in the red bone sorrow than did the other lots.
The higher liver, spleen, heart, and red bone marrow values which the barrow on high molybdenum showed are of interest since they may be indicative of greater copper retention, but more animals would be necessary to establish this. A study of the data for tissue accumulation of labeled copper reveals that in general, the swine seemed to be much more resistant to a modifying effect upon uptake of labeled copper caused by high dietary levels of molybdenum than the rats of Experiment 2 which received the same supplementary level of molybdenum.
The data on alimentary tract distribution and excretion of labeled copper (Table 31) were somewhat erratic, but there did not seem to be any differences caused by the various rations.
By comparing the values for lets Ij, XX and VI (Table 32) it may be seen that the feeding of an additional 1000 parts per million of zinc or 1000 parts per million sine plus 200 parts per million of copper during a period of seven months had very little effect upon the copper and molybdenum concentrations of liver, spleen and kidney, plasma copper level, or femur shaft molybdenum concentration of these swine. The relatively high hide molybdenum value for lot VI may possibly be due to


contamination since the lot VI pen was adjacent to that of lot V (high molybdenum plus high copper) and some of the water used in washing the pens may have drained from let V into lot VI* carrying molybdenum in it which may have come in contact with the hides of these swine* the hide samples were taken from between the shoulders in an attempt to minimise this possibility*
Lot III (high copper) had about a twofold Increase in liver copper, considerably more copper in the hide, and a somewhat higher kidney copper level than the control lot* which Indicated seme cumulative effect of the 208.9 parts per million of copper in the ration of this lot*
Both lot IV (high molybdenum) and lot V (high molybdenum plus high copper) showed a considerable accumulation of molybdenum in the liver* kidneys, spleen and femur shaft after six months on a 0*1 percent molybdenum dietary level* There was an indication that the addition of 200 parts per million of copper to the ration containing 0*1 percent molybdenum may have depressed liver, spleen and kidney molybdenum accumulation, since the values for these tissues for lot V (high molybdenum plus high copper) averaged 32*0, 8*8 and 2k.6 respectively, while for lot IV (high molybdenum) the corresponding values were 66,7, 165 -and 75*2*
The feeding of the high dietary level of molybdenum had a most striking effect upon accumulation of copper in the liver, spleen and kidney of these swine* Lot IV (high molybdenum) showed a threefold increase in liver copper level, and more than a threefold Increase of spleen and kidney copper concentration compared to the levels found in the control lot (lot I) which received the same dietary copper level (8*9 parts


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