THE JOURNAL OF NUTRITION”
OFFICIAL ORGAN OF THE AMERICAN INSTITUTE OF NUTRITION
R i c h a r d H . B a r n e s , E d i t o r Graduate School of Nutrition Cornell University, Savage Hall Ithaca, New York
H a r o l d H . W i l l i a m s E . N e i g e T o d h u n t e r Associate Editor Biographical Editor
e d i t o r i a l b o a r d
G e o r g e M . B r i g g s G e n n a r d M a t r o n e R o s l y n B . A l f i n -S l a t e r
R . M . F o r b e s C l a r a A . S t o r v i c k G e o r g e H . B e a t o n
J u l e s H i r s c h S a m u e l J. F o m o n B . C o n n o r J o h n s o n
H o w e P a u l M . N e w b e r n e W i l l i a m N . P e a r s o n
H e n d e r s o n B o y d L . O ’D e l l F . H . K r a t z e r
H i l l H . E . S ä u b e r l i c h H . N . M u n r o
V O L U M E 9 0
SEPTEMBER - DECEMBER 1966
PUBLISHED MONTHLY BY
THE W ISTAR INSTITUTE OF ANATOMY AND BIOLOGY
PHILADELPHIA, PENNSYLVANIA THE JOURNAL OF NUTRITION® ® Trade mark registered, U. S. Patent Office
Copyright © 1966 by
T h e W i s t a r I n s t i t u t e o f A n a t o m y a n d B i o l o g y
All rights reserved Contents
No. 1 SEPTEM BER 1966
John Bennet Lawes and Joseph Henry Gilbert — Biographical Sketches.
F. ( '. B a w d e n ...... 1
Effect of Age, Vitamin B 6 Deficiency, Isoniazid and Deoxypyridoxine on the Urinary Taurine of the Rat. Nina H. M ercer, Phyllis P. Bowen and Frances A. Johnston ...... 1 3
Effect of Coprophagy on Protein Utilization in the Rat. B. R. Stillings and I.. R. H ackler ...... 1 9
Amino Acid Activation in the Liver of Growing Rats Maintained with N orm al and with Protein-deficient diets. A. M ariani, P. A. M igliaccio, M. A. Spadoni and M. Ticca ...... 2 5
Utilization of D-Amino Acids for Growth by Drosophila m elanogaster L a r v a e . B . W . G e e r ...... 3 1
Mechanism of Suckling Rat Hypercholesterolemia: Dietary and Drug S t u d ie s . R. A. Harris, J. E. M acNintch and F. W . Quackenbush . . 4 0
Effect of Carbohydrate and Fat Intakes upon the Activities of Several Liver Enzymes in Rats, Guinea Piglets, Piglets and Calves. R . L . Baldwin, M. Ronning, C. Radanovics and G. P lan ge ...... 4 7
Dietary Metal-complexing Agents and Zinc Availability in the Rat. Donald Oberleas, M erle E. M uhrer and Boyd L. O’Dell ...... 5 6
Carbohydrate Metabolism and Physical Activity in Rats Fed Diets Con taining Purified Casein Versus a M ixture of Am ino Acids Simulating C a s e i n . Richard A. Ahrens and James E. W ilson, Jr ...... 6 3
Diurnal Rhythms of Tissue Components Related to Protein Metabolism in Norm al and Virus-infected Chicks. Robert L. Squibb ...... 7 1
Copper, Sulfate and M olybdenum Interrelationships in Sheep. R . D . Goodrich and A. D. Tillman ...... 7 6
Correlation of Liver Cytochrom e Oxidase Activity with M itochondrial Cyto chrom e Oxidase and Phospholipid Concentrations in Protein-deficient R a t s . J. N. W illiam s, Jr., R. M. Jacobs and Alice J. Hurlebaus . . . . 81
Effect of Iron on the Growth Rate of Fishes. Martin Roeder and Rachel
/ / . B o e d e r ...... 8 6
Effect of a M ethionine-deficient Diet on Am ino Acid Incorporation in Rat Liver Cell-fractions. E . A . Ke a n ...... 91
Bioassay of Vitam in K in Chicks. John T. M atschiner and E. A. Daisy, Jr. 9 7
Proceedings of the Thirtieth Annual Meeting of the American Institute of Nutrition. Shelburne Hotel, Atlantic City, New Jersey, April 1 2 - 1 6 , 1 9 6 6 ...... 1 0 1 III IV CONTENTS
No. 2 OCTOBER 1966
Influence of Dietary Vitamin A on Carotenoid Utilization, Nitrogen Retention and Energy Utilization by the Chick. P. N. Dua, Elbert J. Day, Henry C. Tipton and James E. H ill ...... 1 1 7
Effects of Dietary M odifications on Response of the Duckling to Aflatoxin. Paul M. Newberne, Gerald N. W ogan and Allen Hall, III ...... 1 2 3
Alterations in Liver Enzyme Activities and Blood and Urine Metabolite Levels during the Onset of Thiamine Deficiency in the Dairy Calf. N. J. Benevenga, R. L. Baldwin and M. Ronning ...... 1 3 1
Comparative Effects of DihydrotachysteroL and DihydrotachysteroL in Chicks. H. Bruce Bosmann and Philip S. Chen, Jr ...... 1 4 1
Effect of an Essential Fatty Acid Deficiency in Rats on the Incorporation in vitro of Palm itate-l-14C and Linoleate-l-14C into Liver Glycero- l i p id s . Kathleen Stitt and Ralph M. Johnson ...... 1 4 8
Cycad Flour Used by Guamanians: Effects Produced in Rats by Long- Term Feeding. M. G. Yang, O. M ickelsen, M. E. Campbell, G. L. Laqueur and J. C. K eresztesy ...... 1 5 3
Protein Requirements of Growing-Finishing Cattle and Lambs. R . L . P r e s t o n ...... 1 5 7
Alterations in the M etabolism of 2- 14C-Thiazole-labeled Thiam ine by the Rat Induced by a High Fat Diet or Thyroxine. M esbaheddin Balaghi a n d W . N . P e a r s o n ...... 1 6 1
Metabolism of a Plant W ax Paraffin (n-Nonacosane) in the Rat. P. E. Kolattukudy and L. Hankin ...... 1 6 7
Requirements for Glucose and Amino Acids in Defined Media for Chick Embryos. R. E. Austic, C. R. Grau and G. C. M atteson ...... 1 7 5
Effect of Dietary Fat, Protein and Cholesterol on Atherosclerosis in Swine. S. A. N. G r e e r , V. W . H a y s , V . C . Speer and J. T. McCall . 1 8 3
Growth Inhibition of Rats Fed Raw Navy Beans (P haseolus vulgaris ). M . L . Kakade and Robert John E va n s ...... 1 9 1
Isolation of a Compound from Alfalfa Lipids that Inhibits Tocopherol Deposition in Chick Tissues. George Olson, W . J. Pudelkiewicz and L. D. M atterson ...... 1 9 9
M etabolizability and Nutritional Im plications of L-Arabinose and D-Xylose for Chicks. P. V. Wagh and P. E. Waibel ...... 2 0 7
No. 3 NOVEMBER 1966
Effect of Protein Depletion on Urinary Nitrogen Excretion in Under nourished Subjects. C. Gopalan and B. S. Narasinga Rao ...... 2 1 3
Effect of Selenium and Vitamin E on the Regeneration of Rat Liver. Tiberius N. Maros, George P. Fodor, Virginia V . Kovacs and Bela K a t o n a i ...... 2 1 9 CONTENTS V
Effect of Different Essential Amino Acid Deficiencies on Amino Acid Pools in Rats. A. J. Clark, Y. Peng and M. E. Swendseid ...... 2 2 8
Digestion of Potato Starch by Larvae of the Flour Beetle, T r i b o l i u m castaneum. Shalom W . A p p l c b a u m ...... 2 3 5
Glucose Milliequivalents Eaten by the Neonatal Pig and Cessation of Intestinal Absorption of Large M olecules (Closure). James G. Lecce 2 4 0
Accentuation of Essential Fatty Acid Deficiency by Dietary Tri-o-cresyl Phosphate. C. R. Seward, G. Vaughan, G. M. Shue and E. L. Hove 2 4 5
Renal Lipid Composition of Choline-deficient Rats. Mona E. Fewster, Joseph F. Nyc and W endell H. G riffith ...... 2 5 2
Uptakes of Zinc, Manganese, Cadmium and Mercury by Intact Strips of Rat Intestine. Benjamin M. Sahagian, I. Harding-Barlow and H. M itchell Perry, Jr ...... 2 5 9
The Conservation of W ater by Young Adult Rats with Restricted W ater Supplies. S. A. Osman, J. D. Smith, L. E. Hanson and R. J. M eade 2 6 8
Dietary Carbohydrates and Possible Pre-lesion Biochem ical Changes in the Aortas of Adult Male Rats. H. F. Sassoon, B. Connor Johnson and Karen Moser ...... 2 7 5
Metabolism of Radioactive Phenylalanine in Rats with Different Dietary Intakes of Phenylalanine. Claude Godin and Gail Dolan 2 8 4
Am ino Acids in Postprandial Gut Contents of Man. William W . Olmsted, E. S. Nasset and M aurice L. Kelley, Jr...... 2 9 1
Utilization of Environm entally Produced High Nitrogen Corn by W eanling Rats and Adult Humans. Shirley Chii-Shya Chen, Hazel M etz Fox, E. R. Peo, Jr., Constance Kies, C. T. Blunn and W . L. C olville ...... 2 9 5
Effect of Vitamin D on 65Zn Absorption, Distribution and Turnover in R a t s . W . M. Becker and W. G. Hoekstra ...... 3 0 1
Effect of Altering the Dietary Cation-Anion Ratio on Food Consumption and Growth of Young Chicks. A. L. Melliere and R. M. F orb es ...... 3 1 0
The Role of Iron in the Copper-Zinc Interrelationship in the Rat. Kenneth E. Kinnamon ...... 3 1 5
Potassium and Creatinine as Indexes of Muscle and Nonmuscle Protein i n R a t s . Kenneth S. K. Chinn ...... 3 2 3
Vitamin K Content of Ground Beef. John T. M atschiner and E. A. Doisy, Jr. 3 3 1
Effects of Zinc Deficiency per se and of Dietary Zinc Level on Urinary and Endogenous Fecal Excretion of 65Zn from a Single Intravenous Dose by Ruminants. W. J. Miller, D. M. Blackmon, G. W . Powell, R. P. Gentry and J. M. Hiers, Jr ...... 3 3 5
T h e J o u r n a l o f N u t r i t i o n G u id e f o r A u t h o r s ...... 3 4 3 VI CONTENTS
No. 4 DECEMBER 1966
Chromatographic Separation and M icrobiologic Assay of Vitamin Be in Tissues from Norm al and Vitam in B6-depleted Rats. V. F. T h i e l e a n d M . B r in ...... 3 4 7
Organ Weights and Water Levels of the Rat following Reduced Food I n t a k e . J. M. Peters and Eldon M. Boyd ...... 3 5 4
Functional and Allometric Descriptions of the Liver and Small Intestine in Genetically Obese Mice. Henry J. Binder, Teodoro Herskovic, Howard M. Spiro and Richard P. Spencer ...... 3 6 1
Arginine-Lysine Antagonism in the Chick and Its Relationship to Dietary C a t i o n s . B. L. O’Dell and J. E. Savage ...... 3 6 4
Effect of Various M ushroom Preparations on Cholesterol Levels in Rats. Takashi Kaneda and Setsuko Tokuda ...... 3 7 1
Effects of Vitamin Bu on the Weights of Certain Organs in the Rat. L . P . Dry den and A . M . H a r t m a n ...... 3 7 7
Relative Concentration of Vitamin B 12 in the Organs of the Male Rat as Affected by its Intake of the Vitamin. L. P. Dryden and A. M. H a r t m a n ...... 3 8 2
Observations on Respiratory Decline in the Pre-necrotic Phase of Dietary Liver Necrosis. E. Bonetti, F. De Stefano and F. S tirp e ...... 3 8 7
In vivo Interactions of Cadmium, Copper, Zinc and Iron in the Mouse a n d R a t . Clara R. Bunn and Gennard Matrone ...... 3 9 5
Changes in Rat Liver Cytochromes b , Ci, and c and M itochondrial Protein in Prolonged Protein Deficiency. J. N. W illiam s, Jr., R. M. Jacobs and Alice J. Hurlebaus ...... 4 0 0
Actinom ycin D Inhibition of Vitamin D- and Dihydrotachysterol-induced Responses in the Chick. H. Bruce Bosmann and Philip S. Chen, Jr. 4 0 5
Determination of the Minimum Dietary Essential Amino Acid-to-Total Nitrogen Ratio for Beef Protein Fed to Young Men. P. C. H u a n g , V. R. Young, B. Cholakos and N. S. Scrimshaw ...... 4 1 6
Effect of Phytate on Iron Absorption in the Rat. J. W . Cozvan, M. Esfahani, J. P. Salji and S. A . A z z a m ...... 4 2 3
Linoleic Acid Requirem ent of the Chick. J. G. Bieri and E. L. P riva l ...... 4 2 8
Influence of the Type of Dietary Saturated Fatty Acid on Lipemia, Coagulation and the Production of Throm bosis in the Rat. S. R e n a u d , C. Allard and J. G. L atour ...... 4 3 3
Effects of M ineral and Vitamin Supplementation on Swim ming Times and Other Parameters Related to Perform ance of Rats on a Low Calorie R e g i m e n . Dirk Tollenaar ...... 4 4 1
Glycogen M etabolism in M eal-fed Rats and Chicks and the Tim e Sequence of Lipogenic and Enzymatic Adaptive Changes. Gilbert A. Leveille 4 4 9
E r r a t u m ...... 4 6 1
T h e J o u r n a l o f N u t r i t i o n G u id e f o r A u t h o r s ...... 4 6 3
I n d e x t o V o l u m e 9 0 ...... 4 6 7 J o h n B e n n e t L a w e s J o s e p h H e n r y G i l b e r t John Bennet Lawes
(Deceviber 28, 1814 — August 31, 1900)
Joseph Henry Gilbert
(August 1, 1817 — December 23, 1901)
— Biographical Sketches
In recalling for the Journal of Nutrition Lawes had already revealed his unex something of the lives and achievements pected talents and interests before 1843, of Lawes and Gilbert, it is wholly fitting when Gilbert joined him, but how far to do so in a single biography for although these talents would have taken him in his they were very dissimilar, unalike in up scientific enquiries without Gilbert, it is im bringing, temperament and outlook, they possible to guess. Gilbert’s career is the were united by their devotion to scientific more easily understood. A studious child, enquiry, and their contribution to knowl he went to the university to study chem edge of the nutrition of plants and animals istry and having done so, when Lawes was truly a joint enterprise. Factually needed a chemist, it was reasonable for one was the employer of the other, but him to take the post. What remains in this is not the relationship evident from explicable is why Lawes with his upbring their many publications, which show ing should have developed an interest in them as scientific colleagues collaborat research and wanted to em ploy a chemist. ing as equals, which they did productively It is idle to speculate on the reasons be and harmoniously for the remarkable cause he also was at a loss him self to ex period of more than half a century. plain his interests, as this revealing quota The interests and abilities of the one tion shows: “As I had no male relations, perfectly com plem ented those of the other. my mother was the only person to in Lawes was essentially a practical man, fluence me in my pursuits, and she was but a very unusual one in being a non violently opposed both to science or busi conformist with an enquiring mind and ness, although at the same time devoted great insight, who was imbued with the to me. Home influence and education all desire that agricultural practices should tended to make me in pursuits an ordin be based on established principles instead ary country gentleman, in politics a con of untested traditions. Gilbert was more servative, in religion an ordinary m ember the academic chemist, meticulous in all of the Church of England accepting as he did, cautious and painstaking, m ethod truth all that they teach. Whereas for ically analyzing and measuring everything some causes to be enquired into and ex to produce the mass of facts and figures plained, I have been largely engaged in he needed before he would commit him manufacturing pursuits, devoted to sci self to any conclusion. The broad concept entific investigation, very liberal in poli of the field experiments that first made tics, and in religion although firmly and Rothamsted fam ous was Lawes’, but their thoroughly believing in the truth of the long continuance and the detailed infor Christian religion and ready to accept it mation they provided over the years re as the guide of my life as far as I can flects the influence of Gilbert. understand it and being at the same time
J. N u t r it io n , 90: ’ 66 3 4 JOHN BENNET LAWES AND JOSEPH HENRY GILBERT a regular attendant at the service of the to set up as a country gentleman. I had Church of England, still I cannot admit no idea or wishes about farming, but the the right of that church or of any other home farm was vacant and therefore I church to teach dogmatically what truths took it. Up to this period,’ I had formed are necessary for my salvation.” From no opinions of my own on any subject.” this, let us look briefly at his early life, This statement we must accept and there knowing that we shall find little in it to is certainly no evidence that he was then account for his scientific curiosity, radical attracted by agriculture; that he had de ideas or business acumen. cided to dabble in chemistry, however, is obvious, for as soon as he arrived at BEFORE 1843 Rothamsted he had one of the best bed John Bennet Lawes was born on 28 room s fitted out as a laboratory, where he December 1814 in the Rothamsted Manor started experim enting, and he spent m uch House, Harpenden, which his ancestors time reading books on chemistry. The had occupied for many generations. His writings of A. T. Thomson, the first pro father, whose friendship with the Prince fessor of M ateria M edica and Therapeutics Regent had proved very costly, died when at University College, London, impressed he was only eight, and his family then Lawes, who sowed many kinds of drug moved into Rothamsted Lodge, which was plants on the farm and extracted from their hom e for several years. After attend them opium and other active principles. ing two preparatory schools where, he He also worked for a time in Thom son’s says, he learned very little and was always laboratory and while there showed his first in mischief and disgrace, he went to interest in comm ercial enterprises; after Eton, where “I learnt just enough to es persuading Thomson to patent a process cape punishment but no more” and “most for making calomel and corrosive subli of my pursuits were more or less mis mate, he turned an old barn on his farm chievous, such as digging m ice out of the into a factory where he made many tons fields and putting them into my tutor’s of these substances. This venture was a house.” He left without regret and went commercial failure, but gave Lawes ex to Oxford University, where he stayed perience that proved useful to him later. for two years “learning little and follow Lawes seems first to have seen a con ing no particular pursuits. I did not go nection between chem istry and agriculture up for my degree.” Although at that time in 1837 from a chance remark to him by Oxford had little to offer a man whose Lord Dacre that bones were useful as a interests were scientific rather than clas manure for turnips on some fields but sical, Lawes did attend some lectures by useless on others. This particularly the Professor of Chemistry and he may roused his interest because he had spent have learnt of the plans for experiments a good deal of money on bones without in the Botanic Gardens to compare crops seeing any reward from applying them grown continuously with the same ones to his fields. At about this time also he grown in rotation. W hile a school boy he was offered free a lot of spent animal showed some interest in chemistry, for charcoal, which he treated with the sul during one of his holidays in Paris he phuric acid he had for making chlorine not only helped to build the barricades and found that the product was an effec during the revolution of 1830, which he tive m anure. thought “great fun,” but also bought vari During the next few years he did many ous chemicals, which “caused such de experim ents with plants in pots and in his struction to m y clothes and the furniture fields, testing the effects of bones, burnt that m y m other got rid of them as quickly bones and mineral phosphates decom posed as possible.” by various acids. This was the birth of W hen Lawes left Oxford at the age of superphosphate, and by 1842 Lawes was 20, the tenant of Rothamsted was insol well enough convinced of its value to take vent, so he and his mother again lived out a patent for it. In 1842 he also mar there. He says, “My education was there ried Caroline Fountaine, an amateur fore supposed to have finished and I was artist of considerable ability. Their son BIOGRAPHICAL SKETCHES 5
Charles, who became well known as an analytical chemistry, but also m a t e r i a athlete and sculptor, was born in 1843 m e d i c a and botany. He learned German as and their daughter Caroline in 1844. a prelim inary to going in 1840 to Giessen, Much against the wishes of his own the then Mecca of chemistry, to work family and his wife, who did not approve under Liebig, with whom he was later to of him entering any trade, but least of disagree so vehemently over the nitrogen all the manure trade, in 1843 he opened nutrition of plants. After taking his Ph.D. a factory to make superphosphate, and degree at Giessen, he returned for a brief so was born the fertilizer industry. His second spell at University College, London, family’s fears that the venture would before he went to Manchester to work on ruin him proved unfounded, for the busi the dyeing and printing of calico. While ness prospered and despite costly lawsuits there he was recommended by Thomson with other manufacturers who infringed to Lawes and in June 1843 moved to his patents, he was able to sell it in 1882 Rothamsted, where he worked until he to the Lawes Chemical Manure Company died in 1901. In 1850, he married Eliza for £300,000. However, at first he had to Forbes Laurie, but she died two years practice strict econom y and for four years later, and in 1855 he m arried M aria Smith, let Rothamsted Manor; it says much for who survived him. He had no children his enthusiasm for research that, with his by either marriage. factory making such demands on his Unlike Lawes, who had many other ac capital, he should nevertheless have paid tivities than agricultural research, Gil Gilbert to conduct the experiments at bert devoted his whole life and energies Rothamsted. to this. He not only supervised the con From 1843 Lawes in effect led two lives. duct of the Rothamsted experiments and During part of the week he was a manu all the chemistry done in connection with facturer, running not only an expanding them, but he also was their main expo fertilizer business, but also factories to nent, both in the written and spoken word. produce citric and tartaric acids. A later Both men were equally willing to demon venture that did not prosper was the grow strate the experiments to visitors but ing of sugar cane, and sugar processing Lawes was less willing than Gilbert to in Australia. During the rest of the week address scientific m eetings or attend social he collaborated with Gilbert in agricultural f u n c t i o n s . research, the activity in which he found the greater satisfaction and to which he 1843 ONWARDS: CROP NUTRITION gave increasingly more of his time as the We have already seen that Lawes had years went by. started to experiment on crop nutrition Joseph Henry Gilbert was born at Hull, before Gilbert joined him. Also, as the Yorkshire, on 1 August 1817. His father first of the large-scale field trials, with was an eminent Congregational minister roots on Barnfield, was laid down in the and his mother was widely known as a spring of 1843, it is unlikely Gilbert helped writer of hym ns and songs for children; of in its design. However, with Gilbert’s ar their four sons and three daughters, he was rival, which is taken as the foundation the only one to become a scientist. His date of Rothamsted Experimental Sta schooling at Nottingham and Mansfield tion, the scope of the work greatly in was interrupted by a shooting accident, creased and its type changed. Results which destroyed the sight of one eye, were no longer simply observed and yields damaged the other and impaired his gen weighed, but everything was now care eral health for some years. He later tri fully measured and analyzed. How this umphed over these disabilities, but they was done with such accuracy in the old delayed his education and he was 24 years barn that served as a laboratory until old before he went to the university, first 1855 is difficult to imagine. for a year at Glasgow and then to Uni The autumn of 1843 saw the start of versity College, London, where in the lab their most famous field experiment, with oratory of A. T. Thomson he first met winter wheat on Broadbalk field, which Lawes. At both places he mainly studied still continues. This, although of different 6 JOHN BENNET LAWES AND JOSEPH HENRY GILBERT design, was of the same general pattern done that established the greater benefits as with the root crop on Barnfield and was from nitrogenous fertilizers. later followed with other m ain agricultural How strongly Lawes felt early on in the crops. The same crop was grown year controversy will be shown by a quotation. after year and usually each plot was given This, too, serves well to indicate his phi the same treatment every year. The losophy and practical approach to re main treatments were no manure; farm search. “The theory advanced by Liebig, yard manure; nitrogen only; minerals that ‘the crops on a field diminish or in only; minerals plus nitrogen. But there crease in exact proportion to the dim inu were additional treatments, testing differ tion or increase of the mineral substances ent kinds and amounts of nitrogen, dif conveyed to it in m anure,’ is calculated so ferent times of applying it, and various seriously to m islead the agriculturist that combinations of inorganic materials. In it is highly important that its fallacies addition to the plots given the same treat should be generally known. The contempt ment annually, Broadbalk contained two which the practical farmer feels for the plots in which the treatments alternated, science of agricultural chemistry arises one getting nitrogen only in the year when from the errors which have been com the other got minerals only, with the pro mitted by its professors. They have en cedure reversed the next year. A unique deavored to account for, and sometimes to feature of the Broadbalk experiment was pronounce as erroneous, the knowledge the installation of drains, one to each plot, which ages of experience have established; to allow the losses of nutrients by leach and they have attempted to generalize ing into the subsoil to be measured. without the practical data necessary to The m ain question that interested Lawes accomplish their end with success. Agri and Gilbert was the relative importance culture will eventually derive the most for the growth of crops of nitrogenous important assistance from chemistry, but manures and minerals, i.e., the constitu before it can propose any changes in the ents of the ash of crops, mostly com established routine of the farm er, it must, pounds of phosphorus, potassium, sodium by a series of laborious and costly ex and magnesium. In seeking to answer this periments, explain this routine in a satis question, they were stimulated not only factory manner. by its practical importance for farming, “Although the experimental results but by Liebig’s assertion that crops could which have been detailed undoubtedly get all the nitrogen they needed from prove that to produce agricultural crops ammonia in the air and to yield, fully of corn, nitrogen must be supplied to the needed to be m anured with minerals only. soil in some form or other, two important The Rothamsted experiments, particularly questions still rem ain unanswered, nam ely, with cereals, soon showed the fallacy of first, what amount of ammonia will be re Liebig’s mineral theory, for yields were quired to produce a given amount of corn? small without organic or inorganic nitro or, in other words, what amount of nitro genous manures and minerals produced gen must the farm er accum ulate in his soil appreciable effects only when nitrogen to obtain each bushel of corn beyond the was also given. However, Liebig was natural produce? Secondly, what are the unconvinced and his adverse criticisms most economical means at his disposal of the Rothamsted experiments led to for securing the necessary supply? The controversy, which became increasingly solution of these questions is within the heated as he minimized the importance reach of careful experiment and calcula of nitrogen while Lawes and Gilbert pro t i o n . ” duced more and more evidence of its Although Liebig questioned the work at paramount importance except with leg Rothamsted, farmers were rapidly im pres umes. There is, indeed, a touch of irony sed by its value and showed their ap in the fact that Rothamsted work began preciation by subscribing to a testimonial by demonstrating the value of superphos fund to Lawes which was used to build in phate, from which Lawes mainly made 1855 the first Rothamsted laboratory. At his fortune, whereas later so much was its opening, Lawes paid public tribute to BIOGRAPHICAL SKETCHES 7
Gilbert, saying: “To Dr. Gilbert I consider alternating dressings of nitrogen and a debt of gratitude is due from myself minerals showed how transient was the and from every agriculturist in Great effect of inorganic nitrogen, for although Britain. It is not every gentleman of his yields were large in the years when nitro attainments who would subject himself gen was given, in the alternate years the to the caprice of an individual, or risk plots yielded little more than those that his reputation by following the pursuit of never received nitrogen. A still more im a science which has hardly a recognized portant feature of Broadbalk was the dem existence. For twelve years our acquain onstration that yields could be as large tance has existed, and I hope twelve more with inorganic as with organic manures. years will find it continuing.” It was on It is difficult now to recapture the reac this occasion that Lawes first mentioned tions at the time to being told that a few his intention to provide for the mainten hundredweights of powder from a factory ance of the work after his death. He im could produce the same results as many plemented this intention by setting aside tons of farmyard manure, but there were from the sale of his manure business the results for all to see and to convince £100,000, which in 1889 he transferred the disbelievers. Although there are a few together with the buildings to the Lawes people who still attribute almost mystical Trustees. Under the Trust Deed a man value to organic manures, later work has agement committee was set up with m em done nothing except strengthen Lawes’ bers appointed by the Royal Society of and Gilbert’s conclusions that the nutri London, the Royal Agricultural Society, tive value of organic manures lies solely the Linnean Society and the Chemical in their content of nitrogen and minerals, Society. This committee continues to be and that any other effects on crop growth the governing body of Rothamsted Experi are indirect, by changing soil structure m ental Station. or the water-holding capacity of the soil. Lawes’ hope for the continuation of his The ability of nitrogen to increase leaf association with Gilbert was m ore than ful growth was plainly evident without filled, for they worked together for an m easurem ent, but Gilbert’s detailed chem i other 45 years after 1855, steadily amas cal analyses also provided much new in sing valuable information about such form ation on the way it affected leaf con various problems as the relative needs of stitution, not only increasing the content different crops for different nutrients of nitrogenous substances, but also of (“for the production of increased growth, other nutrients and of sugars and starch. nitrogenous manures h a d t h e m o s t c h a r They further showed that these large ef a c t e r i s t i c effect upon the cereals; p o t a s s fects on the constitution of leaves was not o n t h e leguminous crops; a n d p h o s p h a t e s reflected in the constitution of the grain, on turnips”); the effects of different nu for although wheat yields were much trients on yield and quality of crop; the increased by nitrogenous m anure, the com interactions between different nutrients; position of grain on plots with and with the different amounts of nutrients taken out nitrogen differed little. From the con from the land by different crops; the in tinuation of the experiments over many teraction between different crops grown years, Lawes and Gilbert also noted that in rotation; the effect of fallow and green- responses to the same manuring varied manure crops on yields of subsequent greatly, with the results depending m uch crops; the effects of different manurial on the weather. In attributing all the regimens on the supply of nutrients in effects of weather on crop growth directly t h e s o il. to nutrition, they were in error, for many From Broadbalk they were able to com pile reflect effects on the incidence of pests a balance sheet showing what happened and diseases, but they drew some shrewd to applied manures, how much came off conclusions. For example, they noted how in the wheat grain and straw, how much nitrogen was leached from the soil during was retained in the soil and how much wet winters and the need to allow for was lost in the drainage. Nitrogen was this in manuring, something many far the m ain nutrient lost, and the plots given mers still fail to do. 8 JOHN BENNET LAWES AND JOSEPH HENRY GILBERT
Their experiment on old pasture, which were grown in burnt soil and kept in an like the one with wheat on Broadbalk enclosed system to prevent any external still continues, was outstanding, not forms of combined nitrogen reaching simply for showing how manuring af them. Everything was analysed in detail fected the yield of hay, but much more and the results showed clearly that neither so for its dramatic demonstration of how legumes nor other plants gained nitrogen differential manuring changed the com from the air. Indeed, in these circum position of the sward. Park Grass ori stances legumes grew less well than other ginally had a rich flora of grasses, plants. The experiments were magnif legumes and weeds, as the unmanured icently done but the precautions taken to plot still has. Fertilizers rapidly altered make the results reliable chem ically made the proportions of these three compo them irrelevant to what happens in field nents; nitrogen suppressed the legumes soils; by burning the soil before use, they and weeds, whereas potash and phos killed any R h i z o b i u m sp. and so not only phate without nitrogen increased the leg destroyed their chances of discovering the umes. Ammonium sulphate soon pro m ain natural source of com bined nitrogen duced a sward that was almost wholly grass, but the species depended on whether in the soil, but set themselves off on m any it was given alone or with phosphate and/ fruitless searches for other explanations or potash, and the extent to which it of the action of legum inous crops. Gilbert acidified the soil. For example, below pH was present at the meeting in 1886 when 4 . 1 , Agrostis tenuis dominated where only Hellriegel and W illgarth reported the sym nitrogen was given, but Holcus lanatus biotic association between R h i z o b i u m a n d where potash and phosphate were added; legumes that leads to nitrogen fixation
between pH 4 and 6 , there were m ore spe in the root nodules. It says much for his cies, with Alopecurus pratensis t h e m o s t receptive m ind and energy that, although common given full manuring but sup over 70, he immediately began a series of planted by Festuca rubra on plots lacking experiments that not only confirmed but
phosphate and potash; above pH 6 , t h e extended the results of Hellriegel and sward was still more mixed and no spe W il l g a r t h . cies dominated, but A. p r a t e n s i s a g a i n Although most of Lawes’ and Gilbert’s was on plots given phosphate and potash. work was done at Rothamsted, they In denying Liebig’s “minerals only” played a leading role in designing experi theory and in their insistence on the para ments at Woburn, where in 1876 the mount importance to crops of nitrogen Royal Agricultural Society of England es m anuring, LawTes and Gilbert were guided tablished a research station, which is solely by the results of their experiments now a part of Rothamsted. The central and observations. They well knew that problem there was to measure the residual the growing of legumes left a residue of value of m anures and of food fed to cattle, nitrogen in the soil for succeeding crops, so that outgoing tenants could be properly and they were not prejudiced against the compensated, but Lawes and Gilbert also idea that plants m ight assimilate nitrogen took the opportunity of duplicating on the from the air. Indeed, when writing in light land there m any of the experiments 1847 about sources of ammonia, Lawes done on the heavy land at Rothamsted. said “by cultivating turnips and the leg At the request of the Government they uminous plants, a large amount of this also undertook a m ajor series of experi substance is collected by them from the ments in which they assessed the ma- atmosphere.” nurial value of sewage. Here it is pos The explanation of the extra nitrogen sible only to indicate the scope of their gained by legum inous plants eluded them, work, and impossible to summarise the but they abandoned the idea of assimila detailed results on crop nutrition they re
tion from the air because of the results ported in more than 1 0 0 publications, of critical experiments done, in conjunc mostly in the Journal of the Royal Ag tion with a visiting American scientist, ricultural Society a n d t h e Philosophical Evan Pugh, on plants in pots. The plants Transactions of the Royal Society. BIOGRAPHICAL SKETCHES 9
ANIMAL NUTRITION enous materials. They advanced various The work of Lawes and Gilbert on ani reasons in support of their ideas, but re mal nutrition is almost as notable as garded as conclusive the results of their their work with crops and fertilizers, and experiments in which pigs fed very differ would have ensured them a place in the ent amounts of protein excreted nitrogen history of science had they done nothing roughly in proportion to the amount they else. There are several reasons why it is were fed and quite independently of their less widely known than their work on m uscular activity. crop nutrition. First, although they stud Lawes and Gilbert were also the first to ied animal nutrition during many years, show that not all proteins were of equal the subject was a m ajor activity only in nutritive value. This they did by feeding the middle years of their collaboration pigs on either lentil meal containing 4% and was not continued at Rotham sted after protein or barley meal containing 2 % . their deaths. Secondly, the results could From the total food eaten, they calculated be obtained only from their published the nitrogen intake and, after the pigs papers, in records of weights and analyti had been on the different diets for a while, cal measurements, not as in the fertilizer experiments where large effects were they measured the amount of nitrogen clearly demonstrated simply by looking at excreted. The pig given lentil meal ex the growing crops. Thirdly, although their creted more than twice as much urea as work produced much new information the one given barley meal, showing that and, equally important, destroyed several the proportions of the total nitrogen in the myths, it did not have the im mediate prac two foods retained and converted into tical consequences of their work on crop pig meat differed greatly. The significance n u t r i t i o n . of their conclusion, however, for long went Their work started in 1848 when they unappreciated by nutritionists, who con compared the fattening capacity of dif tinued to assume that all proteins were ferent breeds of sheep in normal farm ing e q u a l. conditions, by m easuring the ratio of food As with the wheat on Broadbalk field, eaten to live-weight increase, but it soon so with the animals, Lawes and Gilbert at extended to more thorough work, not only tempted to compile a balance sheet, show with sheep, but also with pigs and oxen. ing the fate of food eaten, whether it was At this time the relative importance and excreted or retained in the body and when roles of different constituents of food for retained in what form and whether it was animals was unestablished and a subject used to sustain the animal or to add to of controversy. However, it was widely its weight. This work entailed enormous accepted that the most important factor numbers of dry-matter measurements and was the amount of nitrogenous substances determinations of the ash and nitrogen and that there was need to know only in the food, feces and urine. Probably their this to assess the relative value of differ most laborious piece of work, however, ent foods. Fat in the animal was assumed was to determine the com position of whole by most people to com e from fat or nitrog bodies of animals of different ages and enous substances in the food; in the con in different conditions of fatness. They troversy over this, Lawes and Gilbert separated and weighed the amounts of found themselves on the side of Liebig, different organs or parts in 2 c a l v e s , 2 and their experiments on the fattening of heifers, 14 bullocks, 1 lamb, 249 sheep pigs produced the evidence to support his and 59 pigs. In 1 calf, 2 bullocks, 1 lamb. assertion that fat in the animal body can 4 sheep and 2 pigs, they also analyzed be synthesized from carbohydrate in the each part and organ to find the proportions food. However, in another controversial of water, minerals, fat and nitrogenous subject, the source of energy for muscu substances. This work not only provided lar effort, they were again in conflict with the first factual information on the com Liebig, who maintained that it came position of farm animals and how their from m uscle substance, whereas they con composition changes with age and with sidered it came mainly from non-nitrog degree of fatness, but their results for 10 JOHN EENNET LAWES AND JOSEPH HENRY GILBERT long remained the standard textbook fect of malting on the nutritive value of fi g u r e s . barley. Malt was generally believed to be Interested as they were in both crops the more nutritious, but they disproved and animals, their work extended beyond this by measuring the loss of dry matter the effects of various animal foods on the during malting and showing that, per unit growth and composition of the animals to of dry matter, malted and unm alted barley their effects on the manurial value of the had the same food value. Their at animal’s excreta. They concluded that, tention to detail and desire to put every for the fattening of cattle, provided the thing to the test is well evidenced by their diet was not deficient in nitrogenous sub experiments with condiments, showing stances, richness in digestible carbohy these added nothing to the nutritive value drates was the most important, whereas o f f o o d . for the manure to be valuable the diet needed to be rich in nitrogen. Their wide- THE JUBILEE ranging interests are shown in Bulletin Neither Lawes nor Gilbert sought hon no. 22 of the U.S. Department of Agricul ours but severally and jointly they received ture, published in 1895, where Gilbert them in quantity. Lawes was created a summarized the main points of their work Baronet and Gilbert received a Knight with animals under the following seven hood. Each was given an honorary degree h e a d i n g s : from several universities and was made “(1) The amount of food and of its an honorary member of many academies. several constituents consum ed in relation Each was elected to Fellowship of the to a given live weight of animal within a Royal Society and jointly they were given time. awarded a Royal Medal. The Royal So (2) The amount of food and of its ciety of Arts also jointly awarded them several constituents consum ed to produce its greatest honor, the Albert Gold Medal. a given amount of increase in live weight. A unique occasion was the Jubilee Cele (3) The proportion and relative de bration, held at Rothamsted on 29 July velopm ent of the different organs or parts 1893, when a very distinguished com pany, of different animals. headed by the President of the Board of (4) The proximate and ultimate com Agriculture, gathered to do them honor. position of the animals in different con Unlike the occasion in 1855, when the ditions as to age and fatness, and the Testim onial Laboratory was opened and it probable composition of their increase in was left to Lawes to pay tribute to Gilbert, live weight during the fattening process. this one honored them jointly. Lawes (5) The composition of the solid and was presented with his portrait, Gilbert liquid excreta (the m anure) in relation to with a silver salver, and congratulatory that of the food consumed. addresses were read from many learned ( 6 ) The loss of expenditure of constit societies. The main testimonial took the uents by respiration and the cutaneous ex form of a granite monolith, inscribed “To halations; that is, in the mere sustenance com m em orate the com pletion of fifty years of the living meat-and-manure-making of continuous experiments (the first of m a c h i n e . their kind) in agriculture conducted at (7 ) The yield of milk in relation to the Rothamsted by Sir John Bennet Lawes and food consumed to produce it, and the in Joseph Henry Gilbert.” fluence of different descriptions of food From the address to Lawes, signed by on the quantity and on the composition the Prince of W ales on behalf of subscri of the m ilk.” bers to the Jubilee Fund, who came from Although impressive, this list of their the world over, we may fittingly quote a interests is far from complete. It needs paragraph: “The Memorial which is now supplementing at least to the extent of erected, will, it is hoped, preserve your saying they also compared the feeding joint names in honored remembrance for value of hay and silage, and, at the request centuries to com e, while the portrait will of the Government, they studied the ef hand down to future generations the like BIOGRAPHICAL SKETCHES 11 ness of one of the most disinterested as were published in 1895 as Bulletin no. 22 well as the most scientific of our public of the United States Department of Ag benefactors.” And from his address to Gil riculture. M ajor H. E. Alvord, then chair bert: “If the institution of the various in man of the executive committee of the As vestigations and experiments carried out sociation of American Agricultural Col at Rothamsted has been due to Sir John leges and Experiment Stations, wrote: Lawes, their ultimate success has been in “The lectures com prise the only condensed, a great measure secured by your scien carefully prepared, and authorized review tific skill and unrem itting industry. of the fam ous investigations by Lawes and “A collaboration such as yours with Sir Gilbert for half a century at Rothamsted. John Lawes, already extending over a They constitute an extrem ely valuable and period of upwards of fifty years, is unex truly unique contribution to the literature ampled in the annals of science. I ven of experimental agriculture.” M ajor Al- ture to hope for an extended prolongation vord’s use of the word “condensed” was of these joint labors, and trust that the correct but could be misleading; the Bul names of Lawes and Gilbert, which for so letin runs to 316 pages of not very large m any years have been almost inseparable, print and has 85 tables; its preparation may survive in happy conjunction for was no mean feat for a man nearing 78, centuries to com e.” and the manner of his writing shows no Lawes and Gilbert could not have had falling off in vigour or delight in contro such full and active lives had they not v e r s y . been unusually hale. W riting of Lawes, his Both men remained active till the end colleague R. W arington said: “W hen past of their lives. Lawes died on 31 August 85 he still exhibited few of the infirmities 1900, after a brief illness. The end of of old age;” and of his interests and per their long association was a great blow sonality, W arington said: “He was a keen to Gilbert and although, with his charac observer and knew the experim ental fields teristic perseverance, he kept the Rotham better than any of the Rotham sted workers. sted experiments going for another year, Not the fields only, but the birds and every his health then failed and he died on 23 living thing on the estate. The large December 1901. Like Lawes, he was amount of business he was able to get buried in the churchyard at Harpenden. through was in no small degree due to Their unique partnership did more his calm and cheerful temperament, which than simply produce new information on no disaster seemed to disturb. This quiet, the nutrition of crops and animals. It self-contained temperament sometimes ap revolutionized the manuring of crops and peared as reserve or even shyness, and led it set a tradition for accuracy in agricul to a reluctance to accept public positions tural research. They were modest about and to take part in public functions; but their achievem ents, realising not only the his work doubtless gained by his refusal many problems that remained unsolved, to expend his energy on outside occupa but that other methods than theirs would tions. The reserve we have m entioned was, be needed to solve them. Let Lawes have however, a m ood rather than a character, the last word; writing in 1888 about Lie and disappeared the instant he was ap big’s book published in 1840, on Agricul pealed to by any scientific or benevolent tural Chemistry, he said: “Nearly fifty question. To speak to him of agricultural years have passed since that book was science would at once open the storehouse written. It was a bold work; and for some of thought and lead to a discourse of years afterward everyone could give con ready eloquence, interspersed with shrewd fident opinions upon all subjects relating observations and humorous remarks.” to agriculture — but where are we now? Gilbert, too, showed few infirmities, Have we a foundation laid, and can we for soon after the Jubilee Celebrations he say that such a thing exists as a science visited the U.S.A. to give lectures under of agriculture? Another half-century will the provision made by Lawes in his Trust doubtless show more rapid progress, as Deed for the purpose. His six lectures there are so m any more brains at work on 12 JOHN BENNET LAWES AND JOSEPH HENRY GILBERT the subject in various parts of the world; plenty of hard work and be content with but when we consider that almost every moderate amount of success.” other science contributes its share to form F. C. B a w d e n , M.A., F.R.S .,D irector what we call the science of agriculture, Rothamsted Experimental Station those who follow the pursuit must expect Harpenden, Herts, England Effect of Age, Vitamin B6 Deficiency, Isoniazid and Deoxypyridoxine on the Urinary Taurine of the Rat
NINA H. MERCER,* 1 PHYLLIS P. BOWEN a n d FRANCES A. JOHNSTON Department of Food and Nutrition, New York State College of Home Economics, Cornell University, Ithaca, New York
ABSTRACT The object of the present study was to find the effects of isoniazid (INH) and deoxypyridoxine (DOP) as well as a vitamin B6 deficiency on urinary taurine excretion. Groups of male rats 38 to 47 days of age were fed 5 diets: adequate in vitamin B6, adequate in vitamin B6 with INH added, deficient in vitamin B6, and deficient in vitamin B6 with INH or DOP added. The urinary taurine of male rats fed adequate vitamin B6 remained at about 2m g/100g body weight/day up to 50 to 55 days of age, then increased rapidly to about 7 mg at 75 to 95 days of age. The urinary taurine of rats fed diets deficient in vitamin Be remained at 1 to 2 mg over a 7-week period. The urinary taurine of rats fed diets adequate in vitamin Be plus INH and of those fed diets inadequate in vitamin B6 plus DOP was significantly higher than that of the controls; the taurine also tended to be higher when rats were fed vitamin Be- deficient diets plus INH. The results are contrary to expectation in view of the known inhibitory effects of the 2 compounds on vitamin B6.
The object of the present study was to tion containing no pyridoxine , 4 2 .2 ; a n d learn whether urinary taurine can be used enough sucrose to make 1 0 0 ; thus, the to follow the development of vitamin B 6 basal diet was adequate except for vitamin inhibition caused by isoniazid (IN H ) or B6. In experiment 1, 4 mg pyridoxine- deoxypyridoxine (DOP). Urinary taurine HCl/kg of food were fed to the group proved to be useless for this purpose. Ob which was to have adequate vitamin B 0 servations of value to persons working on but in the other experiments when pyri taurine metabolism, however, were made, doxine -HC1 was added only 2 mg were nam ely: urinary taurine increased with age incorporated to provide adequacy. In ex in male rats fed diets adequate in vitamin periment 1 the group of rats given INH B6, but, for deficient rats, urinary taurine was fed 1.5 g of IN H /kg of food but in the did not increase with age and continued at other experiments 1 g was used. In ex the sam e low level for 7 weeks; and urinary periment 1 the group given DOP was fed taurine excretion increased with the ad 100 mg of DOP/kg of diet; in experiment ministration of the vitamin Bs-inhibiting 5, the amount was increased to 1 g. compounds, INH and DOP. In all 5 experiments the animals were pair-fed to the animal which ate the least. METHODS In experiments 1 and 2, in which the rats Five experim ents were carried out using were arranged in groups of four with each
60, 24, 24, 24 and 16 male rats of the Received for publication December 15, 1965. Holtzman strain, of ages 46, 42, 39, 38 1 Present address: Department of Biochem istry, Uni versity of Alberta, Edmonton, Alberta, Canada. and 47 days, respectively; the mean 2 The casein preparation contained 0.63 m /ig of vita weights on the corresponding days were m in B6/g. The basal diet therefore contained 0.113 mg vitamin Be/kg. 144, 131, 123, 128 and 151 g. In each 3 Salt mixture USP XIV, Nutritional Biochemicals Corporation, Cleveland. experim ent the rats were paired by weight 4 The vitamin mixture supplied in each 45.5 kg when they were divided into groups for (100 pounds) of diet: (in grams) vitamin A (200,000 units/g), 4.5; vitam in D cone (400,000 units/g), 0.25; different treatments. a-tocopherol, 5.0; ascorbic acid, 45.0; inositol, 5.0; choline chloride, 75; riboflavin, 1.0; menadione, 2.25; The basal diet contained: (in per cent) p-aminobenzoic acid, 5.0; niacin, 4.5; thiamine-HC1, 1.0; Ca pantothenate, 3.0; and (in m illigram s) biotin, “vitamin-free” casein ,2 18; glycine, 1 ; 20; folic acid, 90; and vitamin B 12, 1.35. (Vitamin methionine, 0.2; corn oil, 5; salt mix Diet Fortification Mixture in Dextrose, with pyri doxine HC1 omitted, obtained from Nutritional Bio U S P X I V ,3 4; agar, 2; a vitamin prepara chem icals Corporation.)
J. N u t r it io n , 90: '66 13 14 NINA H. MERCER, PHYLLIS P. BOWEN AND FRANCES A. JOHNSTON rat in the group fed a different diet, 3 ani group of rats not receiving pyridoxine mals were pair-fed to the fourth. The HCl. The experiment was continued for mean daily food consumptions per rat 7 weeks following the preliminary week. were 8.5, 9.8, 12.0, 9.2 and 9.8 g, re Taurine was determined in the prelimi spectively. nary week and in weeks 2, 4 and 7. The rats were housed in metabolism Experiment 3. Twenty-four rats were cages. Urine was collected each day, fil fed the basal diet plus pyridoxine-HCl; 12 tered and com bined into 3-day composites of them were also given INH. Urine was for each rat except in experiment 4 when collected for two 3-day periods starting cn the urine of 3 rats was composited each the tenth and seventeenth days. day. The composites were stored in a Experiment 4. Twenty-four rats were freezer. All values for taurine are ex fed the basal diet; INH was added to the pressed as a daily mean for one rat over diets of half of the animals. Urine was a 3-day period except for experiment 4 collected on days 12, 13 and 14. where the values are a daily mean per rat Experiment 5. Sixteen rats were fed for 3 rats. The procedure for the deter the basal diet; DOP was added to the diets mination of taurine in the urine was an of half the animals. They were main
adaptation by Mercer 5 of the method of tained with the diet for 3 weeks. Urine Sorbo (1 ) with a substitution of the ion- was collected for 3 days during each week. exchange column for one described by RESULTS AND DISCUSSION G a r v i n (2). By this procedure amino acids present in the urine because of food Effect of age on urinary taurine. W h e n spillage were removed. The method in male rats 38 to 47 days of age were fed volved the selected passage of the zwit- diets adequate in vitamin B 6 (exps. 1, 2 terion of taurine through an anion and a and 3) urinary taurine, in general, re cation exchange resin and quantitative mained at about 2 m g/24 hours/100 g spectrophometric estimation of taurine in body weight until 50 to 55 days of age;
the effluent utilizing the color reaction the range was from 1.40 ± 0.36 mg 6 t o
between taurine and ninhydrin. 2 . 0 6 m g 7 (table 1). The amounts in Experiment 1. Sixty rats were distrib creased with age from 3.75 ± 0.30 mg to uted equally among 4 groups. One group 7.08 ± 0.65 mg for rats 56 to 58 days and was fed the basal vitam in Bs-deficient diet, 75 to 95 days of age respectively (table 1). the other three were given the basal diet An increase in urinary taurine at 50 to plus pyridoxine-HCl, or INH, or DOP. 55 days may possibly be controlled by They were arranged in 15 groups of four hormones. That sex hormones influence each for control of the food intake. The taurine excretion is suggested by the work experiment was continued for 7 weeks. of Chatagner and Bergeret (3) who ob INH was discontinued after 28 days and served that the decarboxylation of cys- DOP after 17 days. Two groups of four teinesulfinic acid was 2 to 3 times faster were killed each week for biological deter in liver hom ogenates from male than from minations not reported in this paper. Be female rats. Sloane-Stanley (4 ) observed fore the rats were killed, urine was col that the decarboxylation of cysteic acid lected for two 3-day periods from most of was twice as rapid in male rats as in fe the groups. males. These decarboxylations are steps Experiment 2. Twenty-four rats were in the conversion of sulfur amino acids to
divided into 4 groups of 6 each. For one taurine; thus, it might be assumed that week all groups received the basal diet taurine formation follows a similar pat plus pyridoxine-HCl. Thereafter 2 groups tern. Another possible explanation for an were fed the basal vitamin Bs-deficient increase in taurine excretion at 50 to 55 diet with INH added to one of them, and days of age is a decrease in thionase
2 groups were fed diets made adequate 5 Mercer, N. H. 1963 Application of a method for in vitamin B 6 by the addition of pyri the determination of urinary taurine to the urine of doxine-HCl with INH added to one of rats fed vitam in B-deficient diets and diets containing B6 antagonists. Master of Science Thesis, Cornell Uni them. At the end of the fifth week INH versity, Ithaca, New York. 6 s e o f m e a n . administration was discontinued for the 7 Mean for only 2 animals. T A B L E 1 Effect of vitamin B6 deficiency, isoniazid (INH) and deoxypyridoxine (DOP) on the excretion of taurine in the urine of rats 1 C X ft ft X C ) U 01 £ Q 2 ° © I SB t f E > ffi o qj u X C3 ', S V) 3 4 3 4 CD /-s C? rp > t D I CO -C rC ^ CM rH CO CM CM o q CO CM o CM r— i—i o o o u o P fi b 3 3 3 M bC M) 1 3 4 3 4 LD CD CO CD o q ID LD /-v CM CM CO LD /—N T-H CD /-v ^P I »“ rH ID t> +1 ID o q t> CM > tN- l> +1 H o 3 fi b 3 4 X! rP CD CO LD P ^ u o P fi b 3 4 CM /-s CD LD S — W q CO 0) X g 0) t f £ '—^ ^~N _ CM B B 'B 3 4 3 4 q o CM o c q p o »—1 o pH 3 4 3 4 CM 05 rH CM q LD t> o T_l 1-1 +1 +1 ■P 5 0 _ H fi b »-I P o P bfi bfi H O P ' o \ rH o c o (M I> CM o q o rH CM LD C r rH co r> LD +1 3 4 LD CO 05 +1 CD LD _ t-4 H o 3 fi b y s W q rP q 5 X t f o s rH t> q CM CM o CM 00 CM CO o CM q ^P +1 3 4 3 4 CM +1 ID o LD ■*-* O P fi b fH 1 W q LD X t f 0) g s CM O CO co o c o c CO o q D 5 0 CD O O CM CD 3 4 3 4 3 4 q 3 4 CO +1 +1 + 1 LD 3 4 ^P 3 3 bD 3 bfi bfi o P fi b H CM q CO 05 o q o 5 ■£ -5 r-l 3 4 CD O ID CO +1 3 4 LD P ^ O h O O fi b H 3 M 1 Urine was collected from the same rats on succeeding days in all experiments ex ept in experiment 2 Pyridoxine hydrochloride. 3 The numbers in parentheses indicate the number of rats included in the mean. 16 NINA H. MERCER, PHYLLIS P. BOWEN AND FRANCES A. JOHNSTON (cysteine desulfhydrase) activity which amount excreted remains on an approxi would result in a decrease in the conver mate plateau through a 7-week feeding sion of cysteine to pyruvate and glucose; period. Hope suggests two possible sources thus the amount of cysteine available for of persistent small amounts of taurine: m etabolism to taurine would be increased. . . since the liver enzyme disappears in Thompson and Guerrant (5) observed the early stages of deficiency, the taurine that weanling rats exhibited a maximal present in the urine in the later stages is thionase activity; 2 1 days later the enzyme probably dependent upon the activity of activity began to decrease. Whether the the decarboxylase present in nervous tis change in thionase is related to horm onal sue, which persists under these condi secretions is not known. tions. . . . Taurine may also be derived The increase of urinary taurine with a from the cysteamine moiety of coenzyme increase in age should be taken into ac A.” count when designing experiments in Effect of INH on urinary taurine. I N H which the object is to determine whether increased the excretion of taurine in the certain treatments affect the amount of urine of male rats fed diets adequate in taurine in urine. Treatments of rats over vitamin Be for 3 weeks or longer and 55 days of age could be expected to give tended to increase taurine excretion when taurine values of greater magnitude. the diets were deficient in vitamin Be. Effect of vitamin Br, deficiency on uri In one of two experiments (exp. 2) in nary taurine. The amount of taurine in which the diet was adequate in vitam in B«, the urine of male rats fed vitamin B 6-d e fi- when values for weeks 2, 4 and 7 were cient diets was similar to that of those fed combined, the taurine of rats given INH adequate vitamin Bs, until 50 to 55 days was higher than that of counterparts not or age; after that age the urinary taurine receiving the drug (table 1 ) although the of the vitamin B6-deprived animals did increase was of low statistical significance not increase although it increased for rats (P < 0.05).8 In the other experim ent (exp. receiving adequate vitamin B 6 (exps. 1, 3), rats that received INH for 17 to 19 2, 3; table 1). The amount of taurine ex days (table 1 ) excreted significantly more creted by rats deprived of vitamin B 6 r e taurine (P < 0 .0 1 ). mained throughout 4 experiments (exps. In 3 experiments (exps. 1, 2, 4), after 1, 2, 4, 5; table 1) at about 2 m g/kg/100 g the rats had been fed a diet deficient in body weight for rats up to 95 days of age vitamin B6, the urinary taurine excretion (7 weeks of feeding), except in experi tended to be greater for rats fed INH than m e n t 2 when the values at times de for rats that did not receive the drug. In creased to about 1 mg (table 1 ). 2 experiments (exps. 1 . 2 ) at the end of The failure of urinary taurine to in 28 days the amount of urinary taurine ex crease with age for male rats fed vitamin creted by rats given INH exceeded that of Bs-deficient diets may be related to re their partners, although the difference was tarded sexual development or may be at of low statistical significance (exp. 1 , tributed to a limitation in the amount of P < 0.05; exp. 2, P < 0.10). In the third pyridoxal phosphate present. This coen experiment (exp. 4) continued for 2 zyme is needed for the activity of cysteine- weeks only, rats given INH excreted more sulfinic acid and of cysteic acid decarboxy taurine than the controls: 2.57 ± 0.28 lase. Blaschko et al. ( 6 ), using a paper mg/24 hours/100 g body weight and chromatography method have shown that 1.73 ± 0.32 mg, respectively. The level in pyridoxine-deficient rats the decarboxy of significance was low (P < 0.07). The lase activity in the liver is lost and that fact that in all 3 experiments the values taurine disappears from the urine. Later, for rats given INH were higher tends to with an improved method, Hope (7) ob substantiate the belief that a real increase served that after 14 weeks of deficiency, occurred. Futhermore, when the admin some taurine still remained in the urine istration of INH was discontinued (exps. of rats. The present study was not con 1 , 2 ) taurine excretion declined (table 1 ). tinued for as long a time but the addi 8 All tests of significance were made by paired t t e s t s tional observation was made that the except where specified as group t te s t s . FACTORS AFFECTING URINARY TAURINE 17 In unpublished work carried out in this Effect of DOP on urinary taurine. I n laboratory the taurine content of the urine experiment 5, DOP increased the urinary of 4 young men was observed to increase taurine of male rats fed the basal vitamin during INH administration. B6-deficient diet. At the end of 2 weeks Two groups of workers, Marcucci and the increase was considerable (table 1 ) ; M u s s i n i ( 8 ) and Yoshikawa et al. (9 ) re at the end of 3 weeks it was highly signifi ported that INH decreased the urinary cant (P < 0.01). Some supporting evidence taurine of rats. Both groups administered was supplied in experim ent 1 in which the many times the amounts of INH used in mean excretion for taurine for 3 rats in the present study. creased during 10 days when DOP (in a The increase in urinary taurine caused smaller dose than in experiment 5) was by INH may have been brought about by being administered. an increase of coenzyme in the liver. An No studies of the effect of DOP on the increase in the pyridoxal phosphate con amount of taurine present in urine were tent of the livers of the rats fed INH in found in the literature. experiments 2 and 3 occurred and has The cause of the increase in taurine been reported by Sevigny et al. (10, e x p s . may have been an increase in the amount B and C). In experiment B, when the diet of pyridoxal phosphate present in the was adequate in vitam in B6, INH increased livers of the rats. The liver of the rats fed pyridoxal phosphate from 8.1 ± 0.3 to 10.9 DOP in experiment 5 contained more st 0.5 ag/g; when the diet was deficient in pyridoxal phosphate than those of their vitamin B6, INH increased pyridoxal phos counterparts not fed the drug. This ob phate from 2.2 ± 0.1 to 3.6 ± 0.4 ag/g. In servation has been reported by Johnston experiment C, when the diet was adequate e t a l. (16, exp. D ). Stoerk (17) h a s r e in vitamin B6, INH increased pyridoxal ported a similar observation. phosphate from 7.1 ± 0.3 to 10.8 ± 0.5 ag/g. In the present study, some of the ACKNOWLEDGMENTS pyridoxal phosphate in the livers of the rats Technical assistance in the determina fed INH m ay have been present as isonico- tion of taurine by Mrs. Esserline Gatewod tinyl pyridoxal phosphate hydrazone. How is gratefully acknowledged. W e wish to ever Gonnard and Fenard (11), Torchinsky express our thanks to Sister Saint Jeanne (12), Makino et al. (13) and Bonavita de Jesus Sevigny, Ph.D., who conducted and Scardo (14) have shown that the experiment 1 for other purposes, for her pyridoxal phosphate in the hydrazone can contribution of material to use in the pres be used to activate several vitamin B 6-r e - e n t s t u d y . quiring apoenzymes. Possibly INH causes an increase of taurine because of a toxic LITERATURE CITED effect unrelated to its anti-vitamin effect. 1. Sorbo, B. 1961 A method for the deter Possibly, too, INH causes the excretion mination of taurine in urine. Clini. Chim. of taurine from preformed tissue taurine Acta, 6: 87. rather than causing the form ation of addi 2. Garvin, J. E. 1960 A new method for the tional taurine. determination of taurine in tissues. Arch. Biochem. Biophys., 91: 219. Another possible cause of the increase 3. Chatagner, F., and B. Bergeret 1956 Dé in taurine excretion by INH is its action carboxylation de l’acide cystéinesulfinique on the adrenal cortex. Both ACTH and par le foie et le cerveau du rat mâle, du rat cortisone are known to increase the femelle et du rat femelle ovariectomisé. amount of taurine in urine. W hether INH Bull. Soc. Chim. Biol., 38: 1159. 4. Sloane-Stanley, G. H. 1949 Amino-acid increases the excretion of cortical hor decarboxylases in rat liver. Biochem. J., mones is, however, questionable. An in 4 5 : 556. crease in certain cortical horm ones follow 5. Thompson, R. Q., and N. B. Guerrant 1953 ing treatment with INH has been observed Effect of dietary protein and vitamin B6 on by some but not all workers. In addition, hepatic thionase activity. J. Nutr., 50: 161. 6. Blaschko, H., S. P. Datta and H. Harris W i e s e l (15) obtained evidence by experi 1953 Pyridoxin deficiency in the rat: liver ments in vitro that INH markedly retards n-cysteic acid decarboxylase activity and uri the inactivation of cortisone by the liver. nary amino-acids. Brit. J. Nutr., 7: 364. 18 NINA H. MERCER, PHYLLIS P. BOWEN AND FRANCES A. JOHNSTON 7. Hope, D. B. 1957 The persistence of 13. Makino, K., Y. Ooi, M. Matsuda, M. Tsuji, taurine in the brains of pyridoxine-deficient M. Matsumoto and K. Kuroda 1962 Some rats. J. Neurochem., 1: 364. notes on the coenzyme-activity of phospho- 8. Marcucci, F., and E. Mussini 1958 Sulla pyridoxal derivates for the brain glutamic eliminazione di acido cisteico et de taurina decarboxylase. Biochem. Biophys. Res., Com- dopo trattomento con isoniazid. Boll. Soc. m u n ., 9: 246. Ital. Biol. Sperim., 34: 422. 14. Bonavita, V., and V. Scardi 1959 Studies 9. Yoshikawa, K. T., T. Aoki, K. Matoba and on glutamic-oxaloacetic transaminase. The I. Matsumo 1960 Change of the taurine coenzymatic role of the isonicotinylhydra- excretion in the urine by the administration zone of pyridoxal-5-phosphate. Biochem. of INAH to rats. Nara Igaku Zaschi., 11: 71. Pharmacol., 2: 58. 10. Sevigny, J., S. L. White, M. L. Halsey and 15. Wiesel, L. L. 1956 Investigation of the F. A. Johnston 1966 The effect of iso synergism of isonicotinic acid hydrazide and niazid on the loss of pyridoxal phosphate cortisone. I. Effect of isonicotinic acid on from, and its distribution in, the body of the metabolism of cortisone by liver tissue. the rat. J. N utr., 88: 45. Amer. J. Med. Sci., 232: 412. 11. Gonnard, P., and S. Fenard 1962 Cere 16. Johnston, F. A., S. L. White, M. L. Halsey bral glutamic acid decarboxylase and pyri- and J. Sevigny 1966 The effect of deoxy- doxal-5-phosphate hydrazones. J. Neuro pyridoxine on the amount of pyridoxal phos chem., 9: 135. phate in the livers and leukocytes of rats 12. Torchinsky, Y. M. 1963 The mode of in and on leukocyte number, size and type. teraction of the isonicotinoyl hydrazone of J. N utr., 88: 51. pyridoxal phosphate with aspartate-gluta 17. Stoerk, H. C. 1950 Desoxypyridoxine ob mate apotransaminase. Biochem. Biopyhs. servations in “acute pyridoxine deficiency.” Res. Commun., 10: 401. Ann. N. Y. Acad. Sci., 52: 1302. Effect of Coprophagy on Protein Utilization in the R at* 1 B. R. STILLIN G S 2 a n d L. R. HACKLER New York State Agricultural Experiment Station, Cornell University at Geneva, New York ABSTRACT Diets differing widely in protein quality were fed to male weanling rats, and the effect of coprophagy on protein utilization was determined. In a metabolism study in which animals consumed 5 g daily of diets containing 10% protein, preventing coprophagy decreased biological value and nitrogen absorption, and increased urinary nitrogen excretion and metabolic fecal nitrogen. In a growth study in which animals consumed diets containing 10% protein ad libitum, coproph agy prevention resulted in lower protein efficiency ratios and weight gains, but had little effect on feed intake. The practice of coprophagy by the rat to produce 1 0 % protein diets, which were is well known, and Barnes et al. (1 ) have calculated to be isocaloric. Diets were estimated that rats recycle approximately stored in metal containers at 1 ° and used 50% of their feces. The possible effect within a few days in a metabolism ex which this practice might have on nu periment and in 3 months in a growth trient utilization and requirements has s t u d y . been the subject of m any previous reports. The biological value of these diets was The effect of preventing coprophagy in determined in a metabolism experiment in mature rats on protein digestion was and computed essentially according to studied by Barnes et al. (2), who reported the procedure outlined by M itchell (4, 5). that the practice of coprophagy did not Male rats of the Holtzman strain, 21 days influence to any measurable extent the old, were maintained with a 15% casein digestibility of protein. Data on the ef d ie t f o r 2 days and then were assigned fect of coprophagy on nitrogen utilization on a weight basis to one of 9 groups. The and retention are not available. Since the groups, in turn, were assigned at random rat is used in the majority of studies to the 9 diets. Each group contained 4 dealing with protein and amino acid nu animals for which coprophagy was pre trition, it was deemed advisable to con vented in two and allowed in the two re duct studies on the effect of coprophagy maining. Coprophagy was prevented by on protein utilization. fecal collection cups similar to those illus trated by Barnes et al. ( 6 ). EXPERIMENTAL METHODS During the experiment, animals were Proteins known to vary widely in quality maintained in metal metabolism cages were used to study the effect of coprophagy which facilitated separation and collec on protein utilization. The sources in tion of urine and feces. Feed was mixed cluded zein, wheat gluten and whole egg. with an amount of water equal to approxi In addition, 4 samples of heat-treated soya mately 60% of the diet weight and offered protein were used. Each was mixed with to the animals once daily. 1.5 times its weight of water, autoclaved The metabolism study consisted of 4 a t 121° for 5, 40, 120, or 240 minutes collection periods of 7 days each. Prior and then freeze-dried. Two amino acid to each collection period, animals were mixtures were also used, the com positions fed experim ental diets for at least 5 days, and they consumed a constant amount of which are given in table 1. The bal anced am ino acid mixture was essentially Received for publication March 12, 1966. that of Ram a Rao et al. (3), and this was 1 Approved for publication by the Director of the New York State Agricultural Experiment Station, substantially modified to produce the im Geneva, New York, as Journal Paper no. 1476, March balanced mixture. The com position of the 4 , 1 9 6 6 . 2 Postdoctoral Fellow on National Institutes of basal diet is shown in table 2. All pro Health Training Grant 2 G-950, Spec. (1) E. H. Pres ent address: Bureau of Commercial Fisheries, Tech tein sources were incorporated in amounts nological Laboratory, College Park, Maryland 20740. J. N u t r it io n , 90: ’ 66 19 2 0 B. R. STILLINGS AND L. R. HACKLER T A B L E 1 During the first and second periods, Aviino acid com position of amino a c i d d i e ts coprophagy was prevented in one-half the “ B a l a n c e d ” ‘Im balanced” animals and allowed in the remaining A m i n o a c i d a m i n o a c i d a m i n o a c i d half. Throughout the third and fourth d ie t d ie t periods fecal collection cups were switched % o f d iet % o f d iet to animals that had been allowed to prac H i s t i d i n e 0 . 2 5 0 . 5 0 A r g i n i n e 0 . 2 8 0 . 1 4 tice coprophagy during the first 2 p e r i o d s . L y s i n e 0 . 9 0 0 . 0 9 Feces were collected daily and dried at T r y p t o p h a n 0 . 1 1 0 . 2 2 50°. Urine samples were preserved with I s o l e u c i n e 0 . 5 5 0 . 2 7 H 2SO 4 and KF. Samples of feed, feces V a l i n e 0 . 5 5 0 . 2 7 L e u c i n e 0 . 7 0 1 . 5 5 and urine were analyzed for nitrogen by T h r e o n i n e 0 . 5 0 1 . 0 0 the m acro-Kjeldahl procedure. M e t h i o n i n e 0 . 1 6 0 . 0 8 In a second study, the effect of coproph C y s t i n e 0 . 3 4 0 . 0 7 agy on weight gain, feed consumption Phenylalanine 0 . 4 2 0 . 8 4 T y r o s i n e 0 . 3 0 0 . 3 0 and protein efficiency ratio was deter A s p a r t i c 1 . 0 4 1 1 . 0 4 1 mined. Twenty-three-day-old male wean A l a n i n e 0 . 4 8 4 0 . 4 8 4 ling rats of the Holtzman strain were al S e r i n e 1 . 0 0 9 1 . 0 0 9 lotted on a weight basis to 1 0 g r o u p s . P r o l i n e 1 . 9 4 3 1 . 9 4 3 G l y c i n e 0 . 3 1 2 0 . 3 1 2 Groups were then assigned at random to G l u t a m i c 3 . 4 1 1 3 . 4 1 1 the 9 diets used in the first study and to a tenth diet which contained a 2 0 - f o l d T o t a l 1 3 . 2 6 1 3 . 5 3 increase in niacin but which was other wise identical to the zein diet. T A B L E 2 A total of 8 animals was assigned to Composition of 10% protein diets each diet, with coprophagy prevented in % four and allowed in the 4 remaining ani Protein source ‘ • n mals. An attempt was made to nullify D ex trose 1 2 ; possible effects which the physical pres S u c r o s e 1 2 . 0 0 C e l l u l o s e 2-3 4 5 . 0 0 ence of the fecal collection cups might S a l t m i x 4 . 0 0 have on the responses of the animals. Vitam in m ix 5 1 . 0 0 Fecal collection cups with the open end Corn o il6'7 1 0 . 0 0 pointing away from the animals were at Choline citrate 0 . 3 0 tached midway down the tails of animals 1 Protein sources: (% of diet) zein, 10.88; wheat gluten, 12.20; soya assay protein heated 5, 40, 120, allowed to practice coprophagy. Thus, and 240 minutes at 120°, 11.70, 11.44, 11.38, and fecal collection cups were attached to 11.54, respectively; whole egg, 21.48; imbalanced amino acids, 13.53; and balanced amino acids, 13.26. all animals, but in one-half of the animals 2 Obtained from General Biochem icals, Inc., Chagrin Falls, Ohio. the cups were attached in a m anner which 3 Non-nutritive fiber (cellulose type). allowed coprophagy. 4 Hubbell, R. B., L. B. Mendel and A. J. Wakeman 1937 A new salt mixture for use in experimental During the second study the animals diets. J. Nutrition., 1 4 : 2 7 3 . 5 Vitamin mix composition: (m g/100 g diet) thia- were fed ad libitum for 28 days, and feed mine-HCl, 1.0; riboflavin, 1.5; pyridoxine-HCl, 0.5; intake and weight gain data were recorded. Ca DL-pantothenate, 2.0; nicotinic acid, 3.0; biotin, 0.03; folic acid, 0.2; menadione, 0.4; inositol, 7.5; Protein efficiency ratio (PER) was calcu v i t a m i n B 12 (0.1% in m annitol), 4.0; p-aminobenzoic acid, 2.5; ascorbic acid, 0.4; crystalline vitamin A, lated by dividing the grams of weight 2.0 (500 USP units/m g); vitamin D2, 0.2 (500 USP gained by the grams of protein consumed. units/m g); and cZi-a-tocopherol, 12.0; (250 USP u n i t s / g ) . Data from both studies were analyzed 6 Mazola corn oil, Corn Products Company, New Y o r k . statistically by an analysis of variance (7). 7 W hole egg contained 35.2% ether extract; there fore, 2.4% corn oil added to whole egg diet. RESULTS AND DISCUSSION the last 3 days. In the first and third Significant depressions in biological periods, animals consumed 1 0 % p r o t e i n value of dietary proteins were observed diets, whereas in the second and fourth when rats were prevented from practicing periods they were fed an equal amount of coprophagy (table 3). The average bio a 3.4% whole egg diet which was iso- logical value for all diets was reduced caloric and otherwise identical to the basal from 60% to 50% when coprophagy was diet (table 2 ). prevented. Decreases were observed fcr E ffect of coprophagy prevention on protein utilization by w eanling rats fed 10% protein diets ' C/3 rÖ TO rH N itrogen x 6.25. 1 2 2 2 B. R. STILLINGS AND L. R. HACKLER all diets; however, significant differences are conducted, any attempt to arrive at an were found only for diets containing the adequate explanation can be only specu balanced amino acid mixture (P < 0 . 0 1 ), la t iv e . whole egg (P < 0.01), imbalanced amino Preventing coprophagy also resulted in acid mixture (P < 0.05), and soya protein a slight but statistically significant (P < heated 5 minutes (P < 0.05). 0 .0 1 ) depression in nitrogen absorption. With the exception of the imbalanced Although the practical significance of this amino acid diet, significant differences small difference is questionable, the re were found only for diets with the highest sults are in accord with those obtained in biological value. Biological value is known the rabbit by Thacker and Brandt ( 8 ) , to be affected by level of feed intake; who reported that coprophagy prevention however, with the exception of the zein decreased apparent protein digestibility. and imbalanced amino acid diets all ani However, Barnes et al. (2 ) reported that mals consumed 5 g of feed daily. In a protein digestibility in the rat was unin subsequent study in which each animal fluenced by preventing coprophagy. In consumed 4.5 g daily of the imbalanced our study, nitrogen absorption was high am ino acid diets, no significant difference for all diets, which possibly was attribut was found in biological value between the able to the low level of feed intake. Nitro coprophagic and noncoprophagic rats. gen absorption decreased, however, in the The differences obtained in biological soya protein diets as the time of autoclav value between coprophagic and noncopro ing increased. phagic rats were reflected in weight gains The metabolic fecal nitrogen was sig of animals. As shown in table 4, the nificantly increased for all animals in average weight gains during the first and which coprophagy was prevented, with the third collection periods were consistently exception of those which previously had higher for animals practicing coprophagy. been fed the 1 0 % whole egg-protein diet In addition, diets which gave the largest (table 3). Metabolic and endogenous differences in biological value also tended nitrogen were determined in the second to result in wider differences in weight and fourth periods with all animals con g a in s . suming 5 g of the 3.4% whole egg-protein The interaction which was observed be diet. Urinary endogenous nitrogen was tween quality of diet and coprophagy on not significantly affected by coprophagy biological value is difficult to explain; how prevention. Barnes et al. (2) also ob e v e r , 2 possibilities can be presented. served small increases in metabolic nitro First, although all animals were fed equal gen in rats prevented from practicing amounts of feed at the same time each coprophagy. They suggested that nitro day, those rats assigned to the higher gen derived from the intestinal tract m ight quality diets tended to consum e their feed be digested if recycled through the small in less time than those fed the lower intestine, which would result in lower quality diets. Also there may have been a difference between diets in the degree to T A B L E 4 which animals practiced coprophagy. For Effect of coprophagy prevention on average weight example, if coprophagic rats assigned to gain during first and third collection periods of metabolism experiment the whole egg diet consum ed m ore of their feces than similar animals assigned to the C o p r o p h a g y C o p r o p h a g y D ie t wheat gluten diet, greater differences due a l l o w e d p r e v e n t e d to coprophagy would be expected with g / r a t f w e e h the form er diet than with the latter. W heat gluten 2 . 9 2 . 6 The depression in biological value which W h o l e e g g 7 . 2 1 .9 occurred when coprophagy was prevented Soya protein, 5 m in 1 6 . 0 2 . 0 was the result of an increased urinary Soya protein, 40 m in 5 . 3 1 .9 Soya protein, 120 m in 5 . 6 3 . 9 nitrogen excretion (table 3). It appears Soya protein, 240 m in 4 . 4 0 . 5 that some factor(s) in the feces was re Im balanced am ino acid 0 . 6 - 0 . 2 quired for optimal utilization of the ab Balanced am ino acid 5 . 9 1 .8 sorbed nitrogen. Until additional studies iM inutes heated at 121°. COPROPHAGY AND PROTEIN UTILIZATION 2 3 metabolic nitrogen values in rats practic ing coprophagy. .Jf. •x- * * * The results obtained with the zein diet 0 5 t r c i h t j - [ CO *H H H H C O C f (N Ci are not included in table 3 because ani d d d d d d d d mals failed to consume the entire amount CD •r-» of feed offered, and one-half of the ani ctS As shown in table 5, all animals consum •j:- % * CD T-HCNcNCNCOfNaiCDincOCD ing the zein diets lost weight, although oOOcMHHOUNHqiOH none showed the symptoms exhibited in d o 6 d d o d d d d d the first experiment. Only slight differ ences between the 2 zein diets were noted -8 b£TD in weight gain and feed consumed, but 05 CÖ CD COCOCDHb-dOKNCOOJCO A ß pr^cqcqrH^iorHcqoicq animals fed both diets and in which co CL « ^ d o d ^ d (Nh h o c o h prophagy was prevented tended to consum e more feed. From these results a conclu sion cannot be drawn to explain the symp rtTJ A. MARIANI, P. A. MIGLIACCIO, M. A. SPADONI a n d M. TICCA Istituto Nazionale della Nutrizione, Città Universitaria, Rome, Italy ABSTRACT The activity of the amino acid-activating enzymes was measured in the liver of growing rats maintained with a normal and with a protein-deficient diet. The determinations were based on the isotope exchange between ATP and PP labeled with 32 P. The changes in the DNA content of the cell nucleus were also determined. Studies of changes in DNA content, and hence changes in ploidy during growth, indicated that in normal rats the activity of the amino acid-activating enzymes in creased when expressed per average nucleus, whereas it remained constant and un affected by growth when expressed per unit weight of DNA. On the contrary, in rats maintained with a protein-deficient diet, in which the arrest of growth causes an arrest of ploidy, the activity of the amino acid-activating enzymes increased both when expressed per average nucleus and per unit weight of DNA. We suggest that under these conditions a control mechanism is brought into action by changes in the amount of amino acids in circulation. This mechanism is independent of the in crease in the DNA content of cell nucleus, and results, for growing rats maintained with a protein-deficient diet, in a preferential utilization of the amino acids for the synthesis of proteins. Following a prolonged protein fast the content of the average nucleus, in the liver liver of the adult rat exhibits a higher of growing rats kept under our experi activity of all the amino acid-activating mental conditions. In protein-deficient rats enzymes, both when expressed per unit we had found no increase with age of the weight of liver protein and per unit weight DNA content of the average nucleus, of liver DNA-P (1 , 2 ). This may be in whereas in normally growing rats the DNA terpreted as an attempt on the part of the per average nucleus increased as a con liver to control the metabolism of amino sequence of the formation of new poly acids by redirecting the latter from cata ploid cells. We decided, therefore, to ex bolic to anabolic processes. press the results relative to amino acid Previous experiments carried out on activation not only per unit weight of adult rats, with isotopically labeled amino DNA-P, but per average nucleus as well. acids, have indicated that a prolonged The results obtained show that the ac protein fast results in the preferential tivity of the amino acid-activating enzymes utilization of amino acids by the hepatic increases in protein-deficient rats, both cells ( 3 ) . The same result has also been when expressed per unit weight of DNA-P obtained in investigations on rats main and per average nucleus. tained after weaning with a protein-de EXPERIMENTAL ficient diet for a prolonged period (4, 5 ). The present experiments were carried Locally bred male albino rats of the out to determine whether in growing rats Wistar strain were taken immediately a protein-deficient diet results in a similar after weaning and fed ad libitum a diet higher activity of the amino acid-activat containing 25 and 5% casein (normal ing enzymes. and protein-deficient diets, respectively) In view of the changes in the ploidy of (table 1). They were then decapitated 10, the hepatic cells during growth and of the 20 and 30 days later, the liver was im effect of the diet on these changes ( 6 -9 ) , mediately excised, and washed with an it was considered advisable to undertake ice-cold solution of Tris (hydroxymethyl) a preliminary investigation on the DNA Received for publication March 11, 1966. J. N u t r it io n , 90: ’66 2 5 26 A. MARIANI, P. A. MIGLIACCIO, M. A. SPADONI AND M. TICCA T A B L E 1 enate was then filtered through gauze and Composition of experimental diets diluted in a ratio of 1 :2 0 with a 3% solu tion of acetic acid containing 0.2% of Normal Protein- diet deficient methyl green. The cells were counted di diet rectly in a Burker cell counter (1 2 ). The % % DNA determination was carried out on a Casein 25 5 suitable amount of the homogenate by Rice starch 34 44 Sucrose 28.7 38.7 the colorimetric method of Webb and DL-Methionine 0.3 0.3 Levy (1 3 ). The results of the above 2 Salt mixture 1 5 5 determinations were then used to calculate Olive oil 5 5 the average DNA content per cell nucleus. Cod liver oil 1 1 Vitamin mixture 2 1 1 To check this method, we used the method of Chauveau et al. (1 4 ) as modified by 1 Each 100 g of the salt mixture supplied: (in grams) calcium carbonate, 38.140; cobalt chloride, Di Girolamo et al. (1 5 ) (but substituting 0.002; copper sulfate, 0.048; ferrous sulfate, 2.700; 0.25 m sucrose with 0.88 m sucrose) to magnesium sulfate, anhydrous, 5.730; manganese sul fate, 0.445; potassium iodide, 0.079; potassium phos isolate cell nuclei and subjected this prep phate, monobasic, 38.900; zinc chloride, 0.026; sodium chloride, 13.930; (Jones, J. H., and C. Foster. aration to a cell count and DNA determina J. Nutrition, 24: 245, 1943, obtained from General tion as described above. The value thus Biochem icals, Inc., Chagrin Falls, Ohio). 2 Each 1,000 g of the vitam in mixture supplied: (in obtained for the DNA content per cell gram s) p-aminobenzoic acid, 2; vitamin Bi, 0.2; vita m i n B 2, 0.4; vitamin B6, 0.2; Ca pantothenate, 0.8; nucleus was in satisfactory agreement niacin, 2; inositol, 2; folic acid, 0.04; biotin, 0.024; with the corresponding value for the above v i t a m i n B 12, 0.001; vitamin C, 2; menadione sodium bisulfite, 0.2; vitamin E, 1,000 IU; choline chloride, homogenate. 40; vitamin A, 200,000 IU; vitamin D2, 20,000 IU; sucrose to make 1,000 g. RESULTS amino-methane (Tris) (pH 7.4) and KC1 DNA content of the nuclei. The values in concentrations of 0.02 and 0.05 m , re for the number of nuclei, the DNAP con spectively. A suitable amount of the liver tent per nucleus and per gram of fresh was homogenized in the Tris-KCl buffer at tissue are shown in table 2. In neonatal 0° by a motor-driven Potter homogenizer rats, the number of nuclei and the DNAP fitted with a Teflon pestle. content per unit weight of liver were the Determination of enzymatic activity. highest, the DNA content per nucleus be The homogenate was centrifuged in a ing approximately the same as the value Martin-Christ ultracentrifuge at 105,000 calculated (1 6 ) for the diploid cells in x g for one hour. The temperature inside rats. Twenty days after birth, however, the tubes was kept below 4° since the ac the DNAP conent per unit weight of liver tivity of the enzymes activating the amino decreased as a result of cell enlargement, acids decreases at a higher temperature. although the DNAP content per cell nu Suitable equal portions of the supernatant cleus remained unchanged. were then treated with Carbowax 1 (2 0 m ) In rats fed a diet containing 2 5 % of according to the technique described by casein (normal diet, normal growth), ow Pennington (1 0 ). This was carried out ing to the formation of tetraploid and oc- to remove the endogenous amino acids. taploid nuclei, the DNA content per nu The activation of the amino acids was cleus in the hepatic cells increased and then determined by measuring the rate of reached the value of adult rats. However, isotope exchange between 32PP and ATP in rats fed a diet containing only 5% of by the method of De Moss and Novelli casein (protein-deficient diet, strongly re (1 1 ). Incubation was carried out as de tarded growth), the DNA content per nu scribed previously (2 ) . The radioactivity cleus remained statistically the same as was determined with the aid of a gas-flow that at the time of the weaning. end-window counter. The results obtained for the activation Preparation of the suspension for nuclei of the amino acids were then correlated count and DNA determination. The liver both with the nuclei and with the DNA. was homogenized manually for 2.30 min In the first case, we expressed the enzy utes in a Potter apparatus with 4 volumes matic activity per average cell on the of a 0.88 m sucrose solution. The homog 1 Union Carbide Chemical Co., Charleston, W. Va. AMINO ACID ACTIVATION IN LIVER OF GROWING RATS 2 7 assumption that the cytoplasmic mass of F- i n Is - CD CO 0 5 o o i n c o rH 0 0 CD F - CD CD the binuclear cells (1 0 to 20% of the § 9 o rH O O o o o o O Ò o d d o d d hepatic cells of rats) (1 7 ) is twice that of g +1 +1 +1 +1 +1 + i +1 +1 mononuclear cells. To correlate the enzy \ o> f - ^ O CO i n 0 5 cm 05 O t- i n ^ 05 5 9 i n 9 i n expressed per unit weight of DNA refer A , i > CD 0 5 CO ^ rH (M »2 i n CD CO CO CM CM 0 5 to the enzymatic activity per hypothetical +1 +1 +1 +1 +1 +1 +1 z £ 1 diploid cell, i.e., per amount of protoplas Q o o CD ^ CM ^ rH O f - m cm 0 5 ^ C O O CO O rH i n i n c d mic mass corresponding to a diploid rH rH amount of DNA (18). Activation of the amino acids as a $ 0 5 9 i n o q c o 0 5 9 h Jr o o CO H T* r i r i l lÓ function of growth and diet. Figure 1 rH H r i H rH rH rH shows the variation in the enzymatic ac s +1 + 1 +1 +1 + ! +1 +1 + i e» c o CM CM CD »h H r H ^ tivity in rat liver during a period of 30 CO 0 5 rH i n cm N u c l e i 'O 4 O ■h CO CO £ cm c o i n w Cl rH QJ (M CM rH tivity with age and diet. Figure 1 shows hJ ■ 8 cm (N C/5 03 + i + i + i + l +1 + l that the amino acid activation increased < a +1 +1 2 8 t o CO CD 0 5 F - ° 0 0 0 5 0 H S 4 o 00 O O i n F - F - The rats fed the protein-deficient diet, -e W CUD At the age of 58 days, the deficient rats f i A showed a definite increase in the activity f i « . a of enzymes activating the amino acids, « 01 bt) » o -0 0 0 > 00 00 00 CO c o CO : « < CM > c o m c o ^ m the activity being about 90% higher than ; 'S a l in normal rats. 28 A. MARIANI, P. A. MIGLIACCIO, M. A. SPADONI AND M. TICCA Ld 0.600- O z cl X o X Ld 0.500- A / / / 0.400- 0.300- 0.200- 0.100- 1 28 38 48 58 AGE (DAYS) Fig. 1 Variation of the amino acid-dependent 32 PP ±5 ATP rate of exchange/nucleus as a function of age. Each point on the graph is the average of values from a group of 5 rats. Assay conditions: 0.2 ml enzyme preparation (between 2.1 and 4.3 mg supernatant protein in each incubation mixture); 100 /¿moles Tris-KCl buffer (pH 7.4); 50 /¿moles KF; 10 /¿moles ATP, disodium salt, (adjusted to pH 7.4); 10 /¿moles pyrophosphate 32 P; 10 /¿moles MgCk; complete amino acid mixture (adjusted to pH 7.4), 4 /¿moles of each of the following amino acids; alanine, arginine, aspartic acid, cysteine, cystine, glycine, glutamic acid, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Cystine and tyrosine did not dissolve com pletely. Total volume 1 ml. Incubated at 37° for 15 minutes. DISCUSSION value which then remained constant. The In rats fed normal diet (2 5 % casein), increase in the ploidy was in turn ac the growth of the liver in the first month companied by a proportional increase in after weaning is characterized by an in the activity of the enzymes activating the crease in the number and size of cells (see amino acids and presumably also in the table 2 ). This has emerged from the cal level of these enzymes. It appeared, there culation of the number of nuclei per gram fore, that the level of the activating en of liver and in the whole liver. This result zymes is related to a unit weight of DNA does not therefore take into account the corresponding to a diploid set of chromo increase in the binuclear cells during somes and that this level remains constant growth. However, the resulting error was and unaffected by the growth of the ani not large, considering that the cytoplasmic mals. mass of the binuclear cells is about twice In contrast, the animals whose growth as great as that of the mononuclear cells. was strongly retarded by feeding a protein- In the first 10 days after weaning, the in deficient diet, showed during the first 10 crease in the number and size of the cells days after weaning, a slight increase in was accompanied by an increase in the the weight of the liver resulting almost DNA content of the average nucleus and entirely from an increase in the number therefore by an increase of ploidy up to a of the cells, after which cell division was AMINO ACID ACTIVATION IN LIVER OF GROWING RATS 29 LJ 600 - CD z cc X o X L d 500 - d C_ cc d z 400 - D_ Q t cn u 300 - Q- a. z Ld 200 - Ld CL UJ 25 % casein diet Q A------A 100 - 5 % casein diet _]______I______!_ _L 28 38 48 58 AGE (DAYS) Fig. 2 Variation of the amino acid dependent 32 PP ATP rate of exchange/DNAP as a function of age. Each point on the graph is the average of values from a group of 5 rats. Assay conditions as in figure 1. arrested and the cells grew in size, acid activating enzymes of rat liver. Nature. though they remained much smaller than 199: 378. the normal cells. The ploidy did not in 2. Gaetani, S., A. M. Paolucci, M. A. Spadoni and G. Tomassi 1964 Activity of amino crease, as in normal rats, but a consider acid activating enzymes in tissues from pro able increase was observed in the level of tein-depleted rats. J. Nutr., 84: 173. the enzymes activating the amino acids. 3. Gaetani, S., A. Mariani, M. A. Spadoni and This increase does not, therefore, depend G. Tomassi 1961 Distribuzione della C14- on the increase in the DNA content per lisina nel plasma, fegato, cuore e muscolo nucleus, as it does during the growth of gastrocnemio di ratti in deplezione proteica. Boll. Soc. Ital. Biol. Sperim., 37: 1685. the normal rats. Instead, it results from 4. Waterlow, J. C. 1959 Effect of protein de a different control mechanism probably pletion on the distribution of protein syn actuated by a change in the pool of circu thesis. Nature, 184: 1875. lating amino acids, similar to the situa 5. Bendicenti, A., A. Mariani, A. M. Paolucci tion observed in adult rats subjected to a and M. A. Spadoni 1959 L’influenza del contenuto proteico della dieta sulla distribu prolonged protein fast. This increase of zione della S 35-metionina nei tessuti di ratti the enzymes activating the amino acids in accrescimento. Boll. Soc. Ital. Biol. may account for the preferential utiliza Sperim., 35; 1997. tion of the amino acids for the synthesis 6. Wilson, J. W., and E. H. Leduc 1948 The occurrence and formation of binucleate and of proteins, which has been found in rats multinucleate cells and polyploid nuclei in fed a protein-deficient diet after weaning. the mouse liver. Amer. J. Anat., 82: 353. 7. Campbell, R. M., H. W. Kosterlitz 1952 LITERATURE CITED The absence of dietary effects on the DNA 1. Mariani, A., M. A. Spadoni and G. Tomassi content of liver nuclei of the adult rat. Sci 1963 Effect of protein depletion on amino ence, 115: 84. 3 0 A. MARIANI, P. A. MIGLIACCIO, M. A. SPADONI AND M. TICCA 8. Ely, J. O., and M. H. Ross 1951 Deoxy 14. Chauveau, J. Y. Moulé and C. R. Rouiller ribonucleic acid content of rat liver nuclei 1956 Isolation of pure and unaltered liver influenced by diet. Science, 114: 70. nuclei: morphology and biochemical com 9. Umana, R. 1965 Effect of protein malnu position. Exp. Cell Res., 11: 317. trition on the DNA content of rat liver. J. 15. Di Girolamo, A., E. C. Henshaw and H. H. Nutr., 85: 169. Hiatt 1964 Messenger ribonucleic acid in 10. Pennington, R. J. 1960 Amino acid acti rat liver nuclei and cytoplasm. J. Mol. Biol., vating enzymes in muscle. Biochem. J., 77: 205. 8: 479. 11. Demoss, J. A., and G. D. Novelli 1956 An 16. Vendrely, R. 1955 In: The Nucleic Acids, amino acid dependent exchange between eds., E. Chargaff and J. N. Davidson, vol. 32 P-labeled inorganic pyrophosphate and 2. Academic Press, New York, p. 155. ATP in microbial extracts. Biochim. Biophys. 17. Iype, P. T., P. M. Bhargava and A. D. Tas Acta, 22: 49. ker 1965 Some aspects of the chemical 12. Price, J. M., E. C. Miller, J. A. Miller and and cellular composition of adult rat liver. G. M. Weber 1950 Studies on intracel Exp. Cell Res., 40: 233. lular composition of livers from rats fed 18. Petermann, M. L., and M. G. Hamilton various amino acids. Cancer Res. 10: 18. 13. Webb, J. M., and H. B. Levy 1955 A sen 1958 The influence of age, sex, pregnancy, sitive method for the determination of de starvation and other factors, on the cyto oxyribonucleic acid in tissues and micror- plasmic ribonucleoproteins of rat liver. J. ganisms. J. Biol. Chem., 213: 107. Biophys. Biochem. Cytol., 4: 771. Utilization of d -Amino Acids for Growth b y Drosophila melanogaster Larvae 1 * B. W. GEER Department of Biology, Knox College, Galesburg, Illinois ABSTRACT The capacity of Drosophila melanogaster larvae to utilize the D-forms of the essential amino acids for growth in place of their L-isomers was examined. D-Phenvlalanine, D-methionine, and D-histidine, in decreasing order, were the only D-amino acids w hich were readily used for growth by larvae. The D-forms of arginine, lysine, and valine stimulated larval growth when fed with suboptim al quantities of their L-isomers. D-Isoleucine and D-threonine had little, if any, effect on larval growth. At the levels tested, D-leucine and D-tryptophan inhibited the growth of D. melano gaster larvae. Feeding either glycine, L-cystine, or L-tyrosine in a diet containing the L-isomers of the essential am ino acids and L-glutam ic acid stim ulated larval growth. Larvae appear capable of using D-cystine for growth in place of L-cystine. The ability of D. melanogaster l a r v a e , Tetrahymena pyriformis, and the rat to utilize D-amino acids for growth was compared. Points of evolutionary interest were dis c u s s e d . Insects require, in general, the same 10 being tested, were fed as the L-enantio- amino acids in the diet for growth that morph. Cultures were prepared by dis are required by the rat (1 ) . The essential pensing 5 ml of medium in 23-g (6-dram) amino acids are usually supplied in the shell vials, plugging with cotton, and diet as their L-isomers although purified sterilizing by autoclaving. Eggs, which diets often contain mixtures of the d- and had been sterilized by methods described L-isomers of some amino acids. The ca previously ( 5 ) , were added to the culture pacity to utilize D-amino acids in lieu of vials by aseptic techniques. The number their L-enantiomorphs has been examined of eggs was limited so that each culture in 2 insects. Fraenkel and Printy (2 ) contained 40 to 60 larvae when the eggs found that Tribolium confusum utilizes hatched. The eggs were collected from fully or partially the D-forms of methio Riverside-Canton-S hybrid females that nine, phenylalanine, and possibly, lysine, had been mated to Oregon-R males. Lar whereas DeGroot (3 ) found Apis mel- vae of the 3 D. melanogaster strains and lificia capable of utilizing D-methionine, those derived by means of the three-way D-phenylalanine, and, to some extent, d- cross between the strains have been used histidine. in other nutrition studies, ( 5 - 9 ) . Cultures Drosophila melanogaster larvae, in ad were maintained at 23.8 ± 1.1° and 45% dition to the 10 essential amino acids, relative humidity with a 10-hour day and are reported to benefit by supplementation 14-hour night. of the diet with L-cystine and glycine The growth requirement for an L-amino ( 4 ) .2 The present study was conducted to acid and the utilization of its D-isomer find which of the D-enantiomorphic forms were tested by a five-part experiment in of the essential amino acids can be utili which the media were prepared and auto zed for growth by D. melanogaster larvae claved at the same time and the larvae for in place of their L-isomers. The nutritive all cultures were derived from eggs col values of L-cystine, L-tyrosine, and glycine lected over the same period of time. In for larval growth were also examined. one part of the series larvae were fed a METHODS AND MATERIALS Received for publication April 20, 1966. 1 This investigation was supported by National Sci The standard medium (table 1) was ence Foundation Grants GB-2239 and GB-4838. 2 Rudkin, G. T., and J. Schultz 1947 Evolution of the same as that used in previous studies nutritional requirements in animals: amino-acids es sential for Drosophila melanogaster. Anat. Rec., 9 9 : ( 5 -7 ) except that all amino acids, unless 613 (abstract). J. N u t r it io n , 90: ’66 31 3 2 B. W . GEER T A B L E 1 amount of the L-amino acid was fed, but Composition of the standard test medium it was supplemented with a quantity of mg the D-form sufficient to raise the total Amino acids amount of the amino acid to the same L-Arginine • HC1 80 level as in the optimal diet (part 2 ). The L-Cystine 30 diets fed to larvae in parts one and four 840 L-Glutamic acid were supplemented with L-glutamic acid Glycine 40 L-Histidine-HCl 100 to make all diets isonitrogenous. Optimal L-Isoleucine 300 levels of the L-amino acids were the quan L-Leucine 200 tities in the standard diet. This amino 190 L-Lysine-HCl acid mixture had been compared with L-Methionine 80 L-Phenylalanine 130 several others and was found to be the L-Xhreonine 200 most adequate of the mixtures for D. 1,-Tryptophan 50 melanogaster larval growth ( 7 ). 80 L-Tyrosine Larval growth was assessed by several L-Valine 280 criteria: larval dry weight, survival of Other components larvae to pupation and to eclosion, and the Agar 1500 duration of the larval growth period. For Sucrose 1000 Yeast ribonucleic acid 100 dry-weight determinations, larvae from 5 Cholesterol 30 cultures were heat-killed after a 10-day Thiamine HC1 0.2 growth period, dried at 100° overnight, Nicotinic acid 1.2 and weighed. To minimize the weighing Riboflavin-5'-phosphate, Na 1.0 Calcium pantothenate 1.6 error, the larvae from each culture were Pyridoxine-HCl 0.25 dried and weighed together. The number Biotin 0.03 of larvae included in each dry-weight Folic acid 1.0 sample ranged from 200 to 300. Choline chloride 8.0 FeS04 1.0 Survival of larvae to pupation was de CaCl.2 1.29 termined by counting the number of lar MgS04-7H2O 24.6 vae to hatch from eggs in each culture and MnS04 H20 1.29 then scoring daily the numbers of larvae to NaHCOs 100 k h ,p o 4 183 pupate. The number of larvae to become Na2HP04 189 adults was subsequently determined for each culture. The duration of the larval ml Water to 100 growth period was the time in days that elapsed between the inoculation of cultures 1 The vitamins, yeast nucleic acid, cholesterol, choline chloride, agar, sucrose, and all l - and D-amino with eggs and the time of pupation. Each acids except D-cystine. D-isoleucine, D-lysine, and d - culture was scored every day and the num lysine were obtained from Nutritional Biochemicals Corporation, Cleveland. The D-forms of cystine, iso ber of new pupae recorded. From these leucine, lysine, and threonine were purchased from Sigma Chemical Company, St. Louis. The salts were data the mean length of the growth period provided by J. T. Baker Chemical Company, Phillips- for larvae fed the experimental diet was burg, New Jersey. calculated. Each determination of larval diet lacking the amino acid; larvae in a survival and growth period length was second part were fed an optimal level of based upon the observation of at least 400 the L-amino acid; and a diet containing an larvae. equivalent amount of the D-form of the RESULTS amino acid was fed to larvae in a third Readily utilized B-amino acids. Three part. The 2 remaining parts of the series of the D-amino acids replaced their l - were designed to determine whether the counterparts to some extent in the diets of D-counterpart can spare the dietary re D. melanogaster larvae (table 2 ) , D-phe- quirement for the L-amino acid. In part nylalanine being the most effective. Lar four, a suboptimal level of the L-isomer vae fed D-phenylalanine survived to pupa was fed; depending upon the amino acid, tion and to eclosion almost as well and the amount of L-isomer fed was 10 to 20% grew at a rate slightly less rapid than lar of the optimal quantity. In the remaining vae fed an equivalent amount of L-phenyl- part of the experiment, a suboptimal alanine. D-AMINO ACID UTILIZATION BY DROSOPHILA 3 3 T A BLE 2 d-Amino acids that are readily utilized for growth by D. melanogaster larvae Larvae to Supplement Larvae to become Growth Dry wt pupate adults period 1 at 10 days 2 mg/100 ml % % days mg x 100 Phenylalanine-deficient medium None 0 0 130 mg L-Phenylalanine 93.0 86.9 12.9± 1.0 24.4 130 mg D-Phenylalanine 93.0 83.2 13.0 ± 1.0 21.5 15 mg L-Phenylalanine 69.3 66.5 19.4 ± 1 .5 4.3 15 mg L-Phenylalanine and 115 mg D-phenylalanine 88.7 77.5 12.7 ±1 .1 23.6 Methionine-deficient medium None 0 0 80 mg L-Methionine 91.4 79.3 12.2 ± 1.2 21.3 80 mg D-Methionine 71.8 64.6 12.1 ±1 .8 17.0 10 mg L-Methionine 64.3 58.5 18.6 ± 2.5 8.4 10 mg L-Methionine and 70 mg D-methionine 73.0 67.9 12.5 ±1 .8 23.8 Histidine-deficient medium None 0 0 100 mg L-Histidine-HCl 93.2 82.4 12.0 ± 1.1 23.6 100 mg D-Histidine-HCl 9.7 2.6 28.9 ± 3 .3 2.7 15 mg L-Histidine-HCl 84.1 72.2 17.1 ± 1.9 9.6 15 mg L-Histidine-HCl and 85 mg D-histidine HC1 87.5 74.8 12.6 ±1.1 22.2 1 Figures represent mean + s d . - Each figure is the mean of the larvae from 5 cultures. The D-form of methionine was utilized their L-form but failed to promote meas more than 70% as effectively as its l - urable growth when fed as the sole dietary isomer, this being true for all the growth source of the amino acid (table 3 ). characteristics measured. The growth D-Arginine exerted a marked sparing period was as short for D-methionine-fed effect on the dietary requirement for its larvae as for L-methionine-fed larvae, on L-isomer. When D-arginine was fed with the average, but there was more variation a growth-limiting quantity of L-arginine, between larvae. At 10 days, D-methionine- the frequencies of larvae to pupate and to fed larvae were smaller than those fed become adults were several times greater L-methionine and fewer survived to pupa than when larvae were fed only a limit tion and to eclosion. ing amount of L-arginine. Also, the ob D-Histidine replaced L-histidine in the served weights of 10-day-old larvae and the diet of D. melanogaster larvae to a limited lengths of the larval growth periods fur extent. Although larvae fed only D-his- ther evidenced the sparing effect of d - tidine grew very slowly, a small percent arginine for the L-arginine growth require age survived to eclosion. Adults raised ment. with a D-histidine diet were successfully D-Lysine spared the growth requirement mated. D-Histidine exerted a marked spar for its L-isomer. When both d - and L - ing effect on the growth requirement for lysine were fed, nearly twice as many L-histidine when the d- and L-forms were larvae survived to pupation and to eclo fed in the same diet; D-histidine increased sion when fed a suboptimal level of l - the weights of 10-day-old larvae and short lysine. Furthermore, 10-day-old larvae fed ened the larval growth period. d - and L-lysine had weights more than Moderately utilized n-amino acids, d - twice the magnitude of larvae fed L-lysine. Arginine, D-lysine and D-valine stimulated The larval growth period was also much the growth of D. melanogaster larvae shorter when the D-form was present in when fed with suboptimal quantities of the diet with L-lysine. 3 4 B. W . GEER T A B L E 3 D-Amino acids that spare the growth requirement of D. melanogaster larvae for their v-isomer Larvae to Larvae to Growth Dry wt Supplement become at 10 days pupate adults period 1 mg/100 ml % % days mg x 100 Arginine-deficient medium None 0 0 80 mg L-Arginine-HCl 87.8 83.4 12.4 ± 1.1 22.6 80 mg D-Arginine-HCl 0 0 16 mg L-Arginine-HCl 6.5 2.0 29.9 ± 2 .2 1.7 16 mg L-Arginine-HCl and 64 mg D-arginine HCl 40.9 21.6 24.8 ±1 .9 4.2 Lysine-deficient medium None 0 0 190 mg L-Lysine-HCl 87.1 82.9 11.9 ± 1.1 23.0 190 mg D-Lysine-HCl 0 0 20 mg L-Lysine-HCl 44.8 35.5 28.6 ± 2 .0 1.7 20 mg L-Lysine-HCl and 170 mg D-lysine-HCl 73.5 63.8 20.8 ± 2 .0 4.0 Valine-deficient medium None 0 0 280 mg L-Valine 84.8 81.3 12.1 ± 1 .1 22.5 280 mg D-Valine 0 0 30 mg L-Valine 82.8 72.4 1 8 .2 ± 1.8 11.2 30 mg L-Valine and 250 mg D-valine 79.7 75.8 15.5 ± 1 .7 15.6 1 Figures represent mean ± s d . 2 Each figure is the mean of the larvae from 5 cultures. Under the test conditions D-valine ex mances of larvae fed the test diets. The erted only a slight sparing influence on D-preparation of isoleucine was a mixture the L-valine growth requirement. Via of D-isoleucine and D-alloisoleucine; thus bilities of larvae fed either a suboptimal neither diastereomer effectively spared the level of L-valine or the same level of l- L-isoleucine growth requirement. valine supplemented with D-valine were D-Threonine had little effect upon essentially the same. However, the mean growth. Larvae maintained on a sub weight of 10-day-old larvae and the growth optimal level of L-threonine grew as well period were significantly 3 altered by the as those fed the suboptimal amount of l - presence of D-valine in the diet with l- threonine supplemented with D-threonine valine (P < 0.01 for both values). (table 4 ). Unavailable D-amino acids. D-Isoleu- Growth-inhibiting D-amino acids, d - cine can not replace L-isoleucine in the Leucine and D-tryptophan inhibited the diet and it is doubtful whether it spares growth of D. melanogaster larvae at the the growth requirement for L-isoleucine levels fed in the test diets (table 5 ). d - (table 4 ). When considered individually Leucine exerted the greater inhibitory in the differences between larval survival fluence. When larvae were fed D-leucine frequencies, larval dry weights, and growth with a suboptimal quantity of L-leucine, periods are questionable due to a great less than half as many larvae became deal of overlapping of individual deter adults as when fed only the limiting minations. Nevertheless, when diets con amount of L-leucine. The period of larval taining 2 suboptimal quantities of L-iso- growth was lengthened by 6.4 days and leucine and L-isoleucine plus D-isoleucine the 10-day-old larva dry weight was re other than that shown in table 4 were duced by 80% when D-leucine was fed tested, small but consistent differences with L-leucine. were noted between the growth perfor 3 Student’s t test. D-AMINO ACID UTILIZATION BY DROSOPHILA 35 TABLE 4 D-Amino acids that do not effectively spare the growth requirement of D. melanogaster larvae for their L-isomer Larvae to Supplement Larvae to become Growth Dry wt pupate adults period 1 at 10 days 2 mg/100 ml % % days mg x 100 Isoleucine-deficient diet None 0 0 300 mg L-Isoleucine 92.4 84.9 1 1 .6 + 1 .1 23.8 300 mg D-Isoleucine 0 0 30 mg L-Isoleucine 48.1 35.3 19.8 + 3.7 4.0 30 mg L-Isoleucine and 270 mg D-isoleucine 56.5 43.8 19.2 + 3.1 5.1 Threonine-deficient diet None 0 0 200 mg x,-Threonine 89.1 77.4 1 2 .5 ± 1.3 26.1 200 mg D-Threonine 0 0 25 mg L-Threonine 60.3 52.7 1 8 .4 + 1 .8 5.9 25 mg L-Threonine and 175 mg D-threonine 55.8 49.3 16.4 + 2.3 6.3 1 Figures represent mean -+- s d . 2 Each figure is the mean of the larvae from 5 cultures. TABLE 5 d-Amino acids that inhibit the growth of D. melanogaster larvae Larvae to Growth Supplement Larvae to become Dry wt pupate adults period 1 at 10 days 2 mg/100 ml % % days mg x 100 Leucine-deficient medium None 0 0 200 mg L-Leucine 87.3 84.3 12.1 + 1.1 23.3 200 mg D-Leucine 0 0 40 mg L-Leucine 81.7 74.5 1 6 .0 + 1 .6 11.1 40 mg L-Leucine and 160 mg D-leucine 36.6 31.7 22.4 + 5.3 2.1 Tryptophan-deficient medium None 0 0 50 mg L-Tryptophan 86.6 82.3 1 1 .8 + 1 .0 24.6 50 mg D-Tryptophan 0 0 5 m g L-Tryptophan 83.9 69.3 20.4 + 2.4 6.9 5 mg L-Trvptophan and 45 mg D-tryptophan 52.6 41.7 23.5 + 2.5 2.5 1 Figures represent mean J s d . 2 Each figure is the mean of the larvae from 5 cultures. The antagonism of D-tryptophan to D. melanogaster larvae in place of L-cys- larval growth affected the viability, dry tine can only be determined with diffi weight, and growth rate of larvae. D-Tryp- culty because L-cystine is not required for tophan reduced by one-third the frequency larval growth (table 6 ). Addition of l - of larvae to become adults, whereas the cystine to the diet improved larval sur larval growth period was extended by 3.1 vival to the adult stage significantly4 days and the dry weight of 10-day-old lar (P < 0 .0 5 ) but did not shorten the growth vae was reduced to two-thirds that of lar period; in fact, the growth period was ex vae fed only the limiting quantity of L- tended slightly. That D-cystine was uti tryptophan. lized for growth was evidenced by the Cystine, glycine, and tyrosine. Whether D-cystine can be utilized for growth by 4 Student’s t test. 3 6 B. W . GEER improved viabilities of larvae fed either DISCUSSION D-cystine or D-cystine plus a suboptimal D-Methionine and D-phenylalanine are amount of L-cystine as compared with effective replacements for their L-enan- those fed no cystine or a suboptimal tiomorphs in the diets of D. melanogaster, amount of L-cystine. However, only when T. confusum ( 2 ) , and A. mellificia (3 ) . the latter diet was fed was the improve Drosophila, like Apis, is able to utilize ment in larval survival to the adult stage D-histidine to a moderate degree in place significant5 (P < 0 .0 5 ). In the cystine ex of L-histidine. periment there were no significant differ Fraenkel and Printy (2 ) obtained in ences between the growth periods or the consistent results when testing D-lysine in dry weights of larvae fed the different the diet of Tribolium. In one experiment test diets. D-lysine appeared to be effective but in a Addition of glycine to the diet improved second experiment it was an ineffective larval growth (table 7 ). The frequencies substitute for its L-counterpart. D-Lysine of larvae to pupate and to become adults spares the growth requirement for L-lysine were higher; the larvae were one-third when fed to D. melanogaster larvae but it larger at 10 days; and the growth period cannot promote the growth of larvae ap was shortened by 2 days when glycine was preciably when fed as the only source of fed. dietary lysine. D-Lysine is an ineffective L-Tyrosine had little effect on larval nutrient for Apis (3 ). viability or the length of the growth period In the present experimentation D-valine (table 7 ). Larval dry weight, however, and D-arginine spared the dietary require- was increased when L-tyrosine was fed. 5 Student’s t test. TABLE 6 Utilization of cystine for growth by D. melanogaster larvae Larvae to Dry wt Supplement Larvae to become Growth pupate adults period 1 at 10 days : mg/100 ml % % days mg x 100 Cystine-deficient medium None 78.7 68.5 11.6 ± 1.4 23.7 30 mg L-Cystine 88.5 83.2 12.3 ± 1.3 24.3 30 mg D-Cystine 81.6 75.2 11.9 ± 1.2 23.4 5 mg L-Cystine 75.2 69.9 11.8 ± 1.5 25.2 5 mg r-Cystine and 25 mg D-cystine 84.7 81.0 12.5 ± 1 .3 22.8 1 Figures represent mean ± s d . 2 Each figure is the mean of the larvae from 5 cultures. TABLE 7 Utilization of glycine and tyrosine for growth by D. melanogaster larvae Larvae to Supplement Larvae to become Growth Dry wt pupate adults period 1 at 10 days 2 mg/100 ml % % days mg x 100 Glycine-deficient medium None 82.7 61.2 14.3 ± 1.8 16.7 40 mg Glycine 88.5 83.2 12.3 ±1 .3 24.3 Tyrosine-deficient medium None 92.5 82.4 14.0 ± 1.8 17.3 80 mg L-Tyrosine 92.0 84.1 13.5 ± 1 .2 21.7 1 Figures represent mean ± s d . 2 Each figure is the mean of the larvae from 5 cultures. D-AMINO ACID UTILIZATION BY DROSOPHILA 3 7 ments for their L-isomers when fed to the rat. 4 ) D-Phenylalanine is readily uti Drosophila larvae. In addition, a very lized by the rat and Drosophila but is not slight sparing effect for the requirement utilized by Tetrahymena. 5) D-Leucine is for L-isoleucine may be attributed to d - used for growth to a moderate extent by isoleucine. Thus, the D-forms of lysine, the rat but has no growth-promoting ac valine, arginine, and possibly isoleucine tivity for Tetrahymena or Drosophila. 6) can be utilized for growth by Drosophila n-Histidine and D-valine are utilzied to larvae but not to the extent that the lar different extents for growth by the rat vae are able to utilize D-phenylalanine, and Drosophila but are ineffective for D-methionine, or D-histidine. D-Trypto- Tetrahymena. phan and D-leucine inhibit the growth These comparisons raise some questions of D. melanogaster larvae, whereas d - of evolutionary interest. For example, the threonine has no apparent growth influ ability to utilize D-lysine for growth ap ence. Both Apis and Tribolium are in pears to have been lost during the evolu capable of utilizing the D-forms of valine, tion of the mammals. That this is the arginine, isoleucine, leucine, threonine, case is suggested by observations on the or tryptophan for growth in place of their rat (1 1 ), the mouse (1 2 ), and man (1 3 ). L-isomers (2 ,3 ). None of these mammals are able to use Comparisons (table 8 ) between the re D-lysine in place of its L-form. sults of the present experimentation and Also, the capacity to utilize D-tryptophan studies of the protozoan Tetrahymena efficiently appears to be an almost unique pyriformis ( 1 0 ) 6 and of the rat (1 1 ) yield trait of the rat. The mouse (1 2 ) and interesting but only approximate rela chick (1 4 ) utilize D-tryptophan only to a tionships due to the differences in meth slight degree; Drosophilia, Apis, Tribolium, ods and growth criteria used: 1) D-Meth- and Tetrahymena can not utilize D-trypto- ionine is the only D-amino acid that can be phan for growth. readily utilized for growth by all 3 or Tetrahymena does not share the ability ganisms, whereas D-arginine can be used to utilize D-phenylalanine with the in for growth to some degree by each. Several sects or vertebrates. This suggests that differences are notable. 2 ) The rat utilizes the insects and vertebrates might possibly D-tryptophan nearly as well as its L-coun- have inherited the ability to utilize d - terpart for growth, whereas D-tryptophan phenylalanine from a common ancestor. is an ineffective growth-promoting sub The rat and Drosophila share the ability stance for Tetrahymena and Drosophila. to utilize D-histidine for growth, a quality 3 ) D-Lysine is utilized to some degree by not possessed by the mouse (1 2 ) but ex Tetrahymena and Drosophila but not by hibited by Apis. Thus, the capacity to utilize D-histidine is established in certain T A B L E 8 insects but may be rare in mammals. Utilization of D-amino acids for growth of T. That D-valine is utilized by the rat and pyriformis, D. melanogaster, and the rat1 Drosophila but not by Tetrahymena is D-Amino acid Tetrahymena 2 D r o s o p h i l a R a t 3 of questionable significance. Drosophila stands alone among the insects with its A r g i n i n e + + + + H i s t i d i n e — + + + ability to utilize D-valine for growth and I s o l e u c i n e — — the rat holds an equally unique position L e u c i n e — — + among the vertebrates that have been L y s i n e + + + — tested. The ability of the rat to utilize M e t h i o n i n e + + + + + + D-valine for growth is meager (1 5 ), Phenylalanine — + + + + T h r e o n i n e — — — whereas the mouse (1 6 ) and man (1 7 ) T r y p t o p h a n — — + + are unable to use D-valine for growth. The V a l i n e — + growth-promoting activity of D-valine for 1 + + indicates that the D-amino acid is readily Drosophila, as shown in the present ex utilized for growth, -f indicates that the D-amino acid is moderately available for growth, ± shows that perimentation, is limited. utilization of the D-amino acid for growth is meager, — shows that the D-amino acid is not used for growth. 2 Taken from the observations of Kidder and Dewey 6 Elliott, A. M., J. F. Hogg and C. Wu 1952 Utili (10) and Elliott et al. (see footnote 6 in text). zation of D-amino acids by Tetrahymena geleii. F e d e r a 3 T a k e n from a r e v i e w by B e r g ( 1 1 ) . tion Proc., 1 1 : 207 (abstract). 3 8 B. W . GEER The growth-promoting activities of some current study the D-amino acid in question of the D-amino acids, such as D-leucine, would have to be a better source of non- for the rat have been difficult to detect and essential nitrogen than L-glutamic acid, to quantify because of the growth an which was used to make all diets isonitro- tagonisms between certain of the D-forms. genous and which was included in all diets DL-Norleucine, for example, inhibits the in large quantities. Therefore, since the growth response of the rat to D-leucine, L-form of the amino acid was the limiting whereas D-leucine reduces the growth re factor in the test diets and not the quan sponse to D-valine (1 5 ). Presence in the tity of nonessential amino acid, it is more diet of any of several D-amino acids in probable that the growth-stimulating ca hibits the growth-stimulating activity of pacities of the D-amino acids in the cur D-histidine, and the presence en masse of rent experiments were due to the con several of the poorly invertible D-forms version of the D-amino acids to their l - antagonizes the growth promotion of the isomers. rat by the D-isomers of methionine, phe Each of the 3 nonessential amino acids nylalanine, and tryptophan (18, 19). Diets examined in the present investigation im used in the cited studies have in some proved the growth of D. melanogaster lar cases included portions of one or more vae when added to the diet. L-Cystine, al D-amino acids, usually as DL-mixtures, though necessary for optimal larval via other than the one being tested and this bility, is not essential for an optimal rate must be regarded as a limitation and pos of growth by D. melanogaster larvae. Cys sible source of error in the above com tine apparently does not stimulate larval parisons. growth by sparing the methionine require Protozoans, T. pyriformis in particular, ment. The pathway from cystine to are being used more and more in nutri methionine does not appear to operate in tional studies (2 0 ). The amino acid re Drosophila since neither homocysteine nor quirements of Tetrahymena (21) are simi homocystine can replace methionine to a lar to those of the higher invertebrates measurable extent in larval diets ( 5 ). and vertebrates but it should be kept Furthermore, larval growth is not im in mind that there are differences be proved by the addition of methionine in tween the amino acid metabolisms of these quantities exceeding that of the standard groups of organisms, as shown by the comparisons of the previous paragraphs. diet. L-Tyrosine is needed by D. melanogaster Drosophila appears capable of utilizing D-cystine for growth in place of its L-form. larvae to grow at the optimal rate. It is difficult to assess this quality because Whether tyrosine spares the growth re near-optimal development is possible when quirement for phenylalanine in Drosophila cystine is omitted from the diet. However, has not been examined. Golberg and addition of either l- or D-cystine improves de Meillon (24) found Aedes aegypti ca larval viability, D-cystine being slightly pable of growth with a diet lacking either less effective than L-cystine. Experiments phenylalanine or tyrosine but not both of du Vigneaud et al. (2 2 ) showed that of these amino acids, indicating that this D-cystine cannot replace L-cystine for the insect is capable of the interconversion of purposes of growth in the rat diet. phenylalanine and tyrosine. The means by which D-amino acids pro Glycine is necessary for both optimal mote growth are unknown although en larval viability and the optimal rate of zymes capable of converting D-amino acids larval growth of D. melanogaster. Glycine to L-amino acids have been demonstrated has growth-promoting activity for many in rat tissue (1 1 , 2 3 ). The stimulation of of the Diptera. Omission of glycine from growth by D-amino acids when fed with the diets of A. aegypti (2 4 ), Agria affinis suboptimal levels of their L-forms, such as (2 5 ), and Calliphora erythrocephala (2 6 ) in the present study, is possibly due to the retards growth. Phormia regina is the only utilization of the D-amino acids as sources Dipteran studied that does not require of nonessential nitrogen. However, in the glycine for optimal growth (2 7 , 2 8 ). D-AMINO ACID UTILIZATION BY DROSOPHILA 3 9 ACKNOWLEDGMENTS 15. Gerulat, B. F., and C. P. Berg 1960 Growth promotion by D-valine and D-leucine. Arch. Grateful appreciation is extended to Biochem. Biophys., 88: 273. Julia Ricker and Jerrie Tietge for their 16. Bauer, C. D „ and C. P. Berg 1943 The technical assistance. amino acids required for growth in mice and the availability of their optical isomers. J. LITERATURE CITED Nutr., 26: 51. 1. Gilmour, D. 1961 The Biochemistry of In 17. Rose, W. C., R. L. Wixom, H. B. Lockhart sects. Academic Press, New York. and G. F. Lambert 1955 The amino acid 2. Fraenkel, G., and G. E. Printy 1954 The requirements of man. XV. The valine re amino acid requirements of the confused quirement: summary and final observations. flour beetle, Tribolium confusum, Duval. J. Biol. Chem., 217: 987. Biol. Bull., 106: 149. 18. Kamath, S. H., and C. P. Berg 1964 An 3. DeGroot, A. P. 1953 Protein and amino- tagonism of poorly invertible D-amino acids acid requirements of the honey hee ( Apis toward growth promotion by readily inver mellificia L.). Physiol. Comp. Oecol., 3: tible D-amino acids. J. Nutr., 82: 237. 197. 19. Kamath, S. H., and C. P. Berg 1964 An 4. Hinton, T., D. T. Noyes and J. Ellis 1951 tagonism of the D-forms of the essential Amino acids and growth factors in a chemi amino acids toward the promotion of growth cally defined medium for Drosophila. Physiol. by D-histidine. J. Nutr., 82: 243. Zool., 24: 335. 20. Hutner, S. H., and L. Provasoli 1964 Com 5. Geer, B. W., and G. F. Vovis 1965 The ef parative physiology: nutrition. Ann. Rev. fects of choline and related compounds on Physiol., 27: 19. the growth and development of Drosophila melanogaster. J. Exp. Zool., 158: 223. 21. Holz, G. G. 1964 Nutrition and metabo 6. Geer, B. W., and J. G. Ricker 1965 The lism of ciliates. In: Biochemistry and Physi growth effects of carnitine, deoxycarnitine, ology o f Protozoa, vol. 3, ed., S. H. Hutner. and sulfocholine for Drosophila, Neurospora, Academic Press, New York, pp. 199-243. and Saccharomyces. Growth, 29: 405. 22. du Vigneaud, V., R. Dorfmann, and H. S. 7. Geer, B. W. 1966 Comparison of some Loring 1932 A comparison of the growth- amino acid mixtures and proteins for the promoting properties of d- and Z-cystine. J. diet of Drosophila melanogaster. Trans. Biol. Chem., 98: 577. Illinois Acad. Sci., 59: 3. 23. Greenstein, J. P., and M. Winitz 1961 8. Geer, B. W. 1963 A ribonucleic acid-pro Chemistry of the Amino Acids, vol. 1, John tein relationship in Drosophila nutrition. J. Wiley and Sons, New York, p. 320. Exp. Zool., 154: 353. 24. Goldberg, L., and B. de Meillon 1948 The 9. Geer, B. W. 1964 Inheritance of the di nutrition of the larva of Aedes aegypti Lin etary ribonucleic acid requirement of Dro naeus. Biochem. J., 43; 379. sophila melanogaster. Genetics, 49: 787. 25. House, H. L. 1954 Nutritional studies 10. Kidder, G. W., and V. C. Dewey 1951 The with Pseudosarcophaga affinis (Fall.), a biochemistry of ciliates in pure culture. In: dipterous parasite of the spruce budworm, Biochemistry and Physiology of Protozoa, Choristoneura fumiferana (Clem.). I. A vol. 1, ed., A. Lwoff. Academic Press, New chemically defined medium and aseptic- York, pp. 324-400. culture technique. Can. J. Zool., 32: 331. 11. Berg, C. P. 1959 Utilization of D-amino acids. In: Protein and Amino Acid Nutri 26. Sedee, D. J. W. 1954 Qualitative amino tion, ed., A. A. Albanese. Academic Press, acid requirements of larvae of Calliphora erythrocephala (Meigen). Acta Physiol. New York, pp. 57-96. 12. Celander, D. R., and C. P. Berg 1953 The Pharmacol. Neerl., 3: 262. availability of D-histidine, related imidazoles, 27. McGinnis, A. J., R. W. Newburgh, and V. H. and D-tryptophan in the mouse. J. Biol. Cheldelin 1956 Nutritional studies on the Chem., 202: 339. blowfly, Phormia regina (M eig.). J. Nutr., 13. Rose, W. C., A. Borman, M. J. Coon and 58: 309. G. F. Lambert 1955 The amino acid re 28. Hodgson, E., V. H. Cheldelin and R. W. quirements of man. X. The lysine require Newburgh 1956 Substitution of choline by ment. J. Biol. Chem., 214: 579. related compounds and further studies on 14. Morrison, W. D., T. S. Hamilton and H. M. amino acid requirements in nutrition of Scott 1956 Utilization of D-tryptophan by Phormia regina (M eig.). Can. J. Zool., 34: the chick. J. Nutr., 60: 47. 389. Mechanism of Suckling Rat Hypercholesterolemia: Dietary and Drug Studies 1,2,3 R. A. HARRIS,4 J. E. MacNINTCH 5 a n d F. W. QUACKENBUSH Department of Biochemistry, Purdue University, Lafayette, Indiana ABSTRACT The objective of this study was to determine the cause(s) of the hypercholesterolemia which is known to occur in suckling rats. A semi-purified, milk-simulating diet fed to 21-day-old rats upon weaning was found to maintain the hypercholesterolemic condition. With this diet, the mechanism of suckling rat hyper cholesterolemia was investigated by testing the effect of dietary levels of lactose, bulk, polyunsaturated fatty acids, cholesterol and fat. The condition appears to be depend ent upon the high fat content of rat milk but independent of the dietary bulk, polyunsaturates, and carbohydrate source. Isocaloric diets were used to establish the dependency upon the fat content of diet. Feeding experiments suggest that dietary cholesterol is not necessary for maintaining hypercholesterolemia for short periods of time, but isotopic balance studies show that the dam contributes a portion of the cholesterol found in the plasma of suckling rats. Ethyl linoleate, a-p-chlorophenoxy- isobutyrate (CPIB), benzmalecene, and |3-diethylaminoethyl diphenylpropyl acetate hydrochloride were found not to be effective as hypocholesterolemic agents in the suckling rat hypercholesterolemia. L-Thyroxine was active in lowering plasma choles terol in this system, but it increased liver free and total cholesterol. Liver wet weight, dry weight, and protein content increased in response to CPIB. Serum cholesterol levels have been ob EXPERIMENTAL served to be much higher in suckling rats Materials. Ethyl linoleate of 9 7 + % than in weaned rats ( 1 ,2 ) . Hypercholes purity was prepared from safflower o il6 as terolemia also has been observed in suck described previously ( 5 ). Test substances ling rabbits (3 ) and shown to be depend obtained commercially included the fol ent upon the high triglyceride and lowing: ethyl palmitate,7 9 9 + % purity; cholesterol content of rabbit milk ( 4 ). cholesterol-4-14C,8 4.38 mCi/mmole; and Carroll (2 ) observed rat milk to be rela L-thyroxine, cholesterol and sodium glyco- tively low in cholesterol and also noted cholate.9 The other test substances used hepatic biosynthesis of sterol to be sup were : ethyl a-p-chlorophenoxyisobutyrate pressed in the suckling rat. Many drugs (CPIB ethyl ester;10 (3-diethylaminoethyl which normally function as hypocholes diphenylpropyl acetate hydrochloride terolemic agents fail to produce a response (SK&F 525A);n sodium [N-(l-methyl-2,3- in the suckling rat hypercholesterolemia di-p-chlorophenylpropyl) maleamate] benz- *1234567811910 ( 1 )• A better understanding of control mech Received for publication March 24, 1966. 1 Journal paper no. 2831 Purdue University Agricul anisms for plasma cholesterol in the suck tural Experim ent Station, Lafayette, Indiana. ling animal may help to reveal the reasons 2 This study was supported in part by Public Health Service Grants no. HE 08424 and T1 GM 1195. for suppressed hepatic cholesterol biosyn 3 Presented in part before the m eeting of the Federa tion of American Societies for Experimental Biology, thesis and lack of response to certain hy Atlantic City, 1965 (Federation Proc., 24: 1078, 1081, 1965, abstracts). pocholesterolemic agents. The experiments 4 Present address: Institute for Enzyme Research, presented here were designed to test the University of W isconsin, M adison, W isconsin. 5 Present address: Biochemistry Department, Bow role of dietary factors in suckling rat hy man Gray M edical School, W inston-Salem, North Caro l in a . percholesterolemia. Specifically, the con 6 Contributed by Pacific Vegetable Oil Corporation, tributions of lactose, bulk, polyunsatu Richm ond, California. 7 Eastman Organic Chemicals, Rochester, New York. rated fatty acids, cholesterol and fat were 8 Tracerlab, W altham , Massachusetts. 9 Nutritional Biochem icals Corporation, Cleveland. investigated. The response to various hy 10 Ayerst Laboratories, Inc., New York. 11 Smith, Kline and French Laboratories, Philadel pocholesterolemic agents is also reported. p h i a . 40 J. N u t r it io n , 90: ’66 SUCKLING RAT HYPERCHOLESTEROLEMIA 41 malecene);12 13 hydrogenated coconut oil;13 To compare the effects of sardine oil, and sardine oil.14. ethyl linoleate and hydrogenated coconut Diets. The semi-purified diets are oil on hypercholesterolemia, weanling rats shown in table 1. Diet M-47 (4 7 % fat) were fed these fats in diet F for 8 weeks. simulated the dry-weight composition of Drug studies. Since initial studies indi rat milk as described by Luckey et al. (6 ). cated that the degree of hypercholesterol Diets M-24 and M -l 24% and 1% fat, emia in suckling rats from our colony var respectively, were isocaloric with diet M-47 ied greatly between litters but not between since minerals and vitamins were added littermates, the method described by Bizzi to each in constant ratio to carbohydrate et al. (1 ) for the assay of hypocholesterol- and fat calories as described previously emic agents in suckling rats was modified. (5). Lactating females consumed ad lib Litters of 10 rats, 7 days old, were marked itum the colony diet which consisted to divide them into 2 equal groups with chiefly of ground yellow corn, wheat mid respect to body weight and sex; all were dlings and meat meal or diet G when a left with their dams receiving the colony well-defined, semi-purified diet was de diet. Daily, for 7 days, one group of 5 sired. Diet F was similar to diet G but was force-fed, by tube, the compound be 1% cholesterol and 0.5% sodium glyco- ing tested for hypocholesterolemic activity, cholate were added to produce a hypercho and the control group of 5 was similarly lesterolemia. fed an equal volume of saline. All were Dietary studies. Suckling female rats, killed and analyzed at 13 days of age. 19 to 21 days old and weighing 39 to 45 g, Hypercholesterolemic adult rats (2 5 0 - were obtained from our colony of Wistar- 300 g ), obtained by feeding weanling rats strain rats. Groups of 6 weanlings were diet F for 6 weeks, were fed various com fed various diets ad libitum and killed pounds incorporated in the diet for 7 days. after 2 or 5 days. In certain studies the Polyunsaturated fatty acids of rat milk. diet of the lactating females was changed Methyl esters were prepared with diazo at parturition from the colony diet and methane (7 ) from the saponifiable frac diet G with either hydrogenated coconut tion of a chloroform-methanol extract (8 ) oil or ethyl linoleate as the fat source. The of the milk curd obtained from the stom pups of these litters were killed at 21 days achs of 14-day-old suckling rats of several of age. litters from the colony diet. The fatty T A B L E 1 acids were identified by comparison of Composition of diets retention times with those of known stand ards on 2 columns (152-cm and 304-cm D ie t s 1»2 M -4 7 M -2 4 M - l F G [5-ft and 10-ft] diethylene glycol succinate % % % % % on firebrick; Barber-Coleman Model 10 gas F a t 4 7 2 4 i 2 2 chromatograph ). C a s e i n 3 3 2 2 7 2 1 1 5 1 5 Cholesterol-4-14C pellet study. The pro G l u c o s e m onohydrate 4 — — — 7 6 7 7 cedure for producing an isotopic steady L a c t o s e 1 7 4 6 7 5 — — state in the rat was essentially that de Cellulose 5 — — — 1 .7 1 . 7 scribed by Wilson ( 9 ) . A gelatin pellet of Cholesterol — — — 1 . 0 — 100 mg of cholesterol-4-14C (50,000 cpm/ S o d i u m m g) was implanted dorsally under the glycocholate — — — 0 . 5 — skin of a mature female rat; 4 weeks later 1 M inerals and vitam ins w e r e added to each d i e t i n constant ratio to carbohydrate and fat calories (5). the female was mated. One of its pups 2 Diets (see text for complete description: was killed at parturition, before nursing. M-47, 47% fat, simulating dry-weight com position of rat milk Three remaining pups were killed at 15 M-24, 24% fat, isocaloric with diet M-47 M -l, 1% fat, isocaloric with diet M-47 days of age, after 30 minutes of nursing F, semi-purified + 1% cholesterol and 0.5% subsequent to a 1-hour fast, and the milk sodium glycocholate G, sem i-purified curds from their stomachs were pooled Colony, ground yellow com , wheat m iddlings and m e a t m e a l . for analysis. The specific activities of 3 Casein was extracted continuously for 72 hours with boiling ether to remove fat. 12 Merck, Sharp and Dohme, West Point, Pennsyl 4 Cerelose, Com Products Company, Argo, Illinois. v a n i a . 5 Cellu Flour. Chicago Dietetic Supply House, Chi 13 Procter and Gamble Company, Cincinnati. c a g o . 14 M aine Sardine Industry, Bangor, Maine. 4 2 R. A. HARRIS, J. E. MacNINTCH AND F. W. QUACKENBUSH cholesterol from the whole blood, plasma The colony diet, consisting principally and milk of the dam and from the pooled of ground cereal grains, is well supplied plasma of the suckling rats were deter with unsaturated fatty acids. The lipid mined. The percentage of cholesterol in extract from milk curd, pooled from the the plasma of the pups which originated stomachs of suckling rats from the colony, from the female was estimated from suit contained 6.1% of 18:215 and 0.1% of able equations. 2 0 :4 by gas chromatographic analysis. Analysis. Lipids were extracted from When suckling rats were weaned at 20 plasma and tissues as described previously days of age and fed the colony diet, plasma ( 8 ) . Cholesterol was precipitated (after cholesterol fell quickly to near normal val hydrolysis for the total) as the digitonide ues (table 2 ). Diet G with either ethyl which was washed as described by Sperry linoleate or hydrogenated coconut oil pro and Webb (1 0 ). The digitonides were duced comparable responses at 2 days; dissolved in acetic acid for colorimetric de however, linoleate produced lower values termination and in methanol for assay of after 5 days (P < 0 .0 0 1 ). Fasting pro radioactivity. Aliquots of the methanolic duced a linear fall in plasma cholesterol solutions of the digitonides were added to levels over the 5-day period. Diet M-47, 15 ml of a toluene scintillation fluid (0.1 which simulated the dry weight composi g/liter 1,4-bis-2- (5-phenyloxazole)-benzene tion of rat milk, was found to maintain and 4 g/liter 2,5-diphenyloxazole) and as the hypercholesterolemia at a level (table sayed for radioactivity with a liquid scintil 2 ) which is characteristic of 21-day-old lation counter (Packard). Samples were suckling rats. When 2 groups of suckling routinely corrected for quenching with an rats were weaned with colony diet for 48 internal standard of cholesterol-4-1'lC. Pro hours and then forced to either (a ) resume tein was determined by the Folin-Ciocalteu nursing lactating females or (b) consume method as modified by Lowry et al. (1 1 ). diet M-47, both groups returned to a state of hypercholesterolemia after 96 hours RESULTS (suckling 142 ± 4 16 17 m g /1 0 0 m l; diet M-47, Although the plasma cholesterol levels 119 ± 2 mg/100 ml). of both newly weaned and young adult Several alterations in the basic composi rats in our inbred Wistar colony commonly tion of diet M-47 produced no significant range between 50 and 70 mg/100 ml, the effect (table 3 ). These include cholesterol level in suckling rats at 13 days was found addition to the diet at the level found in to be 157 ± 7 m g /1 0 0 ml and at 21 days, rat milk; replacing a portion of the hy 138 ± 10 mg/100 ml (mean of 5-7 and drogenated coconut oil with ethyl linoleate; s e ) . N o increase at 21 days was observed substitution of sucrose for lactose; and when the dam was fed a diet containing the addition of 20% bulk to the diet in hydrogenated coconut oil as the only fat the form of cellulose.” However, replac- source. A decrease in plasma cholesterol 15 Carbon chain length: num ber of double bonds. level between 13 and 21 days is charac 16 s e of the mean for 6 animals. 17 Cellu Flour, Chicago Dietetic Supply House, Chi teristic of this hypercholesterolemia (2 ). c a g o . T A B L E 2 Effect of colony and purified diets on hypercholesterolemia of the suckling rat Plasma total cholesterol Diet1»2 Type of fat 2 days 5 days mg/100 ml mg/100 ml Colony — 6 9 ± 4 3 68 ± 3 G ethyl linoleate 77 ± 3 48 ± 4 G hydrogenated coconut oil 63 ± 4 73 ± 3 Fasting — 61 ± 5 34 ± 1 2 M-47 hydrogenated coconut oil 143 ± 7 13 0 ± 6 1 Suckling rats were weaned with the respective diets and killed after 2 or 5 days. 2 See table 1, footnote 2, for description of diets. 3 Average ± s e of mean for 6 rats/group. SUCKLING RAT HYPERCHOLESTEROLEMIA 4 3 T A B L E 3 Effect of various alterations in diet M-47 upon hypercholesterolemia in the suckling rat Plasma total cholesterol Alteration 1 Added at expense of 2 days 5 days % mg/100 ml mg/100 ml None — — 143 ± 7 2 130± 6 Cholesterol 0.06 — 134 ± 4 142 ± 1 2 Ethyl linoleate 2.0 HCO 3 131 ± 8 152± 10 Sardine oil 24 HCO 187 ± 21 247 ± 2 9 Sucrose 17 lactose 107 ± 8 125 ± 6 Cellulose 4 2.0 — 128 ± 8 139± 7 Cellulose 20 — 133 ± 9 132 ± 8 1 Alterations are based upon diet M-47 (see table 1, footnote 2 for description) with hydrogenated coconut oil as the fat source. 2 Average ± s e for 6 rats/group. 3 HCO, hydrogenated coconut oil. 4 Cellu Flour. ing half of the hydrogenated coconut oil When these same agents were assayed in diet M -47 with sardine oil produced a with groups of 6 or 7 adult rats that had hypercholesterolemic response (P < 0.01 at dietary induced hypercholesterolemia, l - 5 days, compared with diet M -47). How thyroxine and SK&F 525A were very effec ever, the sardine oil and ethyl linoleate tive in reducing plasma cholesterol levels. used in this study produced lower plasma CPIB was less effective and benzmalecene cholesterol levels than hydrogenated coco was without significant effect. Initial and nut oil (191 ± 19, 109 ± 14 and 324 ± final plasma cholesterol levels were : L-thy 64 m g /1 0 0 ml, respectively) after 8 weeks roxine, 342 to 121; SK&F 525A, 272 to when fed at 2% (diet F ) to weaned rats. 109; CPIB, 291 to 193; benzmalecene, Removing half of the fat from diet M-47 265 to 225; and no treatment, 373 to 352 to give diet M -24 did not produce a signifi mg/100 ml. The compounds were fed for cant response, but diet M -l (1 % fat as a 7-day period, thyroxine at 0.003% and hydrogenated coconut oil) reduced plasma all others at 0.15% of the diet. cholesterol to normal (table 4 ). The addi Data from the cholesterol-d-^C pellet tion of bulk to the diet or substitution of experiment showed that 37% of the cho sucrose for lactose did not increase the lesterol in the plasma of the suckling rats,, hypocholesterolemic response. Levels were and 30% of the cholesterol in the pup slightly higher with the sucrose diets, killed at parturition, had originated in the when compared with lactose (P < 0 .0 5 ). dam. The equation used for the plasma Ethyl palmitate (1 0 m g/d ay) and ethyl was: linoleate (10-25 mg/day), force-fed for 7 % Plasma cholesterol from dam = days, had no effect upon plasma and liver specific activity of suckling rats’ plasma cholesterol cholesterol levels of suckling rats (table 100 X —______------5 ). L-Thyroxine was an effective hyper specific activity of milk cholesterol cholesterolemic agent, as shown previously derived from by Bizzi et al. ( 1 ) . However, in the pres % Plasma cholesterol from dam = ent study, L-thyroxine increased both free cholesterol (mg) in plasma originating jqq from milk and total cholesterol of the liver signifi A total cholesterol (mg) in suckling cantly. None of the other drugs lowered rat’s plasma the plasma cholesterol. Benzmalecene sig where nificantly increased plasma free choles Cholesterol (mg) in plasma originating terol. CPIB and SK&F 525A increased from milk = total lipids. CPIB decreased liver choles counts/min in suckling rat’s plasma terol levels, but this decrease was coin cholesterol cident with an increase in liver size. In an specific activity of milk cholesterol other experiment CPIB significantly (P < A similar equation was used to calculate 0.001) increased the liver wet weight, dry the percentage for the pup killed at partur weight and protein content. ition. Calculations were based upon the 4 4 R. A. HARRIS, J. E. MacNINTCH AND F. W. QUACKENBUSH T A B L E 4 Effect of various diets which were isocaloric with diet M-47 upon hypercholesterolemia in the suckling rat Total Added at plasma Diet1 Alteration 2 expense of cholesterol at 5 days % mg/100 ml M-47 None — — 142 ± 4 3 M-24 None — — 148 ± 9 M-l None — — 55 ± 5 M-l Cellulose 4 20 — 61 ± 5 M-l Sucrose 75 lactose 76 ± 4 M-l Sucrose + cellulose 75 lactose 20 83 ± 5 1 See table 1, footnote 2, for description of diets. 2 Alterations are based upon Diet M -l with hydrogenated coconut oil as the fat source. 3 A v e r a g e ± s e for 6 rats/group. 4 C e l l u F lo u r . T A B L E 5 Plasma and liver responses of suckling rats to various test substances Cholesterol found in ÏM. Test substance 1 Level Plasma Liver liver force-fed Body wt lipid Total Free Total Free mg/day mg/100 ml mg/g % Control (saline) 0.028 224 60 2.85 2.32 4.08 Ethyl palmitate 10 0.027 220 58 2.99 2.34 4.31 Control (saline) 0.029 172 50 2.70 2.34 4.54 Ethyl linoleate 10 0.030 157 45 2.80 2.37 4.77 Control (saline) 0.036 179 56 — — ____ Ethyl linoleate 25 0.036 185 53 — — — Control (saline) 0.028 109 34 2.72 2.21 4.30 L-Thyroxine 0.01 0.030 80 2 28 3.51 3 2.83 3 4.69 Control (saline) 0.028 145 41 2.81 2.63 4.38 CPIB 4 10 0.041 3 151 48 2.36 3 2.30 3 5.07 3 Control (saline) 0.028 147 36 2.87 2.58 4.15 Benzmalecene 2 0.032 161 75 2 2.85 2.77 4.26 Control (saline) 0.030 187 49 2.97 2.21 4.54 SK & F 525A 5 2 0.034 180 50 3.39 2.34 5.99 3 1 Five control animals (1 week old) were force-fed saline daily for 1 week. Five littermates received the test substance. 2P between 0.05 and 0.01 (probably significant). 3 P < 0.01 (highly significant). 4 Ethyl a-p-chlorophenoxyisobutyrate. 5 /3-Diethylaminoethyl dephenylpropyl acetate hydrochloride. assumption that the dam was in an isotopic DISCUSSION steady state throughout the experiment. In diet M-47, which simulates the dry The ratios of specific activity of dam’s weight composition of rat milk and which plasma cholesterol to the dam’s whole was found to maintain rats in a state of blood cholesterol and to the dam’s milk hypercholesterolemia, the most important cholesterol, 0.91 and 1.01, respectively, factor appears to be a high fat content. indicated equilibration between milk and Friedman and Byers (4 ) observed hyper tissue cholesterol of the dam. cholesterolemia of the suckling rabbit to SUCKLING RAT HYPERCHOLESTEROLEMIA 4 5 be dependent upon the cholesterol fur The hypercholesterolemia of the suck nished by the milk, and the high fat or ling rat is distinctive in its failure to re triglyceride level of normal rabbit milk spond to either ethyl linoleate or to other was also clearly shown to be necessary for polyunsaturates. Sardine oil, which is maintaining the rabbit in a state of hy known to have hypocholesterolemic prop percholesterolemia. Since the cholesterol erties (1 3 ), produced an increase in content of milk from the rat is much lower plasma cholesterol when fed in diet M-47 than that from the rabbit and since hyper to the suckling rat, perhaps due to the cholesterolemia was sustained with the more pronounced effect of its cholesterol high fat, cholesterol-free diet (M -47 ), the (15 m g /g ). primary factor in hypercholesterolemia of Turnover of cholic acid has been shown the suckling rat appears to be high dietary to be slow in rats fed diets containing lac fat. Dietary and prenatal transfer of cho tose or diets low in bulk (1 4 ). Rats fed lesterol may be important factors for ini diets which tend to promote excretion of tially building up a high plasma choles greater quantities of bile acids appear also terol level in the newborn rat. In the present to have lower serum cholesterol levels study, rats weaned with the colony diet (14—16). Bloomfield (1 7 ) observed the were rapidly cleared of hypercholesterol quantity of bile acid eliminated to correl emia; however, it was restored by chang ate with the quantity of fecal residue pro ing to diet M-47. These results indicate duced by a diet. He concluded that a cho that cholesterol from the milk is not essen lesterol-accumulating diet should be well- tial for this hypercholesterolemia. refined, high in calories, and low in fecal The amount of cholesterol ingested by residue. These are characteristics of rat suckling rats in a 24-hour period can be milk and diet M-47 both of which yield estimated. The cholesterol content of rat slow rates of cholic acid excretion.19 A milk on a dry weight basis is approxi large fecal residue is probably a factor in mately 0.06% (2). Newly weaned rats the rapid lowering of plasma cholesterol (21 days old) fed diet M-47 consume ap when suckling rats are weaned with a proximately 4.2 g of diet/day. If suckling colony diet; this lowering may be related rats consume a comparable number of to the observed increase in cholic acid calories, the milk would provide 2.5 mg of excretion rates on weaning.20 However, cholesterol to each rat/day. Therefore, the presence of lactose and lack of bulk in even though the cholesterol content of milk rat milk were shown in this study not to be is low, the 21-day-old suckling rat con important factors in maintaining hyper sumes more cholesterol daily than is pres cholesterolemia in the suckling rat. Diet ent in his entire plasma.18 The cholesterol- M -l, in contrast with diet M-47, rapidly 4-1JC pellet study showed that approxi diminished plasma cholesterol levels, yet mately one-third of the plasma cholesterol the cholic acid excretion rates were simi of the suckling rats in this experiment lar.21 Therefore, the importance of a slow originated from the female. Evidently rate of cholic acid excretion in maintain both milk consumption and endogenous ing hypercholesterolemia in the suckling biosynthesis contribute significant amounts rat is uncertain. of sterol to the suckling rat hypercholes In agreement with Bizzi et al. (1 ) l - terolemia. thyroxine was found to be an effective The milk of both the rat and the rabbit plasma cholesterol-lowering agent in the is characterized by a high fat content, 40 suckling rat; however, since liver choles to 50% of the dry weight. Lower lipid terol was increased the thyroxine may have levels are found in the milk of species altered the partition of cholesterol be which are known not to have hypercho- tween plasma and tissue. The failure of lesterolemic suckling young. Among those studies only whale milk, in which lipid is 2.4 mg of cholesterol based upon 3.2 ml plasm a/ 100 g, 50 g rat, and 150 m g cholesterol/100 m l plasma. makes up 37% of the wet weight (1 2 ), is Harris, R. A., C. L. Villemez, Jr. and F. W. has a higher level. Plasma cholesterol Quackenbush 1965 Dietary studies on suckling rat hypercholesterolemia. Federation Proc., 24: 1081 (ab levels of suckling whales have not been s t r a c t ) . 20 See footnote 19. reported. 21 See footnote 19. 4 6 R. A. HARRIS, J. E. MacNINTCH AND F. W. QUACKENBUSH other hypocholesterolemic agents to lower 6. Luckey, T. D., T. J. Mende and J. Pleasants plasma cholesterol levels in suckling rats 1954 The physical and chemical charac terization of rat’s milk. J. Nutr., 54: 345. suggests that steps which are rate-limiting 7. Schlenk, H., and J. L. Gellerman 1960 in the lowering in this system are differ Esterification of fatty acids with diazo- ent from those which limit in dietary-in methane on a small scale. Anal. Chem., 32: 1412. duced hypercholesterolemia of adult rats. 8. Harris, R. A., and D. Gambal 1963 Fluo- The increase in liver total lipid with rimetric determination of total phospholipids CPIB may have been the result either of in rat tissues. Anal. Biochem., 5: 479. an accumulation of the drug or of an in 9. Wilson, J. D. 1964 The quantification of cholesterol excretion and degradation in the crease in lipid. The increase in liver wet isotopic steady state in the rat: the influence weight, dry weight and protein content of dietary cholesterol. J. Lipid Res., 5: 409. upon CPIB treatment of suckling rats 10. Sperry, W. M., and M. Webb 1950 A re vision of the Schoenheimer-Sperry method may reflect an increase in synthesis of for cholesterol determination. J. Biol. Chem., drug metabolizing enzymes (18). An in 187: 97. crease in liver weight of CPIB-treated, 11. Lowry, O. H., N. J. Rosebrough, A. L. Farr mature rats has been reported (1 9 ). and R. J. Randall 1951 Protein measure ment with the Folin phenol reagent. J. Biol. Carroll (2 ) observed subnormal choles Chem., 193: 265. terol biosynthesis from labeled acetate in 12. Garton, G. A. 1963 The composition and the suckling rat. This is characteristic of biosynthesis of milk lipids. J. Lipid Res., 4: 237. rats fed diets containing cholesterol (20, 13. Nicolaysen, R., and R. Ragard 1961 21). Evidence recently has been presented Effect of various oils and fats on serum which suggests that bile acids are the meta cholesterol in experimental hypercholesterol- bolites of cholesterol responsible for the emic rats. J. Nutr., 73: 299. 14. Portman, O. W. 1960 Nutritional influ physiological regulation of cholesterol bio ence on the metabolism of bile acids. Amer. synthesis. Bloomfield (17) observed that to J. Clini. Nutr., 8: 462. tal body sterol biosynthesis was independ 15. Danielsson, H., and B. Gustafsson 1959 ent of cholesterol feeding at low dietary On serum cholesterol levels and neutral fecal levels of cholesterol. Fimognori and Rod- sterols in germ-free rats. Bile acids and acids and steroids. Arch. Biochem. Biophys., well (22) observed a suppression of mevalo- 83: 482. nate biosynthesis by bile acids. Since the 16. Wells, W. W., and S. C. Anderson 1959 cholic acid turnover in suckling rats is The increased severity of atherosclerosis in very slow compared with that of adult rabbits on a lactose-containing diet. J. Nutr., 68: 541. animals fed a colony diet,22 hepatic bile 17. Bloomfield, D. K. 1963 Dynamics of cho acids are probably elevated in the suck lesterol metabolism. I. Factors regulating ling rat to the extent that biosynthesis of total sterol biosynthesis and accumulation sterol is suppressed. Therefore, milk cho in the rat. Proc. Nat. Acad., Sci., 50: 117. 18. Conney, A. H., C. Darison, R. Gastel and lesterol as such may not contribute to the J. J. Burns 1960 Adaptive increases in suppression of cholesterol biosynthesis in drug-metabolizing enzymes induced by phéno the suckling rat. barbital and other drugs. J. Pharmacol., 130: 1. LITERATURE CITED 19. Best, M. M., and C. H. Duncan 1964 Hypolipemia and hepatomegaly from ethyl 1. Bizzi, A., E. Veneroni and S. Garattini 1963 chlorophenoxyisobutyrate (CPIB) in the rat. Hypercholesteremia in suckling rats. J. J. Lab. Clini. Med., 64: 634. Atheroscler. Res., 3: 121. 20. Tomkins, G. M., H. Sheppard and I. L. 2. Carroll, K. K. 1964 Acetate incorporation Chaikoff 1953 Cholesterol synthesis by into cholesterol and fatty acids by livers of liver. III. Its regulation by ingested cho fetal, suckling, and weaned rats. Can. J. lesterol. J. Biol. Chem., 201: 137. Biochem., 42: 79. 21. Frantz, I. D., Jr., H. S. Schneider and B. T. 3. Bragdon, J. H. 1952 Spontaneous athero Hinkelman 1954 Suppression of hepatic sclerosis in the rabbit. Circulation, 5: 641. cholesterol synthesis in the rat by choles 4. Friedman, M., and S. O. Byers 1961 Ef terol feeding. J. Biol. Chem., 206: 465. fects of diet on serum lipids of fetal, neo 22. Fimognori, G. M., and V. W. Rodwell 1964 natal, and pregnant rabbits. Amer. J. Phys Cholesterol biosynthesis: mevalonate syn iol., 201: 611. thesis inhibited by bile salts. Science, 174: 5. Quackenbush, F. W., and M. Pawlowski 1038. 1960 Effects of purified linoleic ester on cholesterol in the rat. J. Nutr., 72: 196. 22 See footnote 19. Effect of Carbohydrate and Fat Intakes upon the Activities of Several Liver Enzymes in Rats, Guinea Piglets, Piglets and Calves 1,2 R. L. BALDWIN, M. RONNING, C. RADANOVICS a n d G. PLANGE Department of Animal Husbandry, University of California, Davis, California ABSTRACT The effects of 4 diets containing a wide range of carbohydrate and fat upon the activities of a number of liver enzymes in rats, guinea piglets, calves and piglets were investigated. The results indicated that large adaptive enzymatic changes occurred in rats fed high carbohydrate diets but not in guinea piglets, piglets and calves. The activities of most of the enzymes studied changed in logarithmic relationship to the ratio, carbohydrate calories-to-carbohydrate + fat calories, in the diets indicating that the enzymatic adaptations occurred in response to changes in proportions of energy contributed by carbohydrate and fat rather than to changes in the amounts of these components per se. The results indicated that calves, guinea piglets and piglets have much lower capacities for adaptation to high carbohydrate, low fat diets than rats, due either to species differences in rates of physiological development after birth or to other inherent species differences. The activities of all the enzymes whose functions have been related to fat synthesis were much lower in calves than in rats. The activities of a number of liver en METHODS AND MATERIALS zymes are affected markedly by changes in Male weanling rats of the Sprague the carbohydrate content of rat diets (1, 2). Dawley strain, 3-day-old male albino Several of these enzymatic changes have guinea piglets, 2-day-old male Holstein been investigated in detail and their prob calves and 3-day-old male and female able significance with respect to carbohy Duroc piglets were either purchased com drate and fat metabolism and dietary re mercially or obtained from the Univer quirements for micronutrients (1 -8 ) have sity herds. been discussed. In view of the considerable The rats were fed diets A, B, C and D difficulties that have been encountered in (table 1) upon arrival and were main attempts to develop high carbohydrate, low tained with these diets for 4 weeks prior fat diets suitable for study of the vitamin to killing for sampling. The rates of gain and mineral requirements of young calves of the rats were 4.2, 4.6, 5.0 and 5.1 g / and the apparent metabolic involvment in day with diets A through D, respectively, these difficulties (9 , 10 ), a preliminary and no difficulties with respect to feeding study was made of the effect of carbo and management were encountered. hydrate and fat intakes upon the activities The guinea piglets were fed a commer of several enzymes in calf livers. Only cial guinea pig ration for 2 days after ar minor changes in enzymatic activities rival and then fed the 4 experimental diets were observed when the fat content of the (table 1). Several of the guinea piglets calf diets was varied from zero to 15% fed each diet lost weight during the first (1 1 ), suggesting that the difficulties en few days following the switch to the ex countered in the development of high car perimental diets, but all animals recovered bohydrate diets might be due to a lack of their initial weights within 3 to 4 days. To metabolic adaptation in calves fed these maintain adequate intakes, it was neces- diets. The present study was undertaken to investigate this possibility further and Received for publication April 11, 1966. 1 Supported bv Public Health Service Research to determine whether the apparent lack of Grant no. AM05745 from the National Institute of enzymatic response to high carbohydrate Arthritis and M etabolic Diseases. 2 Taken in part from a Master’s Thesis submitted diets was unique to the young calf. by C. Radanovics. J. N u t r it io n , 90: ’ 66 47 4 8 R. L. BALDWIN, M. RONNING, C. RADANOVICS AND G. PLANGE TABLE 1 Composition of diets 1 Diet A Diet B Diet C Diet D g/100 g of basic diet Glucose monohydrate 2 39.3 32.8 26.2 — Lard — 2.5 5.0 15.1 Non-fat milk solids 34.7 34.7 34.7 34.7 Dried whey 21.2 21.2 21.2 21.2 Non-nutritive cellulose 3 4.8 8.8 12.9 30.0 Vitamin and salt mixes 4 Cal. CHO/Cal. CHO + fat 0.98 0.90 0.80 0.55 1 Diets calculated to be isonitrogenous and isocaloric. 2 Cerelose, Corn Products Com pany, Argo, Illinois. 3 Non-nutritive cellulose was not added to calf and piglet diets which were fed as semi-purified milks (see text). Proportions of other ingredients in diet were not altered. 4 The vitamins and minerals were added to the basic diet and varied with species as follow s: for the rat diets, 0.6 g of a mineral m ix containing 39.8% dipotassium phosphate, 25.4% Ca carbonate, 14.1% sodium chloride, 6.3% dibasic Ca phosphate, 2.3% ferric citrate, 0.63% manganese sulfate, 8.6% magnesium sulfate, 0.9% zinc sulfate, 0.068% potassium iodide, 0.125% copper sulfate, 0.02% zinc chloride, 0.004% cobalt chloride and 1.4% magnesium oxide and 0.3 g of a vitamin mix containing 3,000 IU vitamin A, 375 IU vitamin D, 10 mg dl-a-tocopherol powder (250 IU vitamin E /g) and 0.1 mg menadione, were added/100 g of the basic diets; for the guinea piglet diets: 4.75 g of a mineral mix containing 29.4% dipotassium phosphate, 25.2% Ca carbonate, 18.5% sodium chloride, 10.5% magnesium sulfate, 12.6% Ca phosphate, 2.9% ferric citrate, 0.6% manganese sulfate, 0.08% potassium iodide, 0.03% copper sulfate, 0.03% zinc chloride and 0.01% cobalt chloride and 0.6 g of a vitamin m ix containing 1.6 m g each of thiamine HC1, riboflavin, and pyridoxine • HC1, 4 m g sodium pantothenate, 10 m g niacin, 0.06 m g biotin, 1.0 m g folic acid, 0.004 m g v i t a m i n B i 2, 300 IU vitamin A, 12 mg a-tocopherol, 0.2 mg menadione, 10 mg p-aminobenzoic acid, 200 mg ascorbic acid, 0.004 mg calciferol (vitamin D) and 300 mg choline chloride were added/ 100 g of the basic diets; for the calves and piglets 0.8 g of a salt mix containing 82.5% trace mineralized salt, 9.4% magnesium oxide, 7.6% ferric citrate and 0.5% copper sulfate and 0.3 g of the vitamin m ix used in the rat diets were added/100 g of the basic diets. The trace mineralized salt contained not less than: (in per cent) NaCl, 96.5; manganese, 0.20; iron, 0.16; copper, 0.033- cobalt, 0.01; iodine, 0.007; and zinc, 0.005. sary to pellet the food for the guinea mental diets for an average of 4 weeks piglets. The guinea piglets were fed the ex prior to final sampling at killing. In addi perimental diets for 4 weeks prior to kill tion, the calves were sampled by biopsy ing for sampling. Their rates of gain were during the 4-week feeding period; the re 1.1, 1.0, 2.2 and 3.0 g 7day with diets A sults were similar to those obtained in through D, respectively. The poor growth the final samples. The average rates of responses of the guinea piglets were rec gain of the calves were 0.39 and 0.42 k g / ognized in the course of preliminary ex day with diets A and D, respectively. The periments and were a matter of concern piglets were fed ad libitum and appeared because the poor growth might affect the to respond well to the experimental diets validity of the data. Better growth re during preliminary studies but during the sponse could be obtained by decreasing main study many of the piglets suddenly the carbohydrate content of the diets. However, since one of the goals of the in became sickly and died between the third vestigation was study of the nature and and fourth weeks of the feeding period. extent of the enzymatic adaptations to No specific cause could be determined and high carbohydrate diets and since the poor numerous attempts to control the problem growth may have been a reflection of a were unsuccessful. Therefore, most of the lack of adaptation, the carbohydrate con results reported were determined in sam tent of the diets was not decreased. ples collected after killing of piglets be The calves and piglets were fed 2 of the tween 2 and 3 weeks of age, and only a experimental diets (A and D ) beginning few data are presented. The animals rep at 2 and 3 days of age, respectively. The resented were growing well and appeared diets were prepared as semi-purified milks healthy and vigorous at the time of killing. and fed isocalorically and isonitrogenously Results of routine blood analyses for to the calves in amounts equivalent to hexose, pyruvate, oxyhemoglobin and he usual levels of whole milk feeding. The matocrit did not appear to be affected by calves were maintained with the experi differences in the diets. CARBOHYDRATE AND FAT METABOLISM 49 Liver samples were stored in ice during piglets, piglets and calves fed the experi transport to the laboratory and were pre mental diets are presented in tables 2 to 5, pared for enzymatic analysis according to respectively. With rats, marked decreases methods reported previously (1 2 ). En in the activities of a number of the en zyme assays and auxilliary clinical tech zymes studied were observed in response niques were carried out according to pro to decreases in the ratio of calories from cedures described elsewhere (12, 13). carbohydrate to calories from carbohy Liver slices prepared with a Stadie tissue drate plus fat in the diets (table 2 ). The slicer were incubated under 95% oxygen activity of glucose-6-P dehydrogenase in and 5% C 0 2 in Krebs bicarbonate buffer livers of rats fed diet A (high carbohy with 0.5 uCi of either glucose-l-14C or glu- drate) was 24 times greater than in livers cose-6-14C for 90 minutes in a shaking of rats fed diet D (high fat). The activi water bath. The reactions were stopped ties of the malic enzyme, citrate cleavage with HC1 and the 14C0 2 collected in C 0 2- enzyme, aceto-CoA synthetase, glucose-P free NaOH and counted in a liquid scin mutase, hexokinase, 6-P-gluconate dehy tillation counter. drogenase and pyruvate dehydrogenase were 9.1, 7.0, 5.5, 4.8, 3.8, 2.6 and 2.0 RESULTS AND DISCUSSION times greater, respectively, with the high The enzymes investigated were selected carbohydrate diet (A ) than with the high as representatives of various metabolic fat diet (D ). The activities of most of pathways associated with carbohydrate these enzymes decreased logarithmically and fat metabolism and on the basis of as the ratio of calories from carbohydrate previous reports that their activities were to calories from carbohydrate plus fat in affected by diet composition (1 —7). Glu- the diet decreased (fig. 1 ), apparently an cose-6-P dehydrogenase, 6-P-gluconate de indication that the enzymatic changes hydrogenase and pentose phosphate-me were related to the caloric contributions tabolizing activity were selected as in rather than the amounts, of carbohydrate dexes of the potential activity of the and fat per se in the diets. The general hexose monophosphate pathway. Hexo- nature of the enzymatic changes and the kinase, glucose-6-P isomerase, glucose-P ratios of 14C0 2 produced from glucose-6-14C mutase and glucose-6-phosphatase were and glucose-l-14C were consistent with pre selected because of their relationships to vious reports which indicated that as the the metabolism of glucose-6-phosphate. Fructose-1,6-diP aldolase and glyceralde- hyde-3-P dehydrogenase were selected be cause they function in the Embden-Meyer- hof glycolytic pathway. UDPG pyrophos- phorylase was determined because of its role in glycogen synthesis. Pyruvate de hydrogenase, a-glycerol-P dehydrogenase, glycerol kinase, aceto-CoA synthetase (ex tra-mitochondrial), malic enzyme, malate dehydrogenase (extra-mitochondrial), iso citrate dehydrogenase (extra-mitochon drial), and the citrate cleavage enzyme were selected because of their potential relationships to fat metabolism. Aspartate aminotransferase was studied as a gen eral index. The ratio of 14C0 2 produced from glucose-6-14C and glucose-l-14C by CALORIES FROM CARBOHYDRATE tissue slices was selected as a general CALORIES FROM CARBOHYDRATE PLUS FAT index of the relative activities of the alter Fig. 1 Effect of ratio of calories of carbohy nate glycolytic pathways. drate to calories of carbohydrate plus fat in the The activities of the enzymes observed diet upon the activities of several enzymes in in liver samples obtained from rats, guinea rat livers. 0 5 T A B L E 2 Effect of varying levels of carbohydrate and fat in the diet xvpon the activities of several enzymes in rat livers A 03 (A . . ADI, RNIG C RDNVC AD . PLANGE G. AND RADANOVICS C. RONNING, . M BALDWIN, L. R. w 8 I [K I ! CO k h k q 3 J3 CO pH ro 1 Units of enzym e/gram of tissue. One unit of enzyme is defined as the amount required to convert one /¿mole of substrate/minute at 25° except glucose-6-P isomerase and pyruvate dehydrogenase where one unit causes a change of 1.0 absorbancy unit/minute. 2 Stanard error (N = 6). 3 Pentose phosphate-m etabolizing activity. 4 Ratio of i4C02 produced from respective substrates. CARBOHYDRATE AND FAT METABOLISM 51 fat content of the diet decreased, activity of the hexosmonophosphate pathway, rates of fatty acid synthesis and the activities of enzymes involved in these functions increased ( 1 -6 ) . The results of the study with guinea piglets (table 3 ) were somewhat different from those observed in the rat study. Only one enzyme, glucose-6-P dehydrogenase, decreased in activity as the ratio of calo ries from carbohydrate to calories from carbohydrate plus fat decreased and this change was about two-fold as compared with 24-fold in rats. The decrease ap peared to represent a logarithmic response similar to that observed in the rats (fig. 2 ). The activities of several enzymes tended to increase in the guinea piglet livers as the fat content of the diet in creased. The activities of a-glycerol-P de hydrogenase, glycerol kinase, isocitrate de CALORIES FROM CARBOHYDRATE hydrogenase and aldolase were 2.8, 2.6, CALORIES FRO« CARBOHYDRATE PLUS FAT 2.2 and 2.1 times greater, respectively, Fig. 2 Effect of ratio of calories of carbo with diet D than with diet A. The changes hydrate to calories of carbohydrate plus fat in in the activity of a-glycerol-P dehydrogen the diet upon the activities of several enzymes in ase appeared to increase logarithmically as guinea piglet livers. the ratio of carbohydrate calories to carbo hydrate and fat calories in the diet de genase, the enzymes most affected by the creased (fig. 2 ). The activity of glycerol dietary variations in the guinea piglets kinase increased in a similar fashion until were three of the enzymes affected least the carbohydrate-to-carbohydrate plus fat in the rat. ratio reached 0.80 (diet C ) and then no The results obtained in the calf study further increase was noted (fig. 2 ). The are presented in table 4. The most notable activities of aldolase and isocitrate dehy characteristic of these data was the lack drogenase did not increase until the carbo of differences in the activities of liver en hydrate-to-carbohydrate plus fat ratio had zymes between diets. Glucose-6-P dehydro decreased to 0.90 and thereafter increased genase activity and citrate cleavage en logarithmically as the ratio decreased (fig. zyme activities appeared to be lower and 2 ). These latter observations might sug the activity of a-glycerol-P dehydrogenase gest that there are limits to the extent to appeared to be higher when the fat-con which adaptive changes can occur despite taining diet was fed. The activities of the further changes in diet. The enzymatic remaining enzymes did not appear to be changes observed were consistent with affected significantly. The activities of the tissue slice data obtained with glucose- aceto-CoA synthetase and malic enzyme 1-14C and glucose-6-I4C, in that both indi were below the effective sensitivities of cated very little change in the relative the assays used. The ratio of 14C0 2 pro activities of the hexose monophosphate duced by tissue slices incubated with glu- and Embden-Meyerhof pathways. The cose-6-14C to that from glucose-l-14C was changes in the activities of a-glycerophos- slightly higher when diet D was fed and phate dehydrogenase and glycerol kinase may have indicated that a slight shift in with increasing fat intakes appeared to pathways of carbohydrate metabolism had represent an increased capacity of the occurred. liver for triglyceride synthesis (7 ) . It is The data obtained in the piglet study interesting that a-glycerol-P dehydrogen are presented in table 5. There were fewer ase, glycerol kinase and isocitrate dehydro- animals in this study than in the others 52 Effect of varying levels of carbohydrate and fat in the diet upon the activities of several enzymes in guinea piglet livers . . ADI, RONNING . M BALDWIN, L. R. Q 0) t-» rH CN co CO O q CN CO in q CD rH rH rH CN l> O CO o CO T3 p T3 . AAOIS N G PLANGE G. AND RADANOVICS C. C > C3 03 03 P= 3 h o CO CD o 00 rH o rH rH q CD o q LO LO in o rH q 03 o 00 p T3 CO o S P h cq o CO o (N CN o o LO o LO o rH CD q o q O q o o 1 Units of enzyme/gram of tissue. One unit of enzyme is defined as the amount required to convert one ¿¿mole of substrate/minute at 25° except glucose-6-P isomerase and pyruvate dehydrogenase where one unit causes a change of 1.0 absorbancy unit/m inute. 2 Standard error (N = 6). 3 Pentose phosphate-m etabolizing activity. 4 Ratio of i4C02 produced from respective substrates. CARBOHYDRATE AND FAT METABOLISM 5 3 T A B L E 4 Effect of varying levels of carbohydrate and fat in the diet upon the activities of several enzymes in calf livers D ie t s E n z y m e X SX X sx Glucose-6-P dehydrogenase 2.00 1 0.43 2 1.30 1 0.21 6-P-Gluconate dehydrogenase 1.00 0.17 1.11 0.23 PPMA 3 0.18 0.06 0.20 0.06 Hexokinase — — 0.23 0.08 Glucose-P mutase 73.7 31.1 99.5 21.2 Fructose-1,6-diP aldolase 3.48 0.62 2.75 0.64 Glyceraldehyde-3-P dehydrogenase 14.9 4.40 19.4 8.81 Pyruvate dehydrogenase 0.12 0.04 0.08 0.01 a-Glycerol-P dehydrogenase 0.51 0.21 1.09 0.34 Glycerol kinase 0.14 0.07 0.09 0.03 Citrate cleavage enzyme 0.22 0.09 0.07 0.03 Isocitrate dehydrogenase 12.1 4.47 13.5 3.08 Glucose-6-14C/glucose-l-14C 0.36 4 0.07 0.46 4 0.05 1 Units of enzyme/gram of tissue. One unit of enzyme is defined as the amount required to convert one /imole of substrate/ minute at 25° except pyruvate dehydrogenase where one unit causes a change of 1.0 absorbancy unit/minute. 2 Standard error (N = 6 for diet A and N = 9 for diet D ). 3 Pentose phosphate-m etabolizing activity. 4 Ratio of i4C02 produced from respective substrates. T A B L E 5 Effect of varying levels of carbohydrate and fat in the diet upon the activities of several enzymes in piglet livers Diets Enzyme A D X s x ~x Sx Glucose-6-P dehydrogenase 1.94 1 0.45 2 1.64 1 0.60 2 6-P-Gluconate dehydrogenase 4.49 2.05 4.09 0.48 PPMA 3 1.43 1.20 0.69 0.15 Hexokinase 2.85 0.80 2.91 1.44 Glucose-6-P isomerase 45.2 16.2 31.9 4.96 Glucose-P mutase 3.79 1.45 6.90 3.91 Fructose-1,6-diP aldolase 8.04 4.45 5.28 1.65 Glyceraldehyde-3-P dehydrogenase 34.9 22.9 19.0 8.25 Pyruvate dehydrogenase 3.62 0.36 1.61 1.37 a-Glycerol-P dehydrogenase 2.36 0.82 1.98 0.69 Aceto-CoA synthetase 4.84 1.10 2.76 0.81 Citrate cleavage enzyme 0.39 0.54 0.25 0.09 UDPG pyrophosphorylase 164 44.1 111 29.8 Glucose-6-phosphatase 0.12 0.01 0.12 0.04 Isocitrate dehydrogenase 26.2 10.0 21.4 3.22 Aspartate aminotransferase 4.73 0.81 9.59 1.40 Glucose-6-I4C/glucose-l-14C 0.54 4 0.08 0.56 4 0.07 1 Units of enzyme/gram of tissue. One unit of enzyme is defined as the amount required to convert one /¿mole of substrate/minute at 25° except glucose-6-P isomerase and pyruvate dehydrog enase where one unit causes a change of 1.0 absorbancy unit/minute. 2 Stanard error (N = 3 for diet A and N = 5 for diet D). 3 Pentose phosphate-m etabolizing activity. 4 Ratio of 14C 02 produced from respective substrates. discussed above, because of the difficulties ever, that responses of piglets to high car referred to in the methods and materials bohydrate diets were not large. section. The standard errors (sj) of many The present study was undertaken in of the estimates were fairly large. In view an attempt to determine whether the ap of the difficulties involved in the piglet parent lack of enzymatic responses to study, the data must be considered with changes in carbohydrate and fat intakes caution. They appear to indicate, how observed in preliminary studies with calves 54 R. L. BALDWIN, M. RONNING, C. RADANOVICS AND G. PLANGE was unique to young calves. Further stud The activities of a number of enzymes ies were also required to determine were similar, whereas others were con whether the apparent lack of enzymatic siderably lower in the livers of calves fed response by calves to changes in diet was the high fat diet (D ) than in the livers related to previous difficulties encountered of rats fed the same diet. Many of the in attempts to develop high carbohydrate, enzymes whose activities were lower ap fat-free diets for calves (9, 10). In guinea peared, according to present concepts (1), piglets and piglets as well as calves the to be related to the capacity for synthesis responses to changes in carbohydrate and of fatty acids in liver. The activities of fat intakes appeared to be much more glucose-6-P dehydrogenase and 6-P-gluco- limited than those which occurred in rats. nate dehydrogenase were similar in the These differences may reflect either a lack 2 species; however, the activities of the of ability of neonatal guinea piglets, pig lets and calves to adapt metabolically to pentose phosphate-metabolizing enzymes changes in diet, the existence of adaptive were 10 times lower in calves than in rats, mechanisms that do not involve changes suggesting that the potential for pentose in the activities of liver enzymes, or lack cycle activity in calf livers was lower than of capacity, in these 3 species, for adapta in rat livers. Such a deficiency might be tion to high carbohydrate diets at any age. expected to affect the potential availability There are data that indicate that the ca of NADPH for fatty acid synthesis. The pacity for adaptation to changing physio activities of pyruvate dehydrogenase, cit logical conditions develops slowly in the rate cleavage enzyme and acetyl CoA syn livers of neonatal animals (1 4 ). It is pos thetase were much lower in calf livers sible that the results of the present experi than in rat livers. The low levels of these ment can be explained by suggesting that enzymes in calf livers might be expected the rats were more mature than the other to affect the availability of acetyl-CoA for species studied and thus were capable of fat synthesis. The activity of malic enzyme greater responses to changes in diet. How was very low in calf livers (12) and the ever, guinea piglets, piglets and calves are activity of a-glycerol dehydrogenase was more fully developed at birth than rats and 5 times lower in calf livers than in rat there is evidence that guinea piglets form livers. The activities of these enzymes some adaptive enzymes at an earlier age have also been related to fat synthesis than rats (14, 15). The possibility of (1). These apparent limitations in the alternate adaptive mechanisms in calves, potential capacity for fat synthesis in calf guinea piglets and piglets cannot be ex livers as compared with rat livers became cluded. For example, a greater participa much more prominent when animals fed tion of adipose tissue in fat synthesis in the high carbohydrate diets (A ) were com these species might explain the results ob tained. However, no data are available at pared. Differences in the absolute activi present to support this possibility. ties of enzymes involved in fat synthesis The considerable difficulty that has been were not as great when guinea piglets and encountered in attempts to develop high piglets were compared to rats. carbohydrate, fat-free diets for calves, the high B-vitamin and mineral requirements LITERATURE CITED of calves fed such diets, and clinical data 1. Tepperman, J., and H. M. Tepperman 1965 (9, 10), suggested that metabolic disorders Adaptive hyperlipogenesis-late 1964 model. occurred in calves fed high carbohydrate Ann. N. Y. Acad. Sci., 131: 404. diets. The results of the present study 2. Hill, R., W. W. Webster, N. M. Linazasoro and I. L. ChaikofF 1960 Time of occur indicated that the difficulties encountered rence of changes in the livers capacity to might have been due to a lack of adaptive utilize acetate for fatty acid and cholesterol enzymatic changes required for the con synthesis after fat feeding. J. Lipid Res., version of increased amounts of carbo 1: 150. hydrate to fat in the liver. Guinea piglets 3. Kornacker, M. S., and J. M. Lowenstein 1964 Citrate cleavage enzyme in livers of and piglets also appeared to lack adaptive obese and nonobese mice. Science, 144: capacity as compared with the rat. 1027. CARBOHYDRATE AND FAT METABOLISM 5 5 4. Tepperman, H. M., and J. Tepperman 1963 fed a high carbohydrate low fat diet. Hil- On the response of hepatic glucose-6-phos- gardia, 36: 333. phate dehydrogenase activity to changes in 10. Ronning, M., R. L. Baldwin and B. C. Ten diet composition and food intake pattern. nant 1966 Study of nutritional defects of In: Advances in Enzyme Regulation, vol. 1, a nonfat milk solid and glucose diet for ed., G. Weber. The Macmillan Company, young calves. J. Dairy Sci., 49: 986. New York, p. 121. 11. Baldwin, R. L., and M. Ronning 1966 5. Masoro, E. J. 1962 Biochemical mecha Effects of several constituents of semi-puri- nisms related to the homeostatic regulation fied diets upon carbohydrate and fat metab of lipogenesis in animals. J. Lipid Res., 3: olism in calves. J. Dairy Sci., 49: 688. 149. 12. Benevenga, N. J., R. L. Baldwin and M. 6. Allman, D. W., D. D. Hubbard and D. M. Ronning 1966 Alterations in liver en Gibson 1965 Fatty acid synthesis during zyme activities and blood metabolite levels fat-free refeeding of starved rats. J. Lipid during the onset of thiamine deficiency in Res., 6: 63. the dairy calf. J. Nutr., in press. 7. Howard, C. F., Jr., and J. M. Lowenstein 13. Baldwin, R. L., and L. P. Milligan 1966 1965 The effect of glycerol-3-phosphate on Enzymatic changes associated with the initi fatty acid synthesis. J. Biol. Chem., 240: ation and maintenance of lactation in the 4170. rat. J. Biol. Chem., 241; 2058. 8. Gershoff, S. N. 1964 Effects of dietary 14. Walker, D. G., and G. Holland 1965 The levels of macronutrients on vitamin require development of hepatic glucokinase in the ments. Federation Proc., 23: 1077. neonatal rat. Biochem. J., 97: 845. 9. Benevenga, N. J., and M. Ronning 1965 15. Dawkins, M. J. R. 1966 Biochemical as The effect of certain B-vitamin supplements pects of developing function in newborn on the survival and performance of calves mammalian liver. Brit. Med. Bull., 22: 27. Dietary Metal-complexing Agents and Zinc Availability in the Rat1,2 DONALD OBERLEAS,3 MERLE E. MUHRER and BOYD L. O’DELL Department of Agricultural Chemistry, University of Missouri, Columbia, Missouri ABSTRACT The growth rate of weanling rats was used to determine the effects of phytate, calcium and ethylenediaminetetraacetate (EDTA) upon the physiological availability of zinc. Phytate decreased availability and the effect was augmented by excess dietary calcium. Calcium had no effect in the absence of phytate so that its effect must be mediated through an interaction with phytate. EDTA increased zinc availability when the diet contained phytate but had no significant effect upon the growth rate in the absence of phytate. In vitro experiments showed that zinc phy tate is highly insoluble at the pH range encountered in the small intestine. Addi tion of calcium to the medium produced an even more insoluble complex containing zinc, calcium and phytate. The results suggest that the formation of such com plexes with phytate is the mechanism whereby zinc is made less available and the more complete precipitation of zinc in the presence of calcium explains the effect of excess calcium. In vitro studies with intestinal strips and 65Zn showed that zinc uptake was progressively decreased as the ratio of calcium to phytate was increased. This effect was counteracted in part by the addition of EDTA. It appears that EDTA increases zinc availability by competing with phytate and forming a soluble complex which allows absorption across membranes. The indispensability of zinc in the addition of ethylenediaminetetraacetate nutrition of the rat was demonstrated (E D TA) to soybean protein increases the some 30 years ago, but the requirement availability of zinc to the turkey poult was extremely low and great care was (1 0 ) and the rat (1 1 ) and counteracts necessary in order to produce deficiency the effect of phytate in the diet of symptoms ( 1 ) . The significance of zinc chicks (9 ). in practical diets was not realized until In the experiments described here, the Tucker and Salmon (2 ) showed that zinc rat was used to determine the dietary would prevent or cure parakeratosis in interrelationships of calcium, phytate and swine fed diets composed of natural feed EDTA with respect to zinc availability. stuff s. The fact that zinc deficiency de In vitro studies were also performed to velops in animals fed diets based on soy investigate the mechanism by which these bean protein and corn whereas it does factors affect zinc availability. not occur when the diets are based on animal protein containing the same EXPERIMENTAL amount of zinc has led to the hypothesis Weanling male albino rats were main that the zinc in certain plant proteins is tained in stainless steel cages and allowed not readily available to animals. to consume their respective diets ad libi The addition of phytic acid to diets tum. Distilled water was supplied in glass based on casein or free amino acids has bottles. The animals were weighed been shown to reduce the availability of weekly for 4 weeks. zinc to the growing chick ( 3 - 5 ) the pig (6 ) and the rat ( 7 ) . Under some con Received for publication March 28, 1966. ditions excess calcium decreases the avail 1 Contribution from the Missouri Agricultural Ex periment Station, Journal Series no. 3081. ability of zinc and this is related to the 2 Taken from a thesis by D. Oberleas submitted to source of protein (8 ) . By the use of diets the Graduate School, University of Missouri, in par tial fulfillment of the requirements for the Ph.D. de based on casein with and without added gree. A preliminary account has been presented: phytic acid it has been shown that cal Oberleas, D., M. E. Muhrer and B. L. O’Dell 1965 Federation Proc., 24: 170. cium exerts a deleterious effect only in :5 Present address: Department of Medicine, Wayne State University College of Medicine, Detroit, M ichi the presence of phytic acid ( 7 ,9 ) . The gan, 48207. 56 J. N u t r it io n , 9 0 : ’ 66 ZINC AVAILABILITY 5 7 The basal diet contained: (g /k g ) glu Each incubation vessel contained 0.05 uCi cose hydrate, 700; soybean oil, 100; pro of 65Zn. At least 2.0 ml of buffer medium tein source,4 150; minerals,5 50; methio were used in each vessel and the total nine,6 and vitamin supplement.7 This volume was 2.5 ml. After preliminary diet8 was calculated to contain approxi study of this system the everted sac tech mately 12% of crude protein, 0.8% of nique was discontinued because active calcium, and 0.6% of phosphorus. The transport was not observed. Zinc uptake zinc content as determined by atomic ab by intestinal tissue was determined under sorption spectrophotometry ranged from 6 the same conditions. A normal animal to 8 m g/kg. Phytate phosphorus was de was killed with chloroform and a portion termined by a modification of the method of the small intestine about 30 cm in of Earley (1 2 ) and by this method the length, starting with the jejunum was re purified soybean protein contained the moved to a cold porcelain plate and into equivalent of 2.5 % of inositol hexaphos- a pool of cold isotonic saline. The intes phate. Commercial phytic acid was used tine was cut into segments approximately to supplement the casein diets and was 3 cm in length, freed of adhering tissue, added on the basis of analysis to supply split longitudinally, weighed and placed the equivalent of 1 % of inositol hexa- in the incubation vessel. During this time phosphate. All supplements were added the buffer and tissues were maintained in lieu of glucose, calcium and zinc as near 2 °. After all additions were made the carbonates and EDTA as the disodium the vessels were oxygenated for 15 sec salt. onds, stoppered and then incubated in a For the solubility studies, solutions of Dubnoff water bath for one hour at 37° sodium phytate (7.85 X 10“3m ), calcium with shaking at the rate of 80 oscillations chloride (0.05 m ) and zinc sulfate (0.1 m ) per minute. Following incubation the tis were used. Aliquots were added to a sues were immediately removed, rinsed beaker to give a minimum of 10-4 mole with a stream of cold saline, placed in a of each component in a total volume of tube with 2 ml of concentrated sulfuric 30 ml. In some cases the calcium chlo acid and heated in boiling water for 15 ride concentration was 2 X 10-4 mole. A f minutes. The hydrolysate was counted in ter all additions were made, the pH was a well-type scintillation counter which adjusted upwards to provide a series from gave a counting efficiency of 2.69% . The pH 3 to 9. After standing 24 hours the samples wTere then analyzed for nitrogen samples were centrifuged, the precipitate by the micro-Kjeldahl method. washed with 5 ml of water dried at 60 ° in vacuo, weighed, wet-ashed and ana RESULTS AND DISCUSSION lyzed for phosphate, calcium and zinc. Growth rate and interaction of calcium, Phosphate was determined by the method zinc and phytate. The results of experi- of Fiske and Subbarow (1 3 ), calcium ac cording to the AOAC oxalate-permanga 4 Reprecipitated and purified casein or purified soy nate procedure (1 4 ) and zinc by the bean protein (Promine, Central Soya Company, Chi c a g o ) . Zincon method (15). 5 The following minerals were supplied: (g/kg diet) C a C 0 3, 1 0 .7 4 ; C a H P 0 4 -2 H 20 , 1 6 .7 3 ; M g C 0 3, 1 .0 0 ; Initially, a modification of the everted MgS04-7H20, 1.21; NaCl, 5.00; KC1, 0.84; FePOl gut sac technique (1 6 ) was used to de (soluble), 1.60; KH2P04, 12.67; MnS04 H20, 0.76; CuS04-5H20, 0.064; A1K(S04)2-12H20, 0.0096; KI, termine the effect of calcium, phytate and 0 .0 2 4 ; C o C 12-6H20, 0.0024; and NaF, 0.040. EDTA on zinc absorption in vitro. The 6 One gram of methionine for casein and 3 g for soybean protein diets. buffer had the following composition: 7 Vitamins were supplied at the following levels/ kg of diet: vitamin A, 20,000 IU; vitamin D, 3,000 (g/liter) NaCl, 8.54; KC1, 0.30; fructose, IU; menadione, 10; a-tocopheryl acetate, 30; thiamine- 3.60; 2 - amino - 2 - hydroxymethyl - 1,3- HC1, 16; riboflavin, 16; pyridoxine-HCl, 16; Ca panto thenate, 40; biotin, 0.2; folacin, 5; cyanocobalamin, propanediol (T ris), 0.485; adjusted to pH 0.05; and choline chloride 1,000 mg. The antioxidant, ethoxyquin, was supplied at 100 m g/kg. 6.0 with HC1. The final concentrations of 8 The authors gratefully acknowledge gifts of die the additives were: phytate, 10“3 m ; tary supplements from the following donors: biotin. Hoffmann-LaRoche, Inc., Nutley, New Jersey; folacin, EDTA, 1.3 X 10-6 M, and zinc, 1.3 X 10“6 American Cyanamid Company, Pearl River, New m . Calcium was added to give calcium-to- York; vitamin A, Distillation Products Industries. Rochester, New York; and other vitamins from phytate ratios ranging from 1:1 to 16 :1 . Merck Sharp and Dohme, Rahway, New Jersey. 5 8 DONALD OBERLEAS, MERLE E. MUHRER AND BOYD L. ODELL TABLE 1 Growth rate as affectd by calcium and phytate Casein basal Basal -¡-1% phytate Supplement 0.8% Ca 1.6% Ca 0.8% Ca 1.6% Ca 9 9 9 9 N o n e (1 1 ) 1 3 2 ± 1.3 2 33 ± 0 .7 20 ± 1 .5 7 ± 2 .1 Zn, 55 ppm (7 ) 31 ± 1 .8 32 ± 2 .1 31 ± 1 .8 3 3 ± 1.0 1 Number of animals per treatment. 2 Average weekly gains f o r 4 weeks + s e o f mean. ments which involved 72 rats fed casein- Solubility studies. The solubility ex base diets are shown in table 1. In the periments were designed to investigate the absence of phytate the calcium level had effect of pH and the ratio of calcium and no effect upon growth rate with or with zinc to phytate or of zinc to phytate upon out supplementary zinc. When the diet the quantity of precipitate formed. The contained 1 % phytate and no added zinc, amount of precipitate was calculated by the growth rate of animals fed 0.8 % of the summation of inositol hexaphosphate calcium was about 60% of that of the (3.55 X phosphorus), calcium and zinc zinc-supplemented controls, and those fed as determined analytically. No precipitate 1.6% of calcium gained at about 20% was visible below pH 3.7, but in the pres of the control rate. Analysis of variance ence of zinc a precipitate formed at pH of the groups without zinc showed a 3.8 and above. highly significant interaction between cal As shown in figure 1 with calcium-to- cium and phytate (P < 0.005). The phytate ratios of 1:1 there was only a growth-depressing effects of calcium and trace of precipitate. Zinc phytate was phytate disappeared when a zinc supple much less soluble and at pH 6, 61% of ment was supplied. Thus, the addition of the zinc was recovered in the precipitate. phytate decreased the amount of zinc When equal molar concentrations of cal physiologically available and the results cium, zinc and phytate were present, the suggest that the detrimental effect of ex quantity of precipitate formed was greater cess calcium is mediated through the phy than the sum of the precipitates formed tate present in the diet. The results are when the 2 cations were added to phytate in agreement with earlier observations separately. At pH 6 the precipitate formed made with the pig ( 6 ) , the chick (9 ) and by the 3 ions contained 77% of the zinc the rat (7 ). added. Growth rate and interaction of EDTA, When the calcium concentration was zinc and phytate. In the second experi twice that of phytate a measurable amount ment the basal diet was similar to the of precipitate was formed but only 26% of high calcium (1 .6 % ) diet described above the calcium was removed at the peak, pH and a 2 X 2 factorial design was used. 6. When the molar ratios of calcium, zinc Phytate was added at the 1% and EDTA and phytate were 2 :1 :1 , the precipitate at the 0.1% level. The results, shown in contained 97% of the zinc and 84% of table 2, not only confirm that phytate de creases growth when zinc is limiting (P < TABLE 2 0 .0 0 5 ), but also show that EDTA mark Interaction of EDTA, zinc and phytate when added edly stimulates growth when added to low to a casein-based diet containing zinc diets containing phytate. EDTA 1.6% calcium1 slightly depressed the growth rate of rats Without EDTA Naj EDTA (0.1% ) fed the basal diet, but the effect was not + 1% + 1% statistically significant. The most note Basal Phytate Basal Phytate worthy observation was the highly signifi 9 9 9 9 cant interaction (P < 0.005) between phy 3 2 ± 2 .7 2 8 ± 1.1 26 ± 2 .2 23 ± 1 .8 tate and EDTA, confirming the results 1 Six animals per treatment. obtained with chicks ( 9 ). 2 Average weekly gain for 4 weeks ± se of mean. ZINC AVAILABILITY 5 9 pH Fig. 1 Relation of solubility of metal phytate complexes to pH and ratios of calcium and zinc to phytate. □ ------□ , Ca:phytate (1 :1 ); A ------A, Ca:phytate (2 :1 ); O— -----O, Zn:phytate (1 :1 ); X------X, Ca:Zn:phytate (1:1:1); • ------•, Ca:Zn:phytate (2:1:1). the calcium originally present. Hoff-Jorg- phytate to make zinc unavailable, the enson (1 7 ) has shown that the equilib phytate must be present in sufficient quan rium between calcium and phytate is tity to overcome the effect of dissociation reached almost instantaneously, and that and dilution in the stomach and to assure the solubility product of pentacalcium formation of an insoluble compound in phytate is between 1 0 "19 and 1CT23. Vis the intestine before absorption occurs. ual observations made in this study indi In the in vitro experiments described cate that the zinc and zinc-calcium phy here the ratio of zinc to calcium was ex tate equilibriums are also established tremely high for an animal diet. A con rapidly. ventional diet might contain 1% calcium, If these results can be projected to a 30 m g/k g of zinc, and 1% of phytate, physiological situation, it would be ex molar ratios of 5 4 0 :1 :3 3 . Although the pected that zinc salts of phytate in the in vitro studies do not accurately repre food would be dissociated at the pH of sent a physiological situation, they clearly gastric juice, and at the pH of the small show that in the presence of excess cal intestine to exhibit minimum solubility. cium more zinc is precipitated by phytate. Because of the solubilizing effect in the This offers an explanation for the ob stomach and the subsequent dilution of served effect that calcium decreases zinc ions, it might be expected that the zinc availability when added to a diet contain- would be nearly as available from zinc phytate as from other salts fed in low 9 Green, J. D., J. T. McCall, V. C. Speer and V. W. concentration. Green et al.9 have demon Hays 1962 Effect of complexing agents on utiliza tion of zinc by pigs. J. Animal Sci., 21: 9 9 7 ( a b strated this fact in swine. In order for s t r a c t). 60 DONALD OBERLEAS, MERLE E. MUHRER AND BOYD L. ODELL Fig. 2 Uptake of 65Zn by rat jejunal tissue (per mg of N) as a percentage of the control in each replicate (7 replicates). The control contained no calcium or phytate. X represents mean at each ratio, and the vertical lines represent variation as determined by the least squares method. ing phytate but falls to do so in the ab 65Zn activity per mg of tissue nitrogen and sence of phytate. the values plotted are the averages of the Tissue uptake of 65Zn. The studies with percentage uptake based on the control in intestinal strips were conducted in an at each replicate. The control vessels con tempt to more nearly duplicate physiolog tained neither calcium nor phytate in the ical conditions. Preliminary work indi medium. Another series which contained cated that zinc was not actively absorbed graded levels of calcium but no phytate by the everted sacs according to the cri did not differ from the controls. A cal- teria of Dowdle et al. (1 8 ). However, cium-to-phytate ratio of 16:1 was included 65Zn was taken up by the tissue in a man in most replicates but since it did not dif ner similar to that reported for magne fer from the 8 :1 ratio the values were not sium (19). The zinc accumulated was not easily removed by washing and thus TABLE 3 it did not appear to be a simple adsorp Effect of EDTA on the uptake of 65Zn by tion phenomenon. The process was not intestinal tissue in vitro 1 affected by a nitrogen atmosphere, or by Radioactivity 2/m g N the addition of sodium cyanide, sodium Ca: phytate Increase ratio Without With due to iodoacetate, copper, cadmium, or ATP to EDTA EDTA EDTA the buffer medium. Neither was the rate 104 /id 104 a d % of uptake affected by the zinc status of C ontrol 23.6 22.8 - 3 . 5 the animal. As used here the term uptake 1:1 9.8 10.3 5.1 does not imply true absorption but sug 2 :1 6.4 7.6 18.8 gests the movement of zinc into cells of 4 :1 3.6 4.1 13.9 8:1 2.7 3.5 29.6 the intestinal mucosa. 16:1 3.5 4.1 17.1 The effect of increasing the calcium-to- 1 The medium contained 1 0 - 3 m phytate, 1.3 x lO -6 phytate ratio upon the uptake of 63Zn by m zinc and, when added, 1.3 x 10-6 m EDTA. The intestinal strips is shown in figure 2. These molar concentration of calcium chloride varied as shown in the first column; the control contained data include the results of 7 replicate ex neither calcium nor phytate. 2 Average radioactivity of 65Zn (3 trials), expressed periments. Uptake was estimated by the as microcuries times 104. ZINC AVAILABILITY 61 plotted. The coefficient of correlation be 4. Maddaiah, V. T., A. A. Kurnick and B. L. tween zinc uptake and calcium-to-phytate Reid 1964 Phytic acid studies. Proc. Soc. Exp. Biol. Med., 391. ratios was — 0.639, a value which is statis 115: 5. Likuski, H. J. A., and R. M. Forbes 1964 tically highly significant (P < 0 .001). Effect of phytic acid on the availability of The effect of EDTA in the in vitro sys zinc in amino acid and casein diets fed to tem is shown in table 3. EDTA was added chicks. J. Nutr., 84: 145. in an equimolar ratio with zinc. In pre 6. Oberleas, D., M. E. Muhrer and B. L. O’Dell liminary experiments it was shown that 1962 Effects of phytic acid on zinc avail ability and parakeratosis in swine. J. Ani higher concentrations of EDTA decreased mal Sci., 21: 57. the uptake of 65Zn by tissue in vitro, sug 7. Likuski, H. J. A., and R. M. Forbes 1965 gesting that excess EDTA may under some Mineral utilization in the rat. IV. Effect circumstances actually decrease zinc avail of calcium and phytic acid on the utiliza ability. When the calcium-to-phytate ratio tion of dietary zinc. J. Nutr., 85: 230. 8. Forbes, R. M. 1960 Nutritional interac in the medium was 2 :1 or greater the tions of zinc and calcium. Federation Proc., uptake of zinc by the tissue was increased 19: 643. from 14 to 30% by addition of EDTA. An 9. O’Dell, B. L„ J. M. Yohe and J. E. Savage increase of this magnitude could make a 1964 Zinc availability in the chick as af significant contribution toward the zinc fected by phytate, calcium and ethylendi- aminetetraacetate. Poultry Sci., 415. requirement of an animal fed a sub-opti 43: 10. Kratzer, F. H., J. B. Allred, P. N. Davis, B. J. mal level of zinc in the presence of phy- Marshall and P. Vohra 1959 The effect tate. of autoclaving soybean protein and the The results presented here indicate that addition of ethylendiaminetetraacetate on dietary phytate decreases the physiological the biological availability of dietary zinc for turkey poults. J. Nutr., 68: 313. availability of zinc to the rat. Excess cal 11. Forbes, R. M. 1961 Excretory patterns and cium also decreases availability but only bone deposition of zinc, calcium and mag in the presence of phytate. There appears nesium in the rat as influenced by zinc de to be a three-way interaction among zinc, ficiency, EDTA and lactose. J. Nutr., 74; calcium and phytate. Similar results have 194. 12. Earley, E. B. 1944 Determining phytin been obtained by Byrd and Matrone (2 0 ). phosphorus. Ind. Eng. Chem. (Anal. Ed.), The chemical complexation of zinc by 16: 389. phytate, particularly in the presence of 13. Fiske, C. H., and Y. Subbarow 1925 The calcium to form an insoluble and non colorimetric determination of phosphorus. absorbable compound is believed to be the J. Biol. Chem., 66: 375. 14. Association of Official Agricultural Chem mechanism by which zinc is made less ists 1960 Official Methods of Analysis, available to animals. It appears likely that ed. 9. Washington, D. C. EDTA improves availability in competition 15. Rush, R. M., and J. H. Yoe 1954 Colori with phytate by forming a soluble zinc- metric determination of zinc and copper with 2 carboxy-2'-hydroxy-5'-sulfoformazyl- EDTA complex which when presented to benzene. Anal. Chem., 26: 1345. the absorption sites in the intestine allows 16. Wilson, T. H., and G. Wiseman 1954 The the absorption of zinc. The possibility that use of sacs of everted small intestine for the total zinc-EDTA complex is absorbed the study of the transference of substances cannot be eliminated. Results with from the mucosal to the serosal surface. J. Physiol., 123: 116. chickens (2 1 ) show that EDTA can be ab 17. Hoff-Jorgenson, E. 1944 Investigations on sorbed, but work with rats suggests that the solubility of calcium phytate. Danske very little is absorbed (2 2 ). Vidensk. Selsk., Mat-fys Medd., 21: 1. 18. Dowdle, E. B., D. Schachter and H. Schenker LITERATURE CITED 1960 Active transport of 59Fe by everted segments of rat duodenum. Amer. J. Phys 1. Todd, W. R., C. A. Elvehjem and E. B. Hart 1934 Zinc in the nutrition of the rat. iol., 198: 609. Amer. J. Physiol., 107: 146. 19. Schachter, D., C. B. Dowdle and H. Schenker 2. Tucker, H. F., and W. D. Salmon 1955 1960 Active transport of calcium by the Parakeratosis or zinc deficiency disease in small intestine of the rat. Amer. J. Physiol., the pig. Proc. Soc. Exp. Biol. Med., 88: 198: 263. 613. 20. Byrd, C. A., and G. Matrone 1965 In 3. O’Dell, B. L., and J. E. Savage 1960 Effect vestigations of chemical basis of zinc-cal- of phytic acid on zinc availability. Proc. cium-phytate interaction in biological sys Soc. Exp. Biol. Med., 103: 304. tems. Proc. Soc. Exp. Biol. Med., 119: 347. 62 DONALD OBERLEAS, MERLE E. MUHRER AND BOYD L. O’DELL 21. Koike, T. I., F. H. Kratzer and P. Vohra 22. Foreman, H. 1960 Metal Binding in Medi 1964 Intestinal absorption of zinc or cal- cine. eds., M. J. Seven and L. S. Johnson. cium-ethylenediaminetetraacetic acid com J. B. Lippincott Company, Philadelphia, p. plexes in chickens. Proc. Soc. Exp. Biol. 82. Med., 117: 483. Carbohydrate Metabolism and Physical Activity in Rats Fed Diets Containing Purified Casein Versus a Mixture of Amino Acids Simulating Casein ' RICHARD A. AHRENS a n d JAMES E. WILSON, JR. Human Nutrition Research Division, Agricultural Research Service, United States Department of Agriculture, Reltsville, Maryland ABSTRACT The effects of varying energy intake levels on physical activity and glucose metabolism were studied in young growing rats. Male rats 28 days of age were fed for 31 days diets containing either casein or an amino acid mixture simulating casein at 2 levels of calorie intake. Physical activity was measured and 14C recovery from injected glucose-l-14C, -6-I4C, and -U-I4C was determined as percentage of dose in expired CO2 , feces, and urine, from one to 24 hours after giving each rat his daily ration. The average revolutions per day run was higher for calorie-restricted animals, but there was no significant difference in activity due to substituting the mixture of amino acids for casein, although casein-fed rats tended to be more active. This in crease in physical activity caused by calorie restriction was due to a significant differ ence in daytime activity, as all rats were equally active in the dark. There were no significant differences due to calorie level or nitrogen source in 14CC>2 recovery from glucose-6-14C and glucose-U-14C, but amino acid-fed rats converted more glucose-l-14C to 14C02 as measured cumulatively 6 and 23 hours after injection. Calorie-restricted rats expired less glucose-l-14C as 14C02 during the first 6 hours after injection, but this effect was not evident after 23 hours. There was a trend toward lower 14C recovery from glucose-U-I4C in urinary citrate of amino acid-fed rats, although urinary citrate excre tion was unaltered by diet. These data indicate a greater utilization of alternative pathways to the glycolytic scheme and tricarboxylic acid cycle for metabolism of glucose in amino acid-fed rats and several possible explanations are discussed. Work reported from this laboratory in a starved. Caloric restriction has been im previous paper ( 1 ) showed that for young plicated in the activity of pathways of car rats fed 2 levels of nitrogen and at 2 levels bohydrate metabolism. Lee and Lucia (4) of calorie intake, there were higher nitro reported a decrease in the direct oxidation gen gains when rats in the high calorie of glucose with caloric restriction to the group received the nitrogen as casein extent that severe restriction caused the rather than as a mixture of amino acids hexose monophosphate pathway to dis simulating casein. Rats of the same age appear irreversibly. Benevenga et al. (5 ) fed diets providing similar nitrogen intakes noted that a starvation-refeeding regimen at a lower calorie level showed no signifi produced significant increases in pentose cant advantage in nitrogen storage for phosphate-metabolizing enzyme activity. casein over amino acids. The dependence The present paper reports the effect in of nitrogen storage on calorie intake raised rats of substituting casein for a mixture of a question as to the effect the substitution amino acids simulating casein as measured of casein for amino acids might have on by 14C-labeled glucose metabolism at either energy metabolism. of 2 calorie intake levels. To study the Calorie intake level has been reported to effect, if any, of the dietary regimens on affect the physical activity of the rat, but voluntary activity the rats were allowed the nature of this effect is uncertain. access to activity cages. Hughes (2 ) reported increased physical activity in food-deprived animals, but Received for publication March 16, 1966. Fabry et al. ( 3 ) interpreted their oxygen- 1 Preliminary results of the investigation were re consumption data as indicating reduced ported to the American Institute of Nutrition at the 50th Annual Meeting of the Federation of American activity in animals that were intermittently Societies for Experimental Biology, Atlantic City, 1966. J. N u t r it io n , SO : ’66 63 6 4 RICHARD A. AHRENS AND JAMES E. WILSON, JR. EXPERIMENTAL paratus with their daily ration one hour Specific-pathogen-free male rats2 ob before 14C injection and remained there for tained at 21 days of age were housed 23 hours after injection where all 14C lost individually and fed a stock diet3 for one in C 0 2, feces, and urine was recovered. All week before being fed their particular ex solid samples were dried, ignited in an 0 2 perimental regimen. A control group of bomb, and 14C0 2 was trapped and counted 6 rats was killed to determine initial car as Ba14C0 3. Respired 14C0 2 was collected cass content of calories and nitrogen and at 30-minute intervals for the first 6 hours, to furnish data for correcting initial weight and again determined at 11 and 23 hours. of the experimental animals to the ingesta- All samples were counted at infinite thick free basis. On the basis of these data, ness in a thin-window gas-flow counter initial live weight was multiplied by 91.9% with an anticoincidence correction.4 Total to obtain ingesta-free carcass weight. Con C 0 2 recovery was determined and all spe tents of the gastrointestinal tract were re cific activities were converted to total ac moved and the carcasses homogenized in a tivity and expressed as percentage of dose Waring Blendor before analysis (6 ). injected as suggested by W ang ( 7 ) . This Four diets were used in this study: 1) avoids the dilution effects of endogenous high calorie level with casein as the nitro materials on specific activity pointed out gen source; 2 ) high calorie level with an by Wood (8 ) . L-amino acid mixture simulating casein as Aliquots of the 14C-labeled glucose sam the nitrogen source; 3 ) restricted calories ples used were ignited in an 0 2 bomb and with nitrogen provided as casein; and 4 ) Ba14C0 3 was recovered and counted. The restricted calories with nitrogen provided counts recovered in 1 uCi were 92% of the as amino acids. Diets at the high calorie theoretical amount (i.e., 2,042,810 dpm vs. level contained 3% nitrogen. Restricted- 2,220,000 dpm ), close enough so that the calorie diets were formulated with in difference could be explained by small er creases in proportion of protein or amino rors in measuring initial volume, determin acids, vitamins, salts, and roughage so ing counter efficiency,5 converting infinite that, when calorie intake was reduced from to zero thickness values, etc. Eight of the the high calorie level by approximately experimental animals were killed within one-third, intakes of those nutrients were minutes of removal from the respiration the same as the intakes at the higher cal apparatus. In these 8 animals the carcass orie level. The diets and care of the ani I4C as actually determined was 27.9% ± mals have been described earlier ( 1 ). 3.9 6 of the injected dose, whereas the 14C The casein and amino acids were assayed recovered in respiratory C 0 2, feces, and for purity as reported earlier ( 1 ). The rats, urine was 68.4% ± 5.4 of the total indi 6 per group, were housed in a well-venti cating that about 96% of injected 14C could lated room at 2 8 °, 50% relative humidity, be accounted for by actual measurement. and 12 hours per day of darkness and light. Urine was fractionated into a variety of After being fed the experimental diets for components prior to counting for 14C. Ether 2 weeks all rats were assigned at random and chloroform extracts were made to de to rotating-treadmill activity cages for 3 termine radioactivity present as lactic acid weeks and reading of revolutions turned or ketone bodies. Urea C 0 2 was released were taken at 12-hour intervals coinciding and 14C was counted following treatment of with the darkness/light changeover. Dur the urine with urease (9 ) . Oxalate was ing this 3-week period 1-nCi doses of high precipitated as calcium oxalate (1 0 ) and specific activity glucose-l-14C, glucose-6-14C, sugars were precipitated as the osazones and glucose-U-14C were injected intraperi- (1 1 ). Amino acids were absorbed and sep toneally at weekly intervals into each rat arated into acidic, neutral, and basic frac in a pattern of reversal (i.e., 2 rats in each 2 Lew strain from Microbiological Associates, Beth- group received glucose-1-14C the first week, esda, Maryland. 3 D & G Research Animal Laboratory Diet, Price- 2 rats received glucose-l-14C the second Wilhoite Company, Frederick, Maryland. week, etc.). Each 1-nCi dose involved the 4 All samples were counted for long periods of time on a Sharp “Wide-beta” gas-flow counter having a injection of 0.036 to 0.060 mg of glucose. low background. 5 See footnote 4. All rats were placed in the respiration ap 6 Standard error of the mean. ENERGY METABOLISM ON CASEIN AND AMINO ACID DIETS 6 5 tions by the use of Amberlite IR-4 (1 2 ) physical activity due to substituting casein following hydrolysis of the urine with HC1. for amino acids as the nitrogen source, but Creatinine was isolated by the method of there was a consistent tendency for the Owen et al. (1 3 ). Non-amino organic acids casein-fed rats to be more active, which were determined with paper chromatog bordered on significance. An analysis of raphy after first passing the sample through variance, however, showed a highly signifi a Dowex 50 column to remove amino acids cant effect (P < 0.01) of calorie intake on and salts. The strips were developed in a voluntary physical activity. This increase 4 :1 :5 (v /v ) upper phase N-butanol: acetic in activity due to caloric restriction appears acid: water solvent system and were to reflect an abolition of quiescence rather sprayed with 0.04% bromophenol blue in than an elevation of peak activity. During 95% ethanol. Spots were identified from the 12 hours of darkness when the rat is standards, cut from the paper, ignited, and normally most active ( 3 ) , there was no Ba14COa was determined. The amount of difference in physical activity between citric acid present was determined by titra groups. The 12 hours of light caused no tion of the eluted material with a dilute appreciable change in the activity of the base. calorie-restricted animals, but the illumina This study was conducted according to a tion caused a significant reduction in activ randomized complete block design where ity for rats fed at the high calorie level blocks were composed of those 4 animals, (P < 0 .0 1 ). Thus, rats receiving the high one per group, which received the 3 labeled calorie intake had about 70% of their ac glucose moieties in the same order of in tivity at night, whereas calorie-restricted jection. Analyses of variance were con animals had nearly the same activity, day ducted treating the investigation as a 2 X or night. Food deprivation appears to influ 2 factorial. ence voluntary activity of the rat in a manner which overrides normal diurnal RESULTS AND DISCUSSION variation. The pattern of physical activity Physical activity. The effect of the and the eating pattern appear to be related. dietary treatments on physical activity is Carcass gains. The calorie, nitrogen, shown in table 1. Diets were given once and ingesta-free weight gains are given in daily just after the darkness to light table 2. Calorie, nitrogen, and weight gains changeover. Rats receiving the restricted- were significantly reduced (P < 0.01) by calorie intake level consumed all of their calorie restriction. Rats receiving casein at diet during the first hour after feeding. the higher calorie level gained more weight Rats fed the casein diet at the high calorie (P < 0.05) than amino acid-fed rats; al level ate all of their diet during the first though the change in nitrogen source had 8 hours after feeding. Rats fed the amino no statistically significant effect on calorie acid diet at the high calorie level ate all of gain due to greater variability in the calorie the diet given them, but took 12 hours or gain data, there was a trend toward greater more to do so. At either level of calorie calorie gain in casein-fed rats, similar to intake there was no significant effect on the trend in our earlier report ( 1 ) . An TABLE 1 Diurnal variation in revolutions turned in activity cages over a 3-week period by rats fed nitrogen either as amino acids or as casein, at 2 levels of calorie intake N Intake 2 Mean voluntary physical activity source 1 N Calories 1 2 -hr light 1 2 -hr dark 24-hr total 9 h e a l r e v r e v r e v AA 8.59 1400 1379 ±456 3 4143 ±701 5523 ±838 Casein 8.59 1425 1981 ±465 4230 ± 736 6211 ±997 AA 8.93 982 4584 ±1023 3961 ±417 8544± 1153 Casein 8.94 1040 6074 ±1169 4964 ±1050 11,038 ±1158 1 A A = amino acid mixture simulating casein. 2 Mean dietary intake/rat over the entire 31-day experimental period. 3 se of mean; 6 rats/group. 66 RICHARD A. AHRENS AND JAMES E. WILSON, JR. TABLE 2 Weight gains, calorie and nitrogen storage of rats fed casein or a mixture of amino acids simulating casein at 2 calorie intake levels N Intake 2 Weight Nitrogen Calories stored stored source 1 N Calories gains 3 9 k c a l 9 9 k c a l AA 8.59 1400 7 8 ± 3 .2 |4 3.35 ± 0 .2 1 113.7 ± 11.4 C asein 8.59 1425 8 7 ± 1 .6 | * 3.59 ± 0 .0 7 129.7 ± 9.4 AA 8.93 982 25 ± 3 .5 1.59 ± 0.15 14.8 ± 5.5 C asein 8.94 1040 33 ± 2 .3 1.8 7 ± 0 .1 1 26.3 ± 3.3 1 AA = amino acid mixture simulating casein. 2 Mean dietary intake/rat over the entire 31-day experimental period. 3 Gain in ingesta-free carcass weight over the 31-day period. 4 se of mean; 6 rats/group. * Adjoining means significantly different (P < 0.05). analysis of variance showed a significant sults can best be explained by examining difference between casein and amino acid- the pattern of 14C0 2 recovery plotted against fed rats in nitrogen gain per gram of digest time. Figure 1 shows the I4C0 2 recovery ible nitrogen intake (P < 0.05), with casein- from glucose-l-I4C from the second to the fed rats being more efficient, and there was twenty-fourth hour after feeding. An anal a trend toward greater nitrogen storage ysis of variance (table 4 ) of the percent per gram of gross nitrogen intake in these age of injected glucose-l-14C recovered as rats. With large groups in the earlier study, ,4C 0 2 after 6 hours shows a significant ef differences in gross nitrogen storage were fect of calorie level (P < 0.05). This agrees found to be significant. with the earlier report of Lee and Lucia Radiorespirometry studies. There was ( 4 ) , who used a 3.5-hour collection period, no significant effect of energy level or nitro that calorie-restricted animals have a less gen source on the percentage of injected active hexose monophosphate shunt. This dose recovered in expired 14COz, feces, or measurement of activity of the hexose urine during the 23 hours following injec monophosphate shunt does not appear to tion of glucose-6-14C and glucose-U-I4C reflect accurately the total amount of glu (table 3 ). An analysis of variance shows cose metabolized by this route, at least for that the nitrogen source in the diet had a the casein-fed rats, since rats fed casein significant effect (P < 0 .0 5 ) on the per at the high calorie intake showed a reduced centage of injected glucose-l-14C recov i4C0 2 release from the sixth to twenty-third ered as 14C0 2 (table 4 ). This is in agree hour after injection, whereas calorie- ment with our earlier report of differences restricted animals fed casein maintained a in nitrogen storage at the high calorie in steady rate of 14C0 2 production. As a re take level, since a change in the activity of sult, the percentage of injected glucose-1- the pathways of carbohydrate metabolism ,4C metabolized to 14C02 after 23 hours was would be expected to alter protein metabo unaffected by calorie intake. The effect of lism as well. The significantly greater re changing nitrogen source in the diet on covery of 14C0 2 from glucose- 1-I4C in the i4C0 2 production was significant at 6 hours amino acid-fed rats indicates greater use after injection (P < 0.01) as well as after of alternative pathways of carbohydrate 23 hours (P < 0.05). An examination of metabolism in these animals ( 8 ). The time the mean squares gives an indication of the diet was fed since the last injection did the greater magnitude of the effect of not appear to affect the metabolism of the source of dietary nitrogen on glucose-l-14C labeled glucose, as block effects were not metabolism as compared with the effect of significant. Despite reports of changes in calorie restriction (table 4 ). An analysis the activity of the hexose monophosphate of variance of 14C0 2 recovery after 6 hours shunt due to calorie restriction (4, 5) our from glucose-6-14C and glucose-U-14C i4C0 2 recovery totals indicated no difference showed no effect due to calorie level or due to varying the calorie level (tables 3, 4). nitrogen source, similar to the total 23-hour The explanation for this difference in re observations. Percentage of injected ,4C from radioactive glucose recovered in expired “ C02, feces, and urine during the first 23 hours following injection ^ p ^ * Z £ o £ in u o h 'u C U NRY EAOIM N AEN N AIO CD DIETS ACID AMINO AND CASEIN ON METABOLISM ENERGY a VII Ç+ 1 + 1+ 1 CO CO CD CO if O O CO * •S O OO Ò ^ q ^ c i in i c ^ q ^ i c o c o c i c '£* T-H O C Q I « 666ò 6 6 6 ” ori r io c o c ^ £L_±_ ^ o d •Sì. d i r Il IIII £ II CD£ O^ CD s O Hto O) ^ CJs oj in co in ci in ci in co OC CO c oc CO o CO c oi n cici ci ci t> I> l> r-H T-H m rn + 1I!+ 1+ 1 in ^ t> co in i> CO rH CN ^ inCD CO inci CO Cj co q ^ CO t> o o o ò co ^ o c co o co* q c t> t> o c q q ÒHHO c ic iC c D oi n *c C oC 03 O D in [> rH q CD q rH q *9 ^ q + 1+ 1II + 1 + 1+ 1+ 1+ 1 ri ^ co ^ ri ^ ACOGIO) r Hr HC Oc i Il II II II + 1+ 1II I I co mco in II II II II Il II II II OCd m o ci OO o rF i> co co co t> Il II II II rFrF G) O < V < OS ò co ò t> <- lo h q q q q q q < < d o h < co co CD Cd u 1 I *3 ^ » i •S s .2 -7 3 § . •§T3 <3s < ^ 5 OS 4) fi cu O « “ p ÜS. £ « V CtJ>> o rH (N ‘3 cd o c o * r—H° ^ 9 t> o <, rH T3 ^ 0) < +ig -M i f - Oj- fi co < 3 5 ® 5 3 i ^ rt> aoaoy rats. laboratory How metabolism. on effect an had have hc i ta ms cmol floe for followed commonly most that is which ob differences the that the so to according schedule and same manner same fed the were study in present the in rats all ever, rather night at fed been had rats the if asn 15) eotd vdne o greater for evidence reported ) 5 Tepperman (1 Parson com . ) 1 a ( diet fed acid rats amino than parable rapidly more diets atclr ceue f ae n feeding, and care of schedule particular might this and morning, early the than the receiving rats in night and day tween nrae eoe oohsht sut ac shunt monophosphate hexose increase acid- amino hexose our the the in be of shunt to use appear greater monophosphate the not does for This reason rats time. in of monophosphate deposition fat hexose greater and the shunt of activity n n hu o less. or hour one in evd r ai wti te ofns f this of confines the within valid are served e rt, hrfr, ic fse etn by eating faster since therefore, rats, fed and Hollifield and ) 4 (1 Tepperman and their consumed level calorie high a at diets as t etitdlvl fitk (als 3 (tables intake of level restricted a at rats ocp ta vlnay ciiy elcs the reflects activity voluntary that concept altered been have might intake calorie high h csi-e rt wud e xetd to expected be would rats casein-fed the period shorter a in food their eat to trained ference between casein- and amino acid-fed the amino and casein- addition, between ference In animals. these in tivity nay ciiy 01), t a little had it , ) glu- 1 .0 injected 0 from < 14C 2 recovery 0 on P ( effect activity vol increased untary restriction calorie Although n 4) hr alrt cnue ter diets their consumed rats all where ) 4 and i a n sgiiat fet n outr ac voluntary on effect significant no had in af casein for was substituting by metabolism most fected glucose-l-,4C Further more, period. 23-hours a over cose-l-14C animal. the in metabolism energy of state ecnae f netd 4 rcvrd n the 14C in recovered injected of percentage tivity. simulat acids amino of mixture a diet the lhuh itr tetet a n effect no had treatment dietary -U-14C.Although -6-14C and glucose-l-I4C, of istration n aen Ti cag i irgn source nitrogen in change This casein. ing urine during the 23 hours following admin following hours 23 the during urine xrtd n rn we guoe61C or glucose-6-14C when urine in excreted percent greater a 14C urinary excretion, on g o te netd oe 01) was ) 1 .0 0 < P ( dose injected the of age C02 eoey aa niae a iia dif similar a indicated data 2 recovery 0 4C h dfeecs n hscl ciiy be activity physical in differences The e bevd ale ta rt fd casein fed rats that earlier observed We hs dt ae o cnitn wt the with consistent not are data These rnr U excretion. UC Urinary al 3 it the lists 3 Table 7 6 68 RICHARD A. AHRENS AND JAMES E. WILSON, JR. T A B L E 4 Mean squares of the analyses of variance for 14C02 recovery and urinary N 14C 0 2 r e c o v e r e d U rin a r y N S o u r c e o f Labeled carbohydrate administered e x c r e t io n v a r ia tio n Glucose- Glucose- G lucose-l-14C U-14C 6-14C (%/23 hr) 2 (%/23 hr) 2 ( % /6hr) 2 (mg/24 hr) ! Blocks 354.17 514.33 233.32 148.00 479 E = energy level 13.78 29.10 0.95 425.97* 16,970** S = nitrogen source 485.55 308.24 688.33* 1,226.23** 1,883* ES 397.97 256.04 89.12 10.28 170 Error 238.60 199.87 84.08 74.67 446 ** Significant (P < 0.01). * Significant (P < 0.05). Fig. 1. Effect of calorie intake and nitrogen source on percentage of 14C from intra- peritoneally injected glucose-l-14C recovered as 14C02. glucose-U-14C was given than when glu metabolized by the same route. In frac cose-1 14C was the injected material. Urea tionating the urinary 14C activity, less than 14C accounted for only about 2 % of urinary 5% was found in oxalic acid and between 14C from glucose-6-I4C or glucose-U-14C. 5 and 10% was found in the ether extract, Urease released about 20% of the urinary creatinine, osazone, and dicarboxylic 14C contributed by glucose-l-14C, indicating amino acid fractions. The quantitatively that the bulk of the 14C excreted was in greatest source of urinary 14C was found to other end products of metabolism. be citrate which accounted for about 30% Urinary 14C excretion from glucose-6-14C of the urinary label and the percentage of or glucose-U-14C was similar, indicating injected glucose-U-14C that was recovered that the terminal 5 carbons of glucose were as urinary citrate is reported in table 3. ENERGY METABOLISM ON CASEIN AND AMINO ACID DIETS 6 9 Although no significant differences could converted to succinate (2 0 ) in rather large be established due to the high variability in quantities (2 1 ). Succinate and a-ketoglu citrate-HC excretion, a clear trend was evi tarate thus formed could be metabolized dent indicating greater 14C recovery in via the tricarboxylic acid cycle. Gupta urinary citrate when casein was the nitro et al. (2 2 ), however, observed that free gen source rather than a mixture of amino amino acids did not disappear from the acids simulating casein. The mean citrate digestive tract more rapidly than intact excretion in the urine of rats consuming protein unless the protein was relatively the 4 diets was nearly the same, ranging insoluble (zein). This would indicate that from 0.96 ± 0 .1 1 m Eq/day with the high amino acid absorption in rats fed casein or calorie diet containing casein to 1.10 ± amino acids is not different. 0.12 m Eq/day with low calorie diet con 2 ) The NH4 citrate supplement added to taining casein. Beilin and Steenbock (1 6 ) amino acid diets to increase their N con earlier reported wide variation in citraturia tent might be metabolized by the tricarbox regardless of dietary changes. ylic acid cycle and utilized for gluconeo The specific activity of urinary cit genesis, resulting in inhibition of glycolysis rate following glucose-U-14C administration and causing glucose to be diverted to alter tended to be greater from rats fed casein native pathways of metabolism. A quanti than from those fed amino acids (table 3 ). tatively great effect would not be expected, If this trend is assumed to be real and however, since the daily intake of N H 4 urinary citrate is assumed to be representa citrate by amino acid fed rats was about tive of tricarboxylic acid cycle intermedi 0.16 g. ates throughout the body, it appears that 3 ) There are a number of recent reports more of the 14C from glucose is passing in the literature that would suggest that the through the glycolytic scheme and into the amino acid mixture used in this study, de tricarboxylic acid cycle in the casein-fed spite the fact that it simulates casein, does rats. This is consistent with the conclusion not contain adequate glutamic acid, gluta from the radiorespirometry data that rats mine, asparagine and arginine to support fed amino acid diets derived a significantly maximal weight gains of rats ( 2 3 -2 6 ). larger portion of their energy from direct Most workers who have fed “adequate oxidation of glucose than casein-fed rats levels” of dispensable and indispensable did. However, the possibility exists that amino acids still obtain growth rates some the diammonium citrate in the amino acid what less than they obtain with protein- diets diluted the 14C activity in body citrate containing diets and thus the possibility of pools since the dietary intake of citrate was deficiencies of unknown factors such as about 2 m Eq/day and urinary output was strepogenin, postulated by Woolley (2 7 ), about 1 mEq/day. The explanation for cannot be eliminated. Schwartz et al.7 have apparently greater use of the glycolytic indicated preliminary evidence for the ex scheme and tricarboxylic acid cycle in glu istence of a previously unrecognized growth cose metabolism of casein-fed rats is not factor in 15% casein diets. The metabolic clear. There are several possible explana adaptation in direct oxidation of glucose tions, including the following: observed in this study may be a conse 1) A dilution of the tricarboxylic acid quence of a deficiency of either known or cycle intermediates with intermediates unknown substances. from rapid amino acid breakdown might The trend toward increased non-protein cause increased gluconeogenesis and a re calorie storage (presumably fat) in young sulting inhibition of glycolysis causing glu rats fed amino acid rather than casein diets cose to be diverted to alternative pathways reported in an earlier paper ( 1 ) , might be of metabolism. The normal route of glu due to the increased direct oxidation of tamic acid breakdown involves the forma glucose on amino acid diets and might be tion of a-ketoglutarate (1 7 ), whereas pro an adaptation to a deficiency. pionic acid formed in the degradation of methionine and threonine (1 8 ), serine, 7 Schwarz, K., J. C. Smith and T. A. Oda 1966 Factor G, an agent promoting growth of animals on alanine, valine, and isoleucine (1 9 ) can be amino acid diets. Federation Proc., 25: 542 (abstract). 70 RICHARD A. AHRENS AND JAMES E. WILSON, JR. LITERATURE CITED 14. Tepperman, J., and H. M. Tepperman 1958 Effects of antecedent food intake pattern on 1. Ahrens, R. A., J. E. Wilson, Jr. and M. hepatic lipogenesis. Amer. J. Physiol., Womack 1966 Calorie and nitrogen stor 193: 55. age from diets containing purified casein 15. Hollifield, G., and W. Parson 1962 Meta versus a mixture of amino acids simulating bolic adaptations to a “stuff and starve” casein. J. Nutr., 88: 219. feeding program. I. Studies of adipose tis 2. Hughes, R. N. 1965 Food deprivation and sue and liver glycogen in rats limited to a locomotor exploration in the white rat. Ani short daily feeding period. J. Clin. Invest., mal Behav., 13: 30. 41; 245. 3. Fabry, P., R. Petrasek, E. Horakova, E. 16. Beilin, S. A., and H. Steenbock 1952 Vita Konopasek and T. Braun 1963 Energy min D and citraturia. J. Biol. Chem., metabolism and growth in rats adapted to 194: 311. intermittent starvation. Brit. J. Nutr., 17: 17. Dianzani, M. U. 1955 Content and distri 295. bution of pyridine nucleotides in fatty liv 4. Lee, M., and S. P. Lucia 1961 Some rela ers. Biochim. Biophys. Acta, 17: 391. tionships between caloric restriction and 18. Fruton, J. S., and S. Simmonds 1958 body weight in the rat. II. The metabolism Special aspects of amino acid metabolism. of radioactive glucose and the activity of In: General Biochemistry, ed. 2. John Wiley some TPN-linked enzymes in the liver. J. and Sons, New York, pp. 790-795. Nutr., 74: 249. 19. Feller, D. D., and E. Feist 1962 Metab 5. Benevenga, N. J., W. J. Stielau and R. A. olism of alanine, serine, and valine in adipose Freedland 1964 Factors affecting the ac tissue. Metabolism, 11: 448. tivity of pentose phosphate-metabolizing en 20. Lardy, H. A., and J. Adler 1956 Synthesis zymes in rat liver. J. Nutr., 84: 345. of succinate from propionate and bicarbonate 6. Womack, M., M. W. Marshall and H. E. by soluble enzymes from liver mitochondria. Hildebrand 1964 Utilization of wheat glu J. Biol. Chem., 219: 933. ten by adult rats of two ages. J. Gerontol., 21. Friedberg, F., J. Adler and H. A. Lardy 1956 19: 45. The carboxylation of propionic acid by liver 7. Wang, C. H. 1961 Metabolism studies by mitochondria. J. Biol. Chem., 219: 943. radiorespirometry. In: Advances in Tracer 22. Gupta, J. D., A. M. Dakrowry and A. E. Methodology, vol. 1, ed., S. Rothchild. Ple Harper 1958 Observations on protein di num Press, New York, pp. 274—290. gestion in vivo. I. Rate of disappearance of 8. Wood, H. G. 1955 Significance of alter ingested protein from the gastrointestinal nate pathways in the metabolism of glucose. tract. J. Nutr., 64; 447. Physiol. Rev., 35: 841. 23. Rogers, Q. R., and A. E. Harper 1965 9. Sumner, J. B. 1926 The isolation and Amino acid diets and maximal growth in the crystallization of the enzyme urease. J. rat. J. Nutr., 87: 267. Biol. Chem., 69; 435. 24. Breuer, L. H., Jr., W. G. Pond, R. G. Warner 10. Harrison, H. E., and H. C. Harrison 1955 and J. K. Loosli 1964 The role of dispens A micromethod for determination of serum able amino acids in the nutrition of the rat. calcium. J. Lab. Clin. Med., 46: 662. J. Nutr., 82: 499. 11. Noggle, C. R. 1957 The identification and 25. Ranhotra, G. S., and B. C. Johnson 1965 the quantitative determination of carbohy Effect of feeding different amino acid diets on drates. In: The Carbohydrates, ed., W. growth rate and nitrogen retention of wean Pigman. Academic Press, New York, pp. ling rats. Proc. Soc. Exp. Biol. Med., 118: 608-610. 1197. 12. Cannan, R. K. 1944 The estimation of the 26. Hepburn, F. N., and W. B. Bradley 1964 dicarboxylic amino acids in protein hydrol The glutamic acid and arginine requirement ysates. J. Biol. Chem., 152: 401. for high growth rate of rats fed amino acid 13. Owen, J. B., B. Iggo, F. J. Scandrett and diets. J. Nutr., 84: 305. C. P. Stewart 1954 The determination of 27. Woolley, D. W. 1945 Observations on the creatinine in plasma or serum, and in growth-stimulating action of certain proteins urine; a critical examination. Biochem. J.. added to protein-free diets compounded with 58: 426. amino acids. J. Biol. Chem., 159: 753. Diurnal Rhythms of Tissue Components Related to Protein Metabolism in Normal and Virus-infected Chicks * 1 * ROBERT L. SQUIBB Laboratories of Disease and Environmental Stress, Bureau of Biological Research, Rutgers, The State University, New Brunswick, New Jersey ABSTRACT Diurnal changes in protein metabolism were studied over a 72-hour incubation period in Newcastle disease virus-infected chicks. Protein, DNA, RNA and free amino acid levels were determined, where applicable, in liver, muscle and serum at 4 daily intervals. The data, which included non-infected controls, were plotted in relation to clock hours. Significant diurnal rhythms in all 3 tissues indicated the presence of periodicity. In the controls, liver DNA was highest in the evening but maximal values of RNA, protein and the free amino acids occurred during the day. While the free amino acid pool in the liver was significantly depressed by the New castle disease virus (NDV), in the serum there was an increase in values. The NDV also caused a significant desynchronization in relation to clock hours of the rhythmic patterns in the liver and to a lesser extent in muscle. The effect in the liver was noted within 12 hours post-inoculation. Serum patterns in the NDV chicks, however, re mained synchronized during the incubation period of the disease cycle. The effect of these diurnal rhythms on experimental error was discussed. An earlier report from these laboratories cle in 4-week-old chicks during the first 72 (1 ) described significant diurnal oscilla hours (incubation stage) of an NDV in tions of total protein and the nucleic and fection. free amino acids in avian liver during the PROCEDURES active involvement stage of a Newcastle disease virus (N D V ) infection. The pat A standard reference diet (5 ) was fed terns of the oscillations differed in control ad libitum to approximately 400 one-day- and infected tissues, but in both cases the old White Leghorn cockerels of known patterns suggested a relationship to clock breeding for a 4-week period. At the end hours. The fact that diurnal oscillations of that time average weekly body weights, of tissue components were observed in compared with a reference curve, indicated both normal and NDV-infected animals that the chicks had met their genetic po raised the question whether there would tential for growth with this particular ref be a similar effect during the incubation erence diet. Thus standardized, the birds stage of NDV. were divided into 2 groups: Group 1 served The phenomenon of diurnal rhythms as non-infected controls; those in group 2 has been known for many years. Halberg were injected in the leg muscle with NDV (2 ) , Simmonet (3) and Biinning (4) have (0.1 ml/bird of a 10~3 concentration of made extensive reviews of the literature. virus). In our laboratories 50% mortality However, as far as is known, none of the in 8 days can be expected from this virus research has been directed toward a si concentration. The groups, housed in multaneous comparison of diurnal changes separate isolated rooms, were continued in protein metabolism, in the normal as on the same management procedures that well as the diseased states, in several tis prevailed prior to this time, namely, con sues that reflect the body’s major protein stant artificial light and temperature, and stores. the same caretaker who entered the rooms The present paper reports the results at the same time each day. The standard of an experiment undertaken to study Received for publication March 3, 1966. diurnal changes in several parameters of i Supported in part by Contract no. DA-49-193-MD- 2694, U. S. Army Medical Research and Development protein metabolism in liver, blood and mus C o m m a n d . J. N u t r it io n , 9 0 : ’ 66 71 72 ROBERT L. SQUIBB reference diet and water were continued ad libitum. Starting at time of NDV inoculation (0 8 0 0 ), 8 birds were selected at random from each treatment group and killed at 0800, 1500, 2000 and 2400 hours for 3 consecutive days, the incubation period of the NDV ( 5 ) . The same personnel par ticipated in the tissue sampling throughout the experiment. At each killing period body and liver weights were recorded and individual samples of serum, liver and pectoral muscle were obtained and stored in glass containers under deep refrigera tion. Depending on the tissue, the samples were analyzed individually for DNA, RNA, total protein and 7 free amino acids (the leucines were not separated). To equalize possible analytical errors, each period and treatment were equally represented in any one series of biochemical analyses; standardized reference tissues were also included. Total protein was determined by the biuret method and was standardized by the Kjeldahl procedure. DNA and RNA were determined by the modified method described by Wannemacher et al. (6 ) and the free amino acids by the procedure Fig. 1 Body and liver weights of normal of Squibb (7 ) . chicks and diurnal changes in protein and nu The data for the muscle and liver were cleic and free amino acids of several tissues. first calculated in terms of milligrams per gram of fresh tissue and then in terms of ( < 3% ) and protein ( < 5% ) and the free DNA; the serum was expressed as milli amino acid pool ( < 5% ). grams per 100 milliliters. All values were The diurnal changes of individual free then plotted for each sampling period as amino acids in muscle, serum and liver of a percentage of initial control values. The the controls are shown in figure 2. In oscillations thus represent deviations from some cases the magnitude of change ex the initial period. These were statistically ceeded 40% . Significance levels ranged treated according to Snedecor (8 ). from < 5 % to < 1% but changes in ala nine and the leucines in the liver were not RESULTS significant. Figures 1 and 2 show that in the con The effects of NDV on the various para trol groups increments in body and liver meters are shown in figures 3 and 4. The weight met normal expectations, as de body weight increase was similar to that termined from a standard reference curve, of the controls during the first 60 hours during the 3-day observation period. In but in the final 12 hours, just prior to muscle the DNA did not change; RNA and active involvement of the NDV ( 5 ) , there protein fluctuated significantly ( < 5% ) was a depression of weight gains. How the first 36 hours, whereas the free amino ever, there was a significant ( < 1 % ) in acids oscillated significantly ( < 5 % ) the crease in liver weight and a concomitant entire trial period. The diurnal fluctua significant ( < 1 % ) increase in liver as tions in serum free amino acids were sig percentage of body weight. Oscillations of nificant ( < 1% ), as were those of the serum protein and free amino acids, as in total protein ( < 5% ). In the liver there the controls, were significant ( < 1% ) but were significant daily fluctuations of DNA there was no significant difference between EIURNAL RHYTHMS, PROTEIN METABOLISM AND DISEASE 7 3 acids or protein. Although the data were calculated wherever possible in terms of DNA, the same rhythm patterns were ob served when values were plotted as milli grams per gram of tissue. DISCUSSION This study throws light on the phenom enon of periodicity and its effect on para meters representative of a highly dynamic process — protein metabolism — in nor mal, rapidly growing animals as well as in those under disease stress. The subject is one that has been ignored by many dis ciplines. Significant diurnal fluctuations, some of more magnitude than others, occurred in all 3 tissues examined. That dietary in take is not involved is evidenced by an earlier report from our laboratories (1 ) Fig. 2 Diurnal changes in several free amino acids in normal chick tissues. control and NDV values. Except for RNA ( < 1 % ) , lysine ( < 5 % ) and histidine ( < 1% ), fluctuations in the liver no longer were significant. In comparison with con trols, the NDV caused a significant (< 1 % ) linear decline in all free amino acid levels in the liver. Most of the free amino acids in NDV- infected muscle, serum and liver showed significant ( < 1 % ) diurnal changes, but in the liver and muscle the rhythms dif fered from those in the controls. The NDV caused a desynchronization of patterns (defined as a statistically significant di vergence from control values at specific sampling hours). In muscle the RNA and free amino acid patterns were also de Fig. 3 Body and liver weights of NDV-in- synchronized. However, no desynchroni fected chicks and diurnal changes in protein and zation was evident in the serum free amino nucleic and free amino acids of several tissues. 7 4 ROBERT L. SQUIBB Using the amplitudes of the oscillations as indicators, DNA, RNA and protein syn thesis were more dynamic in liver than in muscle. The free amino acids, however, were equally dynamic in all 3 tissues. When values for control and NDV chicks were compared with respect to clock hours, it was apparent that disease stress brought about a desynchronization of all para meters in the liver within 12 hours post inoculation. Not only were the rhythms desynchronized; by the end of the 72-hour incubation period the free amino acid levels were also linearly depressed. The free amino acid pattern in the muscle was also desynchronized but not to the same extent as in the liver. However, the rhythms in the sera of control and in fected groups continued in synchroniza tion, with the curve for the NDV chicks slightly higher than that for the controls. Other analyses of the data (1 5 ) re vealed that despite the significant diurnal changes in the size of the free amino acid pool in both control and infected birds, the ratios of the individual free amino acids within the pool to total pool size remained remarkably constant. Examination of data for individual birds indicated that for the specific parameters under study, less within-group variability occurred in the evening hours than in the daytime. This low variability, reported Fig. 4 Diurnal changes in several free amino previously for the chick ( 1 ) , may also be acids in NDV-infected chick tissues. observed when data reported by Halberg that showed rhythms also occurred during et al. (8 ) and Frei and Ritchie (1 1 ) are the active involvement stage of NDV, a examined for this phenomenon. This sug time when inanition is acute. Halberg gests that variables associated with genetic et al. (9 ) also have reported that rhythms differentiation are more manifest during of several metabolic processes in mice daylight hours. persisted even though the animals were Although body weights fluctuated starved. slightly, the net gain in the controls dur Of particular interest were the diurnal ing the 72-hour period was significantly changes observed in liver nucleic acids and higher, which would be expected. As body protein. Highest levels of DNA in the non- weights changed in these groups, so did infected birds occurred in the evening and liver weights, thus maintaining a constant lowest in the daytime; similar observations ratio expected for the age period studied. have been reported for various tissues of The increase in liver weight of the NDV a number of species (1 , 1 0 -1 4 ). RNA chicks confirms an earlier observation of and protein synthesis in the liver, however, our laboratories (5 ) and also correlates were highest during daylight hours, with a previously observed increase in which is in line with Halberg’s observa nitrogen retention during the incubation tions (1 2 ) in mouse liver wherein the peak period of the NDV cycle (1 6 ). The latter for RNA preceded that of DNA. is believed to be a part of the defense DIURNAL RHYTHMS, PROTEIN METABOLISM AND DISEASE 7 5 mechanism, which includes antibody pro 5. Squibb, R. L. 1963 Nutrition and bio duction. chemistry of survival during Newcastle dis ease virus infection. I. Liver nucleic acid, The data in the present study indicate protein and lipid patterns in chicks. J. that diurnal rhythms of various tissue con Nutr., 81: 48. stituents can occur with considerable mag 6. Wannemacher, R. W., Jr., W. L. Banks, Jr. nitude even though all conditions of en and W. H. Wunner 1965 Use of a single tissue extract to determine cellular protein vironment, genetics, feeding and manage and nucleic acid concentrations and rate of ment are carefully controlled. Periodicity amino acid incorporation. Anal. Biochem., phenomena therefore must be recognized 11: 320. and considered in the design and interpre 7. Squibb, R. L. 1963 Thin-layer chromato graphic separation and quantitative deter tation of experiments with intact systems. mination of several free amino-acids of avian When periodicity is suspected, then treat liver. Nature, 199: 1216. ment results must be re-examined for their 8. Snedecor, G. W. 1957 Statistical Methods. Iowa State College Press, Ames. patterns since desynchronization may be 9. Halberg, F., J. H. Galicich, F. Ungar and L. a major or a contributing effect. As dem A. French 1965 Circadian rhythmic pi onstrated here, it is possible to obtain a tuitary adrenocorticotropic activity, rectal linear depression of values and desynchro temperature and pinnal mitosis of starving, dehydrated C mice. Proc. Soc. Exp. Biol. nization from the same treatment. Re Med., 118: 414. search toward an understanding of the 10. Dzickanowski, D., and A. Nowak 1962 biological significance of desynchroniza Studies on the control of the mitotic activity tion is indicated. Certainly this phenom in white mice. II. Diurnal variations in the mitotic activity of epidermis and intestinal enon changes biological relationships. glands and in the level of liver glycogen. Act. Physiol. Polon., 13: 700. ACKNOWLEDGMENTS 11. Frei, J. V., and A. C. Ritchie 1964 Diurnal variation in the susceptibility of mouse epi The nucleic and free amino acid analy dermis to carcinogen and its relation to DNA ses were carried out by Marie Utzinger and synthesis. J. Nat. Cancer Inst., 32: 1213. Carol Mundy. 12. Halberg, F. 1960 The 24-hour scale: A time dimension of adaptive functional or LITERATURE CITED ganization. Persp. Biol. Med., 3: 491. 13. Ritchie, A. C., J. V. Frei and H. Shinozuka 1. Squibb, R. L. 1964 Nutrition and bio 1963 The duplication of deoxyribonucleic chemistry of survival during Newcastle dis acid and epidermal carcinogenesis. Acta ease virus infection. IV. Diurnal changes in Union Int. Centre Cancer, 29: 579. protein, nucleic and free amino acids of 14. Horvath, G. 1963 Natural occurring var avian liver. Nature, 202: 1138. iations in rat liver DNA content. Nature, 2. Halberg, F. 1959 Physiologic 24-hour peri 200: 26. odicity: General and procedural considera 15. Squibb, R. L. 1966 The nature of the free tions with reference to the adrenal cycle. amino-acid pool in avian tissues. Nature, Z. Vitamin-Hormon Fermentforsch., 10: 225. 209: 710. 3. Simonnet, H. 1964 Rythmes et cycles bi- 16. Sanslone, W. R., and R. L. Squibb 1962 ologiques chez les organismes animeux. Avian disease virus and nutrition relation Ext. Biol. Med., 53: 266. ships. III. Effect of Newcastle disease virus 4. Biinning, E. 1964 The Physiological Clock. on nitrogen retention in the immature fowl. Academic Press, New York. J. Nutr., 76: 86. Copper, Sulfate and Molybdenum Interrelationships in Sheep R. D. GOODRICH 1 a n d A. D. TILLMAN Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 2-3 ABSTRACT The experiment was designed to study the interrelationship of copper, molybdenum and sulfur in ruminant nutrition. Eighty lambs were used in the repli cated, factorially arranged experiment, which involved 2 levels of copper (10 and 40 ppm), molybdenum (2 and 8 ppm), and sulfate-sulfur (0.10 and 0.40%). Response criteria were growth rate, hematology, and blood and liver mineral levels. Gain and efficiency were reduced by increasing the sulfur level in diets containing 2 ppm of molybdenum; however, this was not true when the diet contained 8 ppm of molyb denum, indicating that molybdenum alleviated the detrimental effects of sulfate. Hemoglobin concentration, erythrocyte counts and plasma protein, copper and calcium were not affected by treatments. Three-way interactions existed for hematocrit, plasma phosphorus and liver molybdenum. Hematocrit was lowered when sulfate-sulfur was increased from 0.10 to 0.40% except in the diet containing 10 ppm of copper and 8 ppm of molybdenum. Plasma phosphorus was also lowered by feeding 0.40% sulfate- sulfur except when 40 ppm of copper and 8 ppm of molybdenum were included in the diet. The three-way interaction for liver molybdenum was caused by decreases in liver molybdenum when 2 ppm of molybdenum and 10 or 40 ppm of copper were fed; feeding of 0.40% sulfate-sulfur, 8 ppm of molybdenum and 40 ppm of copper resulted in slightly higher final liver values, whereas high values were obtained for sheep fed 0.40% sulfur, 8 ppm of molybdenum and 10 ppm of copper. Liver copper was de creased by feeding 0.40% sulfate-sulfur or 8 ppm of molybdenum and increased by feeding 40 ppm of copper. Liver iron was increased as the sulfate-sulfur level was in creased from 0.10 to 0.40%. Correlations among the plasma and liver values were also discussed. Interrelationships among copper, sulfate and 8 ppm of molybdenum and 0.10 and and molybdenum have been demonstrated 0.40% of sulfur. The first trial was initi many times since Dick ( 1 ) reported that ated in the late autumn of 1964 with 40 the limiting effect of molybdenum on the lambs, which were grade Rambouillets and copper nutrition of sheep was dependent averaged 27.1 kg, and the second trial was on the sulfate level of the ration. Other initiated in the early spring of 1965 with 40 results have shown that excess levels of Rambouillet X Suffolk crossbred lambs copper, molybdenum or sulfate also exert averaging 33.9 kg. Lambs in the first trial independent effects (2 ) . As only a few were fed for 66 days and in the second for experiments have been conducted for the 60 days. Blocking on location in the barn specific purpose of studying the effects of was done in both treatments. improper ratios of the 3 minerals, the study The lambs, which were wormed with a reported herein was conducted to study the phenothiazine-lead arsenate bolus 14 days interrelationships of copper, sulfate and prior to the start of the experiments and molybdenum when 2 levels of each in all placed in individual pens with slatted possible combinations were fed to sheep. floors, were fed the basal ration during this adjustment period. At the end of the ad EXPERIMENTAL PROCEDURE justment period, initial weights were taken A replicated, randomized block design after feed and water had been removed for with a 23 factorial arrangement of treat 17 hours. All animals then were fed their ments was used so that all possible combi nations of 2 levels of copper, molybdenum Received for publication February 21, 1966. 1 Present address: Department of Animal Hus and sulfur were fed to growing lambs. bandry, University of Minnesota, St. Paul, Minnesota. 2 Department of Animal Science. Levels were 10 and 40 ppm of copper, 2 3 Approved by the Director. 76 J. N u t r it io n , 9 0 : ’66 COPPER, SULFATE AND MOLYBDENUM INTERRELATIONSHIPS 7 7 TABLE 1 wright et al. (5 ) and plasma calcium by Composition of the basal purified diet the method of Kramer and Tisdal (6 ) with modifications for citrated plasma as de g / 1 0 0 g diet Cornstarch 1 34.40 scribed by Harrison (7 ) and with a Perkin- Dextrose 24.40 Elmer Atomic Absorption Spectrophotom Cellulose 2 30.00 eter, Model 303, using methods suggested Urea 3 4.20 Corn oil4 1.00 by the manufacturer. Other procedures in Polyethylene resin 5 1.00 cluded plasma phosphorus (8), plasma pro Choline chloride 0.10 tein (9 ) and plasma molybdenum and liver Vitamins A and D 6 0.02 k2c o 3 2.22 copper, iron and molybdenum (1 0 ). The CaHPO, 1.32 data were subjected to analysis of variance. MgSO, 0.12 MgC03 • Mg (OH ) 2 ■ 3H20 0.27 RESULTS AND DISCUSSION Na2S04 0.25 NaCl 0.62 As the replication X treatment and block-within-replication X treatment inter m g / 1 0 0 g diet actions were insignificant (P > 0 .0 5 ), the FeS04 42.50 MnSOfHjO 11.50 results of the 2 trials were combined and Na2B407 12.50 are shown in table 2. Main effects which ZnS04-7H,0 15.00 are also a part of a significant interaction CuC03-Cu(OH)2 1.75 Na2Mo04-2H20 0.50 are presented in the footnotes to table 2, CaF2 0.20 but are discussed only in relation to the second factor. Sulfur level X molybdenum f i g / 1 0 0 g diet KI 15.00 level interactions were found for gains Cr2(S04)j 40.00 (P < 0 .0 5 ) and feed efficiency (P < 0 .0 1 ): CoC12-6H20 45.00 When the sulfur level was increased from Na2Se04 25.00 0.10 to 0.40% in diets containing 2 ppm 1 This diet contained 0.10% sulfur, 10 ppm of of molybdenum both of these response copper and 2 ppm of molybdenum. All modifications to obtain the experimental diets were made by re criteria were reduced (P < 0 .0 1 ); however, ducing starch in accord with an increase in Na2SC>4 , no such reduction was obtained when the CuC0 3 *Cu( 0 H) 2 or Na2mo0 4 -2 H2 0 . 2 Solka-Floc (B-W 20), Brown Company, Berlin, sulfur level was increased in diets contain New Hampshire. 3 Crystalline urea, courtesy of John Deere Chemical ing 8 ppm of molybdenum, indicating that Company, Pryor, Oklahoma. 4 Mazola, Corn Products Company, Santoquin (Mon 8 ppm of molybdenum partly overcame the santo Company, St. Louis) added to give 0.0125% in depression caused by feeding 0.40% sulfur total ration. 5 Alathon, E. I. DuPont de Nemours, Inc., Wilming This interaction did not exist for feed con ton, Delaware. 6 20,000 IU and 2,500 USP units of vitamins/g. sumption, however; the higher sulfur level reduced (P < 0.05) feed consumption re appropriate experimental rations, composi gardless of level of molybdenum. Other tions of which are shown in table 1. Feed workers (1 1 , 12) have shown that sulfate and water were provided free-choice. Final exerts a protective effect against molyb weights were also preceded by a period of denum toxicity because it reduced absorp 17 hours without food and water. tion and increased urinary excretion of this Blood samples, taken by jugular punc element. Results of the present experiment ture, were obtained at the beginning and indicate that the poor performance caused end of the growth trial. Hemoglobin values by a high level of sulfate was partially alle were determined on the citrated blood by viated by feeding additional molybdenum, the method of Sheard and Sanford (3 ) . a relationship not found by previous Erythrocytes and the percentage of packed workers. cells were measured by the method of Hemoglobin concentration and erythro Schalm (4 ) and the microhematocrit cyte counts were not significantly affected method, respectively. All analyses involv by treatments. A three-way interaction ex ing whole blood were completed soon after isted for hematocrit; the higher sulfur level bleeding, but the plasma was frozen until lowered hematocrit values except in sheep analysis was performed. Plasma copper receiving the diet containing 10 ppm of was determined by the method of Cart copper and 8 ppm of molybdenum. TABLE 2 Effects of sulfur, molybdenum and copper levels on sheep 8 7 Sulfur level, % 0.10 0.40 Molybdenum level, ppm 2 8 2 Copper level, ppm 10 40 10 40 10 40 D L 5 0 d c 0 > [ H r D C O C M ( O C * M < D l d ' o O HrH O rpCOO H CO 1 H C r rH CMrH CO H O r-H rH rHCO OI>rHCDCMC0CM’ CD^rHl>05C0rH^ CO CM O 05O O (M CD IDr-1 CD* Ó iH l>CO* *—ido £ (M 5 0 5 0 t > LD ^ I> rt Offi £ Offi a s .g ci O \ g o O OrO'P H- O H 5 ¿ 0 b rH 0J 05 05 bo.S GO O O O H CC rH rH ih 3 rH rH O rH O rH rH T3 O S t> r rH > t > ^ • ^ C0 qq 1 05 H H H WP C^CD'^COCMCM^ r-P H o o H HH ^ CD ^ t> HH H ICDOHCDH^CDO) CO p 05 •p io§> C s C = § O o o cJ cti ccJ oJ o3 j o aa a a a a a ft O OO MCft 0P r Pr H P S «§ ““ h ^ H a» -H^ ^ o P CM HrH rH H CM rH ID H rH ID rH H rH^ rH O . . ODIH N A D TLMAN TILLM D. A. AND GOODRICH D. R. to £2?fto h I rH rH ID 05 05 ID CO rH > t CO O > rH I I O I rh P CD rH rH rH rH p bo h § § , P £ - a M P M , CM a “ i! rHCO O CO CM05 O t> CD o P a- ■ a & t CM H CO CDID — rH ¡J¡J¡3 « GO «o T3 ¡»S, rH ID 5 5 05 05 05 Pi tí Jí 05 U > > > > a l U 8 ? P o> 0) a P bn bo -a “ CD( q M M > I in CD^ M , CM > l T3 cq CO H >, P S £ H 05 rH 2 ^ 4 s I Gd H S 05 G 1-1 H.Q j 03 ■Ss* ID I> rH * CM # CD HÍ * U CD cm O rH o ’«3 t f t f S G s s “ 1 rP P .8« .o G C/5T3 rH* * c c * ao* CD 'P ^ o ^ 05 05 rH AS g 8 G£ a « 05 s.a s ^ o fP g o H 05 % o <« 05 3 3 O G C5 rG ** o G AC/5 a t f Gbfi « G ro .i-t g ns05 wG w a _ C/5 2 .0 6 o Ü x G a 03 G G C5 2 2 CO CM ft a s o ft o ft s CO rH CM o q O q * ^T> t> IDl> * ^ q q CO CM - * CD050 id Ü U coed t>b: * * TH<* o o G G ft f t f t f t f s s * x-x G ^ ^ ^ o / C/5 c/5 ^ CD O n * d. ^ .d 2 ^ £ d § O 05 v_x* A A - ® L- ’DS G G 05 03 o o C/5 | o 2 O 2 o | g S « ■g,® P3t>: mo rirn C ft0 «C0HrH O^L/rH a . ^ l g c fi/ ftp-Uu CD ' * ^.o O CDCO I> CM -3- i> cd 05 O * CD q . v—' A y A 5N * h | I g £ ' 'ft t> rH s CO vQ C/5 o o d 2 ex S I rG/q \LO ó Z 0) h g W)v_/ bOt> =ico &A rH 05 HO CD CD CDo 05 * CM P< "^ ft O CM P? * •rj rH (M CO(M ^ rH •rj M | a s ^ $ í ali l a ¿ío 05 fnOCOrHCO ft °o ft^ 6-íP-o x l Q o ¿ ' - GOCDC-rH g't'S oü ¿H A| AA o “ CD 05 CO £ * • l COPPER, SULFATE AND MOLYBDENUM INTERRELATIONSHIPS 7 9 Protein, copper and calcium levels in Goodrich and Tillman (1 5 ) observed blood plasma were not affected by treat that dietary sulfate in comparison with ele ments. Plasma phosphorus was affected mental sulfur lowered liver copper even in (P < 0.05) by both copper and sulfur the presence of low levels of molybdenum. levels; the higher level of sulfur decreased As Dick (1 8 ) reported that ferrous sulfide (P < 0 .0 5 ), whereas the higher level of reduced copper absorption, it appears that copper increased (P < 0 .0 5 ) plasma phos insoluble copper sulfide may be formed phorus levels. Also, there was a significant from either sulfate or sulfide, resulting in interaction between sulfur and molyb reduced absorption of copper from the in denum levels on plasma phosphorus level: testinal tract of ruminants. Molybdenum Increasing the molybdenum level from 2 also reduces copper stores (1 9 , 2 0 ). Dick to 8 ppm caused reduced (P < 0.05) plasma (1 8 ) suggested that high intakes of molyb phosphorus levels when 0.10% sulfur was denum reduce copper absorption and in fed but had no effect when the diet con crease copper excretion only in the pres tained 0.40% sulfur. Also, when the level ence of adequate endogenous or exogenous of sulfur was increased from 0.10 to 0.40% sulfate. Results of the present experiment in diets containing 2 ppm of molybdenum indicate that 0.10% sulfur, as sulfate, was plasma phosphorus was reduced (P < 0.01) adequate for molybdenum to exert this but the addition had no effect when the effect. As the effect of elemental sulfur diet contained 8 ppm of molybdenum. upon copper storage appears to be different There was a copper X sulfur X molyb (1 5 ), further work in this area is indicated. denum level interaction (P < 0.05) in Liver iron levels were significantly in plasma phosphorus level: Sheep fed 0.40% creased as the sulfur level of the ration was sulfur had a lower level of plasma phos increased and are interpreted to be a reflec phorus except when the diet contained 40 tion of the influence of sulfate on copper ppm of copper and 8 ppm of molybdenum, metabolism since Matrone (2 1 ) observed increased dietary levels of both copper that during copper deficiency iron can and molybdenum were required to return enter liver tissue. plasma phosphorus to normal. Shirley et Liver molybdenum levels were affected al. (13, 14) reported losses of phosphorus (P < 0.05) by all treatments, and all inter from the bodies of steers or rats to be two actions were significant. The copper X sul to three times normal when the diet con fur X molybdenum interaction was caused tained high levels of molybdenum and low by liver molybdenum being lower when the levels of copper. As increased levels of sul diet contained 0.40% sulfur plus 2 ppm of fate lowers the retention of copper in sheep molybdenum and 10 or 40 ppm of copper, (1 5 ) and adequate copper levels are re in contrast with the small increase ob quired for proper phosphorus metabolism tained when the diet contained 0.40% sul (1 4 ), it appears that molybdenum acts to fur, 8 ppm of molybdenum and 40 ppm correct the effect of a high level of sulfate of copper and a large increase when the and that additional copper was required diet contained 0.40% sulfur, 8 ppm of because the molybdenum level did not com molybdenum and 10 ppm of copper. These pletely counteract the effect of sulfate. data are in agreement with results of other Growth results of the present experiment, workers ( 2 ) , but the reason for the in in which sheep fed 0.40% sulfur and 8 crease in liver molybdenum when 0.40% ppm of molybdenum gained much faster sulfur, 8 ppm of molybdenum and 10 or than those fed 0.40% sulfur and 2 ppm of 40 ppm of copper were fed remains ob molybdenum, support this idea. scure. Plasma molybdenum levels increased as Correlations of various plasma and liver the dietary levels increased and these re mineral levels are shown in table 3. The sults agree with those of Cox and Harris correlations represent within-treatment and (1 6 ) and Gray and Daniel (1 7 ). within-replication calculations and thus The higher level of sulfur or molyb are unbiased by treatment means. Only denum reduced (P < 0 .0 1 ) copper storage the plasma molybdenum level and liver in the liver and increased dietary copper molybdenum coefficient was significant caused increased storage of the element. (P < 0 .0 5 ); however, the data tend to sup- 8 0 R. D. GOODRICH AND A. D. TILLM AN T A B L E 3 8. Hawk, P. B., B. L. Oser and W. H. Summer- Within treatment, within replication correlation son 1954 Practical Physiological Chemis coefficients among some plasma and try, ed. 13. The Blakiston Company, Phila liver values delphia. 9. Stadtman, E. R., G. D. Novelli and F. Lip- mann 1951 Alimentary excretion of phos Variables Correlation Coefficients 1 phorus-32 in steers on high molybdenum and copper diets. J. Animal Sci., 9: 552. Liver Cu and liver Fe 0.11 10. Sandell, E. B. 1959 Colorimetric Determi Liver Cu and liver Mo -0 .1 9 nation of Trace of Metals, ed. 3. Intersci Liver Cu and plasma Cu 0.04 ence Publishers, New York. Liver Cu and plasma Mo -0 .0 3 Liver Fe and liver Mo -0 .0 8 11. Van Reen, R., and M. A. Williams 1956 Liver Mo and plasma Mo 0.49 2 Studies on the influence of sulfur com Plasma Cu and plasma Mo 0.26 pounds on molybdenum toxicity in rats. Arch. Biochem. Biophys., 63; 1. 1 Degrees of freedom are 48. 12. Mills, C. F., K. J. Monty, A. Ichihara and 2 P < 0.01. P. B. Pearson 1958 Metabolic effects of molvbdenum toxicity in the rat. J. Nutr., port the idea (2 ) that lambs with high 65; 129. molybdenum levels have low liver copper 13. Shirley, R. L., R. D. Owens and G. K. Davis and high plasma molybdenum levels and 1950 Deposition and alimentary excretion those with high plasma copper levels also of phosphorus-32 in steers on high molyb denum and copper diets. J. Animal Sci., 9: tend to have high plasma molybdenum 552. levels. 14. Shirley, R. L., R. D. Owens and G. K. Davis 1951 Alimentary excretion of phosphorus- LITERATURE CITED 32 in rats on high molybdenum and copper 1. Dick, A. T. 1952 The effect of diet and diets. J. Nutr., 44: 595. of molybdenum on copper metabolism in 15. Goodrich, R. D., and A. D. Tillman 1966 sheep. Australian Vet., J., 28; 30. Effects of sulfur and nitrogen sources and 2. Underwood, E. J. 1962 Trace minerals in copper levels on the metabolism of certain human and animal nutrition, ed. 2. Aca minerals by sheep. J. Animal Sci., 25; in demic Press, New York. press. 3. Sheard, C., and A. H. Sanford 1929 A 16. Cox, D. H., and D. L. Harris 1960 Effect photo-electric hemoglobinometer. J. Lab. of excess dietary zinc on iron and copper in Clini. Med., 14: 558. the rat. J. Nutr., 70; 514. 4. Schalm, O. W. 1961 Veterinary Hematol 17. Gray, L. F., and L. J. Daniel 1954 Some ogy. Lea and Febiger, Philadelphia. effects of excess molybdenum on the nutri 5. Cartwright, G. E., P. J. Jones and M. M. tion of the rat. J. Nutr., 53; 43. Wintrobe 1945 A method for the determi 18. Dick, A. T. 1956 Molybdenum in animal nation of copper in blood serum. J. Biol. nutrition. Soil Sci., 81: 229. Chem., 160: 593. 19. Cunningham, I. J., K. G, Hogan and B. M. 6. Kramer, B., and F. F. Tisdall 1921 A Lawson 1959 The effect of sulfate and simple method for the determination of cal molybdenum on copper metabolism in cattle. cium and magnesium in small amounts of New Zealand J. Agr. Res., 2; 145. serum. J. Biol. Chem., 47: 475. 20. Dick, A. T. 1954 Studies on the assimi 7. Harrison, G. A. 1957 Chemical Methods lation and storage of copper in crossbred in Clinical Medicine. Their Application and sheep. Australian J. Agr. Res., 5; 511. Interpretation with Techniques of Simple 21. Matrone G. 1960 Interrelationships of iron Tests. J. and A. Churchill Ltd. London, and copper in the nutrition and metabolism p. 366. of animals. Federation Proc., 19; 659. Correlation of Liver Cytochrome Oxidase Activity with Mitochondrial Cytochrome Oxidase and Phospholipid Concentrations in Protein-deficient Rats J. N. WILLIAMS, JR., R. M. JACOBS a n d ALICE J. HURLEBAUS Laboratory of Nutrition and Endocrinology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland ABSTRACT The object of the present study was two-fold: To observe to what extent cytochrome oxidase activity as measured manometrically is correlated with cytochrome oxidase concentration as measured spectrophotometrically and with mitochondrial phospholipid concentration during progressive protein depletion followed by repletion; and to study the influence of methionine fed in a protein-free ration on the maintenance of liver cytochrome oxidase activity. Phospholipids have been shown by others to be necessary for the normal functioning of cytochrome oxidase. Methionine fed in a protein-free ration has been shown to enable the maintenance of certain other liver enzyme and lipid components at higher levels than if methionine is omitted. In pro tein deficiency, rat liver cytochrome oxidase activity and cytochrome oxidase concen tration as measured in isolated mitochondria followed almost identical patterns, both falling to 40 to 50% of normal after 8 weeks and remaining at that level until death of the animals at about 14 weeks. Upon repletion with protein, both activity and con centration of the enzymes returned to normal in a linear fashion within 7 days. Mitochondrial phospholipid concentration follows essentially the same pattern. It appears that, although phospholipid follows the same pattern and is essential for the activity of cytochrome oxidase, the changes in cytochrome oxidase activity are not a reflection of changes in mitochondrial phospholipid in protein deficiency, but more likely they represent a loss of enzyme protein. Addition of methionine to a protein-deficient diet does not protect against loss of cytochrome oxidase, in contrast with its protection of other components of the liver, such as succinic dehydrogenase, ubiquinone, and phospholipids. In an earlier paper (1 ) it was shown dase activity. Moreover, by using a method that approximately 80% of the cytochrome for measuring cytochrome oxidase concen oxidase activity of rat liver cells is lost after tration in whole liver mitochondria ( 6 ) , prolonged protein depletion. In those stud both cytochrome oxidase activity as well as ies 0.30% DL-methionine was included in concentration could be followed. In this the protein-free ration. In subsequent stud way it could be learned whether the losses ies it was found that methionine fed in of cytochrome oxidase activity (1 ) and such a ration protected against the loss of phospholipid observed in protein deficiency certain other enzymes in the electron trans (4 ) were related. In the spectrophoto- port system of liver mitochondria (2 , 3 ). metric assay for cytochrome oxidase con It also was found that liver phospholipid centration the presence or lack of phospho concentration was maintained at more lipid in the preparation would not influence nearly normal levels when methionine was the results since the color produced is due fed in a protein-free ration ( 4 ) . Brierley to the cytochrome a + a 3 hemoprotein moie and Merola (5 ) observed that purified cyto ties of the mitochondria. chrome oxidase loses almost all activity EXPERIMENTAL METHODS after phospholipids are removed by solvent extraction and that the activity is partially Adult male rats of the Sprague-Dawley restored by addition of phospholipid. It strain, previously adjusted for 3 weeks to was thus possible that omission of methio a complete purified diet ( 7 ) , were used nine from the protein-free ration would throughout. They weighed approximately elicit a still further loss of cytochrome oxi Received for publication March 19, 1966. J. N u t r it io n , 9 0 : ’66 81 82 J. N. WILLIAMS, JR., R. M. JACOBS AND ALICE J. HURLEBAUS 300 g at the start of the experiment. Briefly, tures of whole homogenates and mitochon the complete ration (diet R l) consisted of: drial suspensions from control and defi (in per cent) casein, 20; DL-methionine, cient rats were studied to determine 0.3; corn oil, 5; salts N plus molybdate ( 8 ) , whether there was an inhibitor of cyto 6.5; glucose monohydrate, 63.5; choline chrome oxidase in the deficient liver which chloride, 0.2; ¿-inositol, 0.02; and water- might account for the lowered cytochrome soluble vitamin mix in glucose monohy oxidase activity. Also such studies would drate (9), 4.5. Fat-soluble vitamins (7 ) indicate whether normal liver could supply were given weekly to each rat in 2 drops of a substance such as phospholipid, neces corn oil. Four groups of rats were usually sary for the normal operation of cyto used in the studies: Group 1 received the chrome oxidase in liver from protein complete diet ad libitum. Group 2 received deficient animals. Again, amounts of mito the same diet but was pair-fed with group chondrial suspensions equivalent to the 4 (average daily food consumption). Group amount of liver in the whole homogenates 3 received diet R l from which the casein were used. was omitted. Group 4 received diet Rl Finally, a series of studies was performed from which both casein and methionine in which rats receiving diet R l or diet R l were omitted. The food consumption of less casein and methionine (similar to groups 3 and 4 was almost identical. groups 1 and 4 above, respectively) were The general plan of a portion of the stud used. Groups of animals were killed after ies is similar to that described in previous 56 and 100 days of protein deficiency fol papers of this series ( 1 -4 , 7 ). Briefly lowed by 1, 2, 3, 4, and 7 days of protein groups 1 to 4 were fed their respective ra repletion. In these studies cytochrome oxi tions for 102 days, during which time 6 to dase activity, cytochrome oxidase concen 8 animals of each group were killed after tration, and phospholipid concentration, in 24, 56, and 102 days. At 102 days many the liver mitochondria only, were followed. of the animals in the deficient groups were RESULTS moribund from the deficiency. Protein was then added to the diets of groups 3 and 4, In figure 1 are shown the results of the and group 2 was still pair-fed with group 4. first phase of these studies. The food in Rats from group 1 were killed after 22, 52, takes of groups 3 and 4 and those of group and 88 days post-repletion; and rats from 2 (pair-fed controls) were considerably groups 3 and 4 after 10, 52, and 88 lower than normal, reaching about one- days post-repletion. Mitochondrial isola third of normal after 80 to 100 days of tions (10) and cytochrome oxidase assays protein depletion. The food intakes of (11) were performed immediately after re groups 3 and 4 were almost identical. Low moval of the livers, and portions of each ered food intake (group 2) did not influence liver were stored frozen for later DNA ex liver cytochrome oxidase activity. (In other tractions (12) and analysis (13). Portions studies, we have occasionally noticed a de of the mitochondria were mixed with 20 crease in this group maximally to 60 to volumes of 2:1 chloroform-methanol and 70% of normal. The reason for this varia stored at —15° for later extraction of lipids tion from one group of rats to another or by the method of Folch et al. (14). Phos from one study to another is not clear. phorus in aliquots of the lipid extracts was However, in the present studies no signifi analyzed by the method of Fiske and Sub- cant change from normal was observed.) barow (15). The cytochrome oxidase activity of both In some of the studies the cytochrome groups 3 and 4 fell to approximately the oxidase activity of the whole liver homoge same levels, there being no effect upon this nate in 0.25 m sucrose was compared with enzyme of methionine added to the protein- that of mitochondria suspended in 0.25 m free ration. sucrose. In these experiments aliquots of In table 1 a comparison of cytochrome the mitochondrial suspensions were used oxidase activity from whole homogenates which were equivalent to the amount of and isolated mitochondria is presented for liver in the flasks containing the whole each of the groups after 56 days of feeding homogenates. In other experiments mix the experimental diets. These results indi- LIVER CYTOCHROME OXIDASE AND PROTEIN DEFICIENCY 83 cate that there is no difference in activity between the 2 types of enzyme preparations in any of the groups. This indicates that the lowered activity in the protein-deficient groups is not due to the presence of an inhibitor in extramitochondrial liver frac tions but is more than likely due simply to a lowered concentration of the enzyme. This is further substantiated by the results (table 2) which show that mixing of ho mogenates or mitochondria from normal and deficient rat livers had no effect on the activity of the opposite group. Cytochrome oxidase activity as measured manometrically and cytochrome oxidase concentration as measured spectrophoto- metrically in mitochondria gave almost identical results throughout the entire course of protein depletion and repletion (fig. 2 ). Variation between the 2 sets of results was not significant. In the same figure it can be seen that the results for phospholipid changes were also similar to the cytochrome oxidase results. However, as will be pointed out later, this similarity is fortuitous and unless a comparison had been made between cytochrome oxidase ac Fig. 1 Response of cytochrome oxidase (C.O.) tivity and cytochrome oxidase concentra activity of liver homogenates to protein depletion tion, it might have been concluded that the with and without 0.3% DL-methionine, and reple loss in cytochrome oxidase activity was a tion. Protein repletion was begun at the arrows. # = group 1 (ad libitum-fed, normal controls); direct effect of loss of mitochondrial phos A = group 2 (pair-fed controls); B = group 3 pholipid. (protein-deficient rats fed 0.3% DL-methionine); O = group 4 (protein-deficient rats). Statistical DISCUSSION analysis using Student’s t test indicated the fol lowing significant differences (P<0.01) among Although the phospholipid results were the points in the figure: very similar in general to those for cyto chrome oxidase, evidence that the loss of 24 days: 1 versus 3, 1 versus 4, 2 versus 3, 2 versus 4; 56 days: 1 versus 3, 1 versus 4, 2 ver cytochrome oxidase activity in protein defi sus 3, 2 versus 4; 102 days: 1 versus 3, 1 versus 4, ciency is due to loss of enzyme protein 2 versus 3, 2 versus 4. rather than phospholipid associated with the protein is that the response of cyto- TABLE 1 Comparison of homogenate and mitochondrial cytochrome oxidase activities after 56 days of depletion Homogenate cytochrome Mitochondrial cytochrome Group oxidase activity oxidase activity flitters 02/hr/mg liver DNA fditers 02/hr/mg liver DNA 1 (ad libitum-fed normal controls) 27,900 ±2,950 1 24,200 ±2,050 2 (pair-fed controls) 27,800 ±2,100 23,400 ± 4,100 3 (protein-deficient rats fed 0.3% DL-methionine) 8,200 ±2.050 6,970 ±1,640 4 (protein-deficient rats) 9,840 ±820 10,250 ±2,460 1 Sem, 4 rats/group. 8 4 J. N. WILLIAMS, JR., R. M. JACOBS AND ALICE J. HURLEBAUS TABLE 2 Cytochrome oxidase activity of homogenates and mitochondria of controls mixed in equal amounts with those from experimental groups Homogenate cytochrome Mitochondrial cytochrome oxidase activity oxidase activity Groups Avg of Avg of mixed Mixed individual Mixed individual homogenates groups mitochondria groups alone alone filiters 02/hr/mg liver filiters 02/hr/mg liver 1 + 4 48.0 ±5.4 1 4 8 .0 ± 3 .8 49.3 ± 5 .1 41.1 ± 6 .9 2 + 3 39.8 ± 3.9 39.6 ± 5 .8 43.7 ± 3.4 35.2 ± 3 .2 1 SEU, 4 rats/group. lipid would make little difference. It has been shown by Brierley and Merola (5 ) that cytochrome oxidase from which phos pholipids have been extracted gives almost the same spectrum as unextracted oxidase, the only difference being a shift in the 605 mu peak to 603 mu. This slight shift would have negligible influence on the re sults in the spectrophotometric assay for mitochondrial cytochrome oxidase since the 605 mu peak is fairly broad in these preparations (6). Further evidence that the loss of cytochrome oxidase activity in protein deficiency is due to loss of enzyme protein rather than phosphohpid associated with the protein is as follows: Since me thionine was observed earlier to protect against loss of total phospholipids, it would have been expected that, if the loss of cytochrome oxidase activity was due to loss of phospholipid (5), it should have protected against loss of the enzyme ac tivity. Furthermore, in the experiments in which control enzyme preparations were mixed with deficient preparations, some TIME IN DAYS equilibration of phospholipids from the nor mal to the deficient should have occurred. Fig. 2 Response of mitochondrial cytochrome oxidase activity, cytochrome oxidase concentra Thus if the lower enzyme activity in the tion and phospholipid concentration to protein deficient preparations were due to lowered depletion followed by repletion. Protein repletion phospholipid, the activity of the mixed was begun at the arrows. Statistical analysis us preparations should have been higher than ing Student’s t test indicated the following signifi cant differences (P < 0.01) between the protein- the sum of the activities of the individual deficient group (group 4) and the ad libitum-fed preparations. That the lower enzyme ac normal controls ( group 1 ) : day 56, day 100 and tivity of the protein-deficient group was not day 1 post-repletion. due to production of inhibitors by the pro tein deficiency is also indicated by the fact chrome oxidase concentration was almost that the enzyme preparations from the identical to that of the enzyme activity. deficient groups had no influence on the In the spectrophotometric assay for mito activity of those from the control groups. chondrial cytochrome oxidase concentra Thus it appears that the similarities in the tion the presence or absence of phospho loss of phospholipids and cytochrome oxi LIVER CYTOCHROME OXIDASE AND PROTEIN DEFICIENCY 8 5 dase activity in protein deficiency are prob tein depletion. VI. Total phospholipids and ably coincidental. plasmalogens, and protection of phospho lipids by methionine and cystine. J. Nutr., The responses of cytochrome oxidase as 85: 82. observed in the present paper and of suc 5. Brierley, G. P., and A. J. Merola 1962 Stud cinic dehydrogenase (3 ) to uncomplicated ies of the electron-transfer system. XLVIII. protein deficiency are almost identical, Phospholipid requirements in cytochrome oxi dase. Biochim. Biophys. Acta, 64: 205. pointing to perhaps a common regulatory 6. Williams, J. N., Jr. 1964 A method for the mechanism controlling the maintenance of simultaneous quantitative estimation of cyto levels of these 2 mitochondrial enzymes chromes a, b, Ci, and c in mitochondria. Arch. under the stress of protein deficiency. How Biochem. and Biophys., 107: 537. ever, the fact that methionine protected 7. Williams, J. N., Jr. 1961 Response of the liver to prolonged protein depletion. I. Liver against the loss of succinic dehydrogenase weight, nitrogen, and deoxyribonucleic acid. but had no effect on cytochrome oxidase J. Nutr., 73: 199. indicates that such a common regulatory 8. Fox, M. R. S., and G. M. Briggs 1960 Salt mechanism, if it exists, is more complex mixtures for purified-type diets. III. An im than might be suspected at first glance. proved salt mixture for chicks. J. Nutr., 72: 243. Further studies are being carried out at 9. Fox, M. R. S., L. O. Ortiz and G. M. Briggs present concerning such a possible com 1955 Toxicity of ethionine in the young mon regulatory mechanism. chick. Agr. Food Chem., 3-' 436. 10. Schneider, W. C., and G. H. Hogeboom 1950 ACKNOWLEDGMENTS V. Further studies on the distribution of cyto chrome c in rat liver homogenates. J. Biol. The authors wish to thank Mrs. Esther Chem., 183: 123. Hurley and Woodrow Duvall for care and 11. Umbreit, W. W., R. H. Burris and J. F. feeding of the animals in these studies. Stauffer 1959 Manometric Techniques. Bur gess Publishing Company, Minneapolis, p. 173. LITERATURE CITED 12. Schneider, W. C. 1945 Phosphorus com 1. Williams, J. N., Jr. 1961 Response of the pounds in animal tissue. I. Extraction and liver to prolonged protein depletion. II. The estimation of desoxypentose nucleic acid and succinic oxidase system and its component of pentose nucleic acid. J. Biol. Chem., enzymes. J. Nutr., 73: 210. 161: 293. 2. Williams, J. N., Jr. 1963 Response of the 13. Davidson, J. N., and C. Waymouth 1944 liver to prolonged protein depletion. III. Co Tissue nucleic acids. 3. The nucleic acid enzyme Q. Arch. Biochem. Biophys., 101: and nucleotide content of liver tissue. Bio 512. chem. J., 38: 379. 3. Williams, J. N., Jr. 1964 Response of the 14. Folch, J., M. Lees and G. H. Sloane-Stanley liver to prolonged protein depletion. IV. Pro 1957 A simple method for the isolation and tection of succinic oxidase and succinic de purification of total lipides from animal tis hydrogenase by dietary methionine and cys sue. J. Biol. Chem., 226: 497. tine in a protein-free ration. J. Nutr., 82: 51. 15. Fiske, C. H., and Y. Subbarow 1925 The 4. Williams, J. N., Jr., and A. J. Hurlebaus colorimetric determination of phosphorus. J 1965 Response of the liver to prolonged pro Biol. Chem., 66: 375. Effect of Iron on the Growth Rate of Fishes 1,2 MARTIN ROEDER a n d RACHEL H. ROEDER Department of Biological Science, Florida State University, Tallahassee, Florida ABSTRACT To determine the nutritional requirment for iron in fish growth, the effect of daily addition of supplemental ferrous sulfate on the growth of Xiphophorus helleri and Xiphophorus maculatus (the swordtail and the platyfish) was measured by determination of weight gain and hematocrit levels. Both variables increased as a result of the added iron. The effectiveness of the treatment diminished as sexual maturity was approached. Treatment with ferrrous sulfate led to an increase in growth rate even in fish fed maximally with live brine shrimp. Ferric salt was not judged effective. Addition of ferrous iron also significantly decreased the mortality from hatching to maturity. Little is known of the mineral require water was adjusted to between 7 and 8, ments, or of the mineral metabolism of and Anacharis (Elodea) and calcium car fishes. Lovelace and Podoliak ( 1 ) showed bonate blocks were added to the tanks. that calcium is absorbed through the gill Snails were introduced and allowed to of the brook trout, and Phillips and his grow. As soon as possible after swimming co-workers (2 ) have reported dietary re fry appeared in a tank, they were removed, quirements for growth in trout, although and fry of a single brood were separated their main concern was with the organic into groups of equal size, and placed into constitutents of prepared hatchery feeds. newly established aquariums. Fry were They determined calcium, phosphorus, and fed an average of 90 mg of dry food/d ay/ magnesium levels in their diets, but did tank, with a supplement of 300 mg of not systematically vary these. Cheprakova liquid food, containing 50 mg dry weight.3 (3 ) reported some effects of iron salts The dried food contained 45% crude pro on developing eggs of the loach, the sig, tein and 3% crude fat, with a maximal and the perch, but the data reported in fiber content of 4% , and the analysis of that paper are not at all conclusive. the liquid food showed 5% crude protein, In connection with studies in our lab 1% crude fat with a maximal crude fiber oratory concerned with respiratory rate content of 1.5%. The iron content of and other factors in the swordtail ( Xipho these feeds, of the aquarium water, and phorus helleri), the platyfish (Xiphophorus of live brine shrimp was measured, us maculatus) and the hybrid helleri X macu ing a modification of the technique of latus (4), we noted that the addition of Sherman et al. (5), in which alpha-alpha' ferrous sulfate to water in which the fishes dipyridyl is used as the color reagent, and were raised appeared to have a stimulatory which is reported to measure biologically effect on the growth of these fishes, as compared with untreated controls. Ac Received for publication March 24, 1966. 1 Supported by grant GB 2664 from the National cordingly studies were made in which we Science Foundation. 2 We wish to thank Wardley Products, Inc., Long systematically varied the amount and kind Island City, New York, for their courtesy in supplying of iron available to newly hatched fry and dried food diets and numerous other aquarium sup plies. to older fishes, and compared weight 3 Dried food was prepared from: animal liver meal, whale meal, meat meal, menhaden fish meal, crab changes and changes in hematocrit values meal, shrimp meat, salmon egg meal, wheatgerm meal, of treated to untreated animals. dried D a p h n ia , kelp meal, oat meal, wheat flour, corn meal, soya flour, 3% whole egg solids, 3 % dried skim milk, 3 % salt, 2 % mosquito larvae, 2 % ground METHODS aniseed, 1% calcium triphosphate, 1% barley malt and 1% primary dried yeast. Liquid food supplement Fish were maintained in 13.5-liter contained: whole eggs, yeast hydrolysate, 3 % cod liver oil, 2.5% kelp meal, 2 % animal liver meal, aquariums, filled with water withdrawn 0.5% spinach powder, 0.5% gum acacia, 0.5% gum tragacanth, 1% dextrin, water and 0.25% sodium from the Wakulla River. The pH of the bisulphite. 86 J. N u t r it io n , 9 0 : ’66 IRON AND FISH GROWTH 8 7 X. helleri AGE in DAYS Fig. 1 Effect of varying concentrations of ferrous sulfate on the growth of the green swordtail, Xiphosphorus helleri. available iron, as measured by growth re in figure 1. After 60 days, the differences sponses, rather than total iron, which may in weight and in survival were striking, be bound and not available for absorption especially as a higher growth rate in the into the blood of the organism. The aquar tanks containing fewer fish would be ex ium water contained less than one part pected (7). After 100 days the groups per million of iron, making the total con which had received 50 and 100 mg of fer tent of an untreated tank less than 0.2 mg. rous sulfate were divided, and the treat Daily rations of food contained less than ment continued for half, and discontinued 0.07 mg available iron, while an average in the other half. The data are presented feeding of live brine shrimp ( Artemia sp.) in figure 2, and indicate that continued contained only 5 X ICR4 mg. In compari treatment with the salt loses its effective son with the amount of added iron these ness after 100 days. The apparent depress are considered to be negligible levels. Sup ing effect of the iron treatment on the plemental iron salts were added daily at fish in the group to which 100 mg had been the indicated levels, as analyses for solu continued may be explained by noting ble iron showed complete disappearance that in this group no evidence of sexual of the dissolved salt after 4 hours. Copious maturity was noted, whereas in the group precipitates of ferrous hydroxide were ob which had been removed from treatment served in all treated tanks. Hematocrit with 100 mg two of five had already levels were measured using a modification shown marked sexual differentiation. In of the orbital bleeding technique of Riley swordtails, the striking sexual dimorphism (6 ) in heparinized micro-hematocrit tubes. leads to both size and weight differences between the sexes, females being larger EXPERIMENTAL RESULTS and heavier than males. When secondary Initially, 40 fish from 2 broods of sword sex differentiation occurs, then, the data tails were combined, and then separated can no longer be treated as coming from a into 4 tanks, each containing 10 fish. common pool, but would have to be paired Three groups were treated by daily addi for sex. The indication that iron treat tion of varying amounts of ferrous sulfate, ments lose effectiveness was confirmed and one was kept as a control group. The by several attempts to change growth results of this initial experiment are shown rates in adult fish after they had been 88 MARTIN ROEDER AND RACHEL H. ROEDER raised with no iron present; no changes in growth rate or in hematocrit values were observed in any of these experiments. The data from 2 experiments utilizing the platyfish are presented in figure 3. In the lower portion of the figure the curves indicate that after 75 days the growth rate of the control group exceeded that of the 50-mg group, and that the growth rate before this time, as indicated by the slope of the curves, is approximately the same for the control, 25-mg, and 50-mg groups. These data must be treated with caution, as by the 44th day, two of the 25-mg group and three of the control group had died, whereas all of the treated fish still sur vived, and did so to the end of the ex periment. On the upper portion of the figure, 2 groups in which the survival rate was similar are compared. With similar mortalities the growth rate of the treated 40 60 80 100 120 140 160 fish exceeds that of the control group by AGE IN DAYS 6 5 % . Fig. 2 Decreasing effectiveness of ferrous sul Efforts were made in subsequent ex fate treatm ent after 100 days from hatching. The 50- and 100-mg groups were split at 100 days, and periments to balance any changes which treatm ent was discontinued in half of each group. might have resulted from crowding ef- AGE IN DAYS Fig. 3 Effect of daily addition of ferrous sulfate on the growth of the platyfish, Xipho- phorus maculatus. After the 44th day only 3 control fish were left, and only four in the 25-mg treated group, in the lower portion of the figure. IRON AND FISH GROWTH 8 9 fects by removing fish from the various groups in which little or no mortality oc curred, so that the size of the control and experimental groups remained similar throughout the experiment. Difficulties of this sort led us to examine our data in terms of increased survival due to sup plemental iron treatments. These data are presented in table 1, in which all ex periments using both platyfish and sword tails are combined. Utilizing a contingency chi-square test the differences in survival rates of the groups treated with 50 mg and 100 mg are significant, P being less than 0.01, whereas the 25-mg groups gave a P value between 0.1 and 0.2. Accordingly the use of the 25-mg supplement was dis continued as being, at best, submaximal. The addition of five brine shrimp (Ar- temia) is often used to supplement fish feeding regimens, with excellent results reported in terms of fish growth ( 8 ). Ac 4 0 6 0 8 0 IOO 120 140 cordingly a brood of fry was separated into 2 equal groups and one, used as a control, A G E IN D A Y S was fed maximally a diet supplemented Fig. 4 Effect of the addition of ferrous sulfate to swordtails fed live brine shrimp daily. daily with brine shrimp; the other was fed in the same way but daily additions of group, was highly variable, for reasons 100 mg of ferrous sulfate were also made. not clear. These data are presented in Figure 4 shows that until 66 days after table 2. In no case did the hematocrit hatching a stimulation due to added iron value of a control fish exceed the value for was still present.4 Both groups grew faster any in the treated group matched with on this regimen than with a diet with only that control, although on occasion one occasional brine shrimp supplements, but control group might have a higher value it appears that brine shrimp do not supply than that observed in a different experi enough iron, at least in the early growth mental group. Despite the overlap in stages. standard deviations, the differences, when Together with increased growth of the subjected to an analysis of variance, are treated fish we observed an increase in significant at the 5% level of confidence. hematocrit levels, when compared with Similar experiments were made using control groups. In analyzing these data ferric nitrate as the iron source, and add- it became necessary to pool the results of many experiments, as the value of the 4 The ratio of the slope of the treated to the un treated group is: days 40 to 50, 1.7/1.1; days 50 to 66, hematocrit, from control group to control 2 .1 5 /1 .5 6 . TABLE 1 Effect of treatment with ferrous sulfate on survival of platyfish and swordtails from hatching to 120 days T re a tm e n t Initial no. F in a l no. M o rtality % None (control) 59 2 4 58 2 5 m g F e S 0 4 added daily 16 10 38 5 0 m g F e S O ,4 added daily 15 14 8 100 mg FeS0 4 added daily 59 4 5 2 4 Combined treated group 9 0 6 9 2 3 9 0 MARTIN ROEDER AND RACHEL H. ROEDER TA BLE 2 Hematocrit levels of fishes with various treatments 1 Daily treatment Type of fish 100 m g F eS04 50 m g F eS 04 C o n tro l Xiphophorus helleri (green swordtail) 3 8 .0 ± 6.6 2( 1 4 ) 3 40.0 ±4.0(22) 37.2±4.9 (34) Xiphophorus maculatus (platyfish) 3 8 .7 ± 6.8 ( 1 5 ) 32.1 ± 4.3 (16) H y b r id ( helleri X maculatus) 40.22± 1.4(8) 31.7± 0.04(5) 1 We are indebted to Miss Patricia Hopper for the data on the hybrid fishes, and for much of the data on swordtails. 2 M e a n packed cell volume/100 ml ± sd. 3 Numbers in parentheses indicate number of fish. ing the salt at the level of 100 m g/day to ( 9 ) . The rise in hematocrit levels indi tanks containing new-hatched swordtail cates that the iron is both absorbed and fry. At the end of an 84-day period the utilized. Further experiments with che average weight and the rate of growth of lated iron and with possible synergistic the control group and the treated fish effects of other minerals are in progress. were essentially the same, 2 groups of treated fish had average weights of 145 LITERATURE CITED mg, and 2 control groups 130 mg, (144 1. Lovelace, F. E., and H. A. Podoliak 1952 treated vs. 144 control; 146 treated vs. Absorption of radioactive calcium by brook 117 control). W e conclude that this treat trout. Progr. Fish Cult., 14: 154. ment does not appear effective in stimulat 2. Phillips, A. M. Jr., F. E. Lovelace, D. R. Brockway, G. C. Balzer et al. 1954 The ing growth; hematocrit values in both nutrition of trout. Bur. Fisheries. N.Y. groups were not statistically different (35.5 Fisheries Res. Bull. 17, Cortland Hatchery treated vs. 34.9 control). Rep. 22. N.Y. State Dept, of Conservation, Cortland, New York. DISCUSSION 3. Cheprakova, U. I. 1960 Some facts about the influence of iron salts concentration on The observed increases in growth rate the development and survival of fish eggs. and in hematocrit levels indicate that, as Vop. Ikhtiologii, 14: 110. with chicks, iron is an essential nutrient 4. Roeder, M., and R. H. Roeder 1964 The respiration of a graded series of two species for these fish during the period immedi of small whole Xiphophorin fishes. J. Cell. ately following hatching. As sexual ma Comp. Physiol., 63; 115. turity is approached the stimulation due 5. Sherman, W. C., C. A. Elvehjem and E. B. to iron addition to the diet disappears, Hart 1934 Further studies on the avail ability of iron in biological m aterials. J. Biol. and it is absent in adult fish. Absorption C h e m ., 107: 3 8 3 . probably takes place across the gill mem 6. Riley, V. 1960 Adaptation of orbital bleed brane, and is evidently accomplished ing technic to rapid serial blood studies. rapidly, as the disappearance of soluble Proc. Soc. Exp. Biol. Med., 104: 7 5 1 . 7. Breder, C. M., Jr. 1935 The aquarium and iron from solution limits the time of ex research. Bull. N.Y. Zool. Soc., 38 (4): 110. posure. Since ferric iron does not appear 8. Dempster, P. P. 1953 The use of larval to be utilized, it is possible that the and adult brine shrimp in Aquarium fish mechanics of transport bear some relation culture. Calif. Fish Game, 39: 355. 9. Ruch, T. C. and F. D. Patton 1965 Physi ship to the absorption of iron through the ology and Biophysics, ed. 19. W. B. Saun intestinal mucosa in mammalian systems ders Company, Philadelphia, p. 1003. Effect of a M ethionine-deficient D iet on A m ino A cid Incorporation in Rat Liver C ell-fractions 1 E . A. K E A N Biochemistry Department, University of the West Indies, Kingston, Jamaica ABSTRACT An investigation was made into the relationship between nutritive value of a protein-rich food and amino acid incorporation into cell-free preparations from rat liver. A low protein efficiency ratio due in part to a methionine deficiency was associated with increased levels of protein labeling. The effect was observed with methionine -35 S but not with leucine- 14C, and was apparently mediated through alterations occurring predominantly in the pH 5-enzyme fraction. The observations indicated that an increase in the activity of amino acid-activating enzymes may be induced by dietary deficiency of the corresponding amino acids. The amounts and activity of liver com tive deficiency of methionine in the ponents which are involved in protein syn protein. Experiments were carried out to de thesis may vary according to the amounts termine which, if any, of the components and relative proportions of dietary amino participating in protein biosynthesis was acids. Thus, there are reports (1 , 2 ) that affected by methionine deficiency. These amino acid-activating enzymes tested in components were isolated from livers of vitro showed higher activity than normal animals used in the feeding trials. By in liver cell-sap from protein-depleted rats; varying the source of these components in this contrasted with reduced activity in protein biosynthetic systems in vitro, it heart and gastrocnemius muscle. These was found that methionine deficiency led investigators pointed out that the differ to increased incorporation of methionine ential response was in keeping with the into protein, and furthermore that a frac changes in the levels of amino acid in tion containing amino acid-activating en corporation which they observed in the zymes was responsible. respective tissues in vivo. The effects of omitting a single amino EXPERIMENTAL acid from the diet have been explored by A depletion-repletion technique (5 , 6 ) measuring amino acid incorporation in was adopted for the determination of pro preparations of liver microsomes and ribo tein efficiency ratios (PER) of 3 protein- somes (3 , 4 ). Fleck et al. ( 3 ) concluded rich supplements. The depletion period that absence of tryptophan from an amino during which rats were fed a protein-free acid mixture administered to rats one hour diet lasted 10 days; thereafter the supple before killing led to a reduction in the ment was fed along with the protein-free capacity of microsomes for amino acid in diet and weight gains were measured after corporation. On the other hand, Sidransky 7 days. et al. (4 ) force-fed diets devoid of threo Animals and diets. Male albino rats 4 nine to rats for 3 or 7 days, and observed to 5 weeks old were used. They had been that this led to increased leucine incor fed a stock diet2 for 1 to 2 weeks after poration. They concluded that the ribo weaning. Each animal was housed in its somes were predominantly responsible for own metabolism cage and fed ad libitum the observed effect. a diet (protein-free diet) of the following The present work was based on feeding composition: (in per cent) sucrose, 8.8;* 12 trials with rats, which showed that the growth-promoting value of a protein-rich Received for publication February 21, 1966. 1 This work was supported by Public Health Service product from peanut was greatly en Research Grant no. AM 07623, from the National Insti hanced by supplementation with methio tute of Arthritis and Metabolic Diseases. 2 Purina Laboratory Chow, Ralston Purina Company, nine; this implied that there was a rela St. L o u is. J. N u t r it io n , 90: ’66 91 9 2 E. A. KEAN cornstarch, 75; butter, 10; salt mixture, such (15,000 X g supernatant). This prep USP XIV, 4; and vitamin fortification mix aration contained 30 to 40 mg protein/ml. ture,3 2.2. The vitamin mixture contained For tests of amino acid incorporation into per g: vitamin A, 9,000 USP units; vita protein of this fraction, 0.15 ml was in min D, 18,000 USP units; (in milligrams) cubated in 1.0 ml medium containing: a-tocopherol, 5; ascorbic acid, 45; inositol, (nmoles) Tris buffer, pH 7.5, 30; KC1, 80; 5; choline chloride, 75; menadione, 2.25; NaCl, 50; MgCL, 5; ATP, 1.0; creatine p-aminobenzoic acid, 5; nicotinic acid, 4.5; phosphate, 20; GTP, 0.60; 30 ng creatine riboflavin, 1; pyridoxine-HCl, 1; thiamine- kinase; and radioactive amino acid, 0.2 HC1, 1; Ca pantothenate, 3; and (in micro nmoles. grams) biotin, 20; folic acid, 90; vitamin For isolation of the cell-sap, pH 5 frac B12, 1.35. tion and washed microsomes, the method The supplements for which determina was essentially that of Stone and Joshi (7). tions of PER were made are as follows, Pooled livers from 2 or more animals in with the amount fed in the daily ration: the same dietary group were used for these 1) peanut protein,4 1.9 g; 2) peanut pro preparations. Cell-sap so prepared con tein, 1.9 g + L-methionine, 0.05 g; 3 ) egg tained about 25 to 30 mg protein/ml. It albumin,5 1.27 g + hydrogenated coconut was used either as such, or as a source of oil,6 0.60 g. These supplements were pre pH 5-enzyme. The latter was prepared by pared in bulk, as liquid suspensions isoelectric precipitation at pH 5.2. After blended into 4% sucrose solution for redissolving in buffered salt medium, 1.0 palatability. They were compounded to ml for each gram of fiver homogenized, supply as nearly as possible an equal the pH 5-enzyme preparations contained amount of nitrogen and of lipid to each about 10 to 15 mg protein/ml. animal in a test, and were fed as a meas Preparations of washed microsomes were ured volume (usually 10 m l/ra t) daily. derived from the pellet precipitated by Each rat soon learned to consume its daily centrifuging the 15,000 X g supernatant ration within 3 hours. The N intake varied at 105,000 X g for one hour. After dis in different tests between 0.16 and 0.18 g / persal in buffered salt medium 0.5 ml./g day/rat. After the protein supplements liver homogenized, these preparations con had been fed for 7 days, gain in body tained about 20 to 25 mg protein/ml. weight was recorded. PER was calculated Ribosomes were isolated by the second as gain in weight (g ) per N eaten (g ). method of Korner ( 8 ). Livers from 6 to The feeding regimen was continued for 8 animals were used for each preparation. a second week, and then the animals were Ribosomal pellets were resuspended for killed for preparation of liver cell-fractions. use in 0.25 ml buffered salt m edium /g of Preparation and incubation of liver-cell fiver homogenized. These preparations fractions. Rats were killed by cervical contained about 10 mg protein/ml. fracture and decapitated. Livers were re Incubation of recombined fractions. moved and chilled on cracked ice. The Microsome preparation, 0.5 ml was incu subsequent procedures were varied to suit bated with 0.5 ml cell-sap or pH 5-enzyme different experiments in which the follow preparation in a total volume of 2.0 ml, ing fractions were tested either alone or in containing: (umoles/ml) Tris buffer, pH combination: 1) a 15,000 X g superna 7.5, 162.5; sucrose, 62.5; KC1, 25; MgCL, tant; 2 ) a 105,000 X g supernatant — re 5; ATP, 4; GTP, 0.5; creatine phosphate, ferred to as cell-sap, and a pH 5-enzyme 20; 25 ug creatine kinase; and 0.4 nmoles preparation derived from it; 3 ) washed radioactive amino acid. microsomes; and 4) ribosomes. All iso Ribosome preparations, 0.4 ml, were lation procedures were performed at zero incubated either with cell-sap or pH 5-en- to 2 °. For tests on fraction 1, liver was 3 Obtained from Nutritional Biochemicals Corpora minced with scissors and homogenized in tion, Cleveland. 4 Lypro, a spray-dried powder containing about 65% m 2 volumes of 0.25 sucrose. The homog protein and 32% fat. Obtained through the courtesy enate was centrifuged at 15,000 X g for of International Protein Products Ltd., London, Eng la n d . ten minutes and the supernatant contain 5 See footnote 3. 6 Puritan cooking oil, Seprod Ltd., Kingston, ing microsomes and cell-sap was used as J a m a ica . LIVER PROTEIN LABELING IN METHIONINE DEFICIENCY 9 3 zyme preparations, 0.2 ml, in a total Ribonucleic acid was determined by the volume of 1 ml medium of composition method of Fleck and Munro (1 0 ). similar to that used for the 15,000 X g supernatant, with the addition of gluta RESULTS thione, 2.5 nmoles. Values are given as means for appropri All samples were incubated in duplicate, ate groups ± standard error. Significance for 30 minutes with shaking at 38° in air. of difference between groups was deter Each set included a blank which was kept mined by a standard t test and was ac at 0° during addition of fractions and cepted at a level of P = 0.05 or less. deproteinized at zero time. Incubations Nutritive value of protein supplements. were terminated by adding an equal vol Representative values for PER are shown ume of 10% trichloroacetic acid contain in table 1. Each of the 3 values is signifi ing 100 mg of the appropriate nonradio- cantly different from the others. Addition active amino acid. of methionine improved the growth-pro Isolation of protein and measurement of moting value of peanut protein; this im radioactivity. Samples were washed twice plies that the protein was deficient in with 5% trichloroacetic acid, heated at methionine.9 * However, the supplementa 90° for 10 minutes with the same reagent, tion with methionine did not improve pea washed once again with cold 5% trichloro nut protein to the level of egg albumin. acetic acid and once each with ethanol- Experiments with 15,000 X g super- ether ( 3 :1 ) mixture, and with ether. The tant. Table 2 shows results of tests for residue was dissolved in the minimum uptake of leucine-14C with this fraction. volume of 1.0 n NaOH (about 0.1 ml/mg There was no difference between the protein), and reprecipitated with ethanol groups on the basis of these results. It (final concentration > 99% ). The precip was thought that a relative deficiency of itate was washed once each with ethanol, ethanol-ether ( 3 :1 ) mixture and finally TABLE 1 with ether. For determination of radioac PER 1 of protein-rich supplements fed to rats tivity, a weighed amount ( 1 -3 m g) of the Protein supplement fed PER air-dried residue was transferred to glass Peanut protein 10.1 ± 0.31 2( 16) 3 counting vials, 1 . 0 ml of 1 m p-(diisobutyl- Peanut protein -f L-methionine 16.1 ±0.43 (12) cresoxy-ethoxy ethyl) dimethyl benzyl Egg albumin 21.2±0.77 (14) ammonium hydroxide 7 * in methanol was added and the protein dissolved by warm 1 pFT? _ gain in weight ( g) N intake (g) ing at 60° for 3 hours. Scintillator solu 2 Mean + s e . Each mean is significantly different tion was then added and counts deter (P < 0.05) from the other two. 3 Numbers in parentheses represent number of ani mined with a Packard Tri-Carb liquid mals contributing to each value. scintillation spectrometer to a probable er ror of < 2% . Appropriate standards pre TABLE 2 pared from the stock solutions of radio Effect of dietary protein on incorporation of leucine into protein of a 1 5 ,0 0 0 X g active amino acids used (L-leucine-14C,s supernatant of liver L-lysine-14C, and L-methionine-35S) were al ways counted along with experimental U p ta k e o f Protein supplement fed leucine-14C samples; levels of incorporation could thus be calculated as micromoles of amino acid mfimoles/mg protein per milligram of protein counted. Since Peanut protein 0.141 ±0.014 ’ ( 5 ) 2 Egg albumin 0.149 ± 0.011 ( 5 ) analyses of protein were made on all cell fractions used for incubations, calculations 1 M e a n ± s e . The 2 means are not significantly different (P > 0.05). could be made, when required, of total 2 Numbers in parentheses represent number of ani mals contributing to each value. uptake into protein of incubation mixtures and then expressed as millimicromoles per 7 Hydroxide of Hyamine 10-X, Packard Instrument milligram of microsomal or ribosomal pro Company, La Grange, Illinois. 3 Labeled amino acids were purchased from Schwartz tein. Bio-Research Inc., Orangeburg, New York. Analyses. Protein was determined by 9 Analytical data suggested a possible deficiency of lysine, but addition of lysine did not alter the PER the biuret method of Gomall et al. (9 ) . (unpublished observations). 94 E. A. KEAN methionine in the diet might specifically the group fed peanut protein but this affect levels of incorporation of this amino proved to be not significant on the basis acid; hence in another series of experi of either microsomal protein or RNA. On ments, tests were repeated with this frac this basis, it seemed likely that changes in tion using methionine-35S instead of leu- levels of incorporation reflected changes cine-14C. In this case, a group fed peanut of activity in cell-sap. To test this, a com protein showed significantly higher levels mon ribosome preparation obtained from of incorporation than a group fed egg al rats fed the stock diet was incubated with bumin (table 3). cell-sap prepared from rats fed either pea To determine whether microsomes con nut protein or peanut protein + methio tributed to the observed differences in nine. Table 5 shows that the cell-sap from levels of incorporation, preparations of the former group produced higher levels of washed microsomes were made from die incorporation. The difference persisted, tary groups fed either peanut protein or although to a less marked degree, when egg albumin. These were incubated with pH 5-enzyme instead of cell-sap from these a common pH 5-enzyme preparation de 2 dietary groups was tested with a com rived from animals fed the stock diet. Re mon ribosomal preparation (table 5 ). It is sults are shown in table 4. These data clear that at least a part of the dietary in show that there is a slight advantage to fluence on incorporating activity takes ef fect on the pH 5-enzyme fraction. TABLE 3 Amino acid-activating enzymes occur in Effect of dietary protein on incorporation of methionine into protein of a 15,000 X g the fraction designated pH 5-enzyme (11, supernatant fraction of liver 12). Enhanced activity of these enzymes, such as has been reported for liver cell-sap U p tak e of Protein supplement fed methionine-35S from protein-depleted animals (1 ,2 ) might result in increased incorporation. Fur vifimoles/mg protein thermore, it might be expected that the Peanut protein 0 .1 3 4 ± 0 .0 0 9 » (IO ) 2 Egg albumin 0.096± 0.007 (10) effect would be non-specific, applying to other amino acids besides methionine, if 1 Mean + se. The 2 means are significantly differ ent (P < 0.05). the pH 5-enzyme from protein-depleted 2 Numbers in parentheses represent number of ani mals contributing to each value. animals were tested by the criteria used TABLE 4 Effect of dietary protein on incorporation of methionine into protein, using microsomes from 2 dietary groups with a common pH 5-enzyme preparation 1 Protein supplement fed Uptake of methionine-35S mfivioles/mg m/imoles/mg microsomal protein microsomal RNA Peanut protein 0.167± 0.014 2( 1 0 ) 3 1.15 ± 0.11(10) Egg albumin 0.142±0.026 (10) 1.01 ± 0 .0 8 ( 10) 1 A preparation derived from animals fed the stock diet. 2 Mean + se. The difference between the 2 means in each column is not significant (P > 0.05). 3 Numbers in parentheses represent numbers of animals contributing to each value. TABLE 5 Effect of dietary protein on incorporation of methionine into protein, using cell-sap or pH 5-enzyme from 2 dietary groups with a common ribosome preparation 1 Uptake of methionine-35S Protein supplement fed With cell-sap With pH 5-enzyme mfimoles/mg ribosome protein Peanut protein 0.216,2 0.239 0.173,0.172 Peanut protein + m ethionine 0.162, 0.170 0.151, 0.137 1 A preparation derived from animals fed the stock diet. 2 Each entry is a result for cell-sap or pH 5-enzyme derived from pooled livers of 2 a n im a ls. LIVER PROTEIN LABELING IN METHIONINE DEFICIENCY 9 5 TABLE 6 factors occur in cell-sap and are part of Effect of diet on incorporation of lysine into the pH 5-enzyme complex (1 3 , 14). protein using a common ribosome preparation 1 with pH 5-enzyme Dietary influence on incorporation was from 2 dietary groups demonstrated when the 15,000 X g super- nant was used with labeled methionine, D iet U p tak e of ly sin e-14C but not wtih labeled leucine. A possible interpretation is that there was an in m/xmoles/rng ribosome protein crease in the activity of the enzyme meth- Protein-free 0.092,2 0.095 ionyl-sRNA synthetase selectively among the other amino acid-activating enzymes S to ck 0.073, 0.070 in liver cell-sap of animals fed peanut 1 A preparation derived from animals fed the stock diet. protein. 2 Each entry is a result for pH 5-enzyme derived Mentioned earlier was an apparent con from pooled livers of 2 animals. flict between previous observations (3 , 4) in the present work. This proved to be the concerning the effects of diets lacking a case when the pH 5-enzyme was prepared single amino acid on incorporation levels. from groups of rats fed either the protein- The present study did not resolve these free diet or the stock diet for 2 weeks differences, since no significant change in after weaning and was incubated with a the incorporating capacity of microsomes common ribosome preparation from rats was noted. The duration of the dietary fed the stock diet. Table 6 shows that the treatment, as well as the labeled amino pH 5-enzyme from protein depleted rats acid used, may be important in this con was more active in promoting uptake of nection. Sidransky et al. (4 ) did find that lysine. cell-sap from threonine-deprived rats was about 21% more active than that from DISCUSSION controls in stimulating uptake of leucine The protein-rich peanut product gave a by ribosomes; in view of the present ob much lower PER than the reference pro servations, it is interesting to speculate tein egg albumin. To explain this, the what result would have emerged if uptake amino acid balance was considered to be of threonine had been measured in their of primary importance; digestibility stud experiments. ies were not attempted. A dramatic im provement in PER was achieved by adding LITERATURE CITED to the peanut protein enough methionine 1. Mariani, A., M. A. Spadoni and G. Tomassi to equalize the sulphur amino acid con 1963 Effect of protein depletion on amino tent, per 100 g N , of the test and refer acid activating enzymes of rat liver. Nature, 199: 3 7 8 . ence proteins. Hence it appeared very 2. Gaetani, S., A. M. Paolucci, M. A. Spadoni likely that a relative deficiency of methio and G. Tomassi 1964 Activity of amino nine existed in the peanut protein, al acid-activating enzymes in tissues from pro though the possibility has to be borne in tein-depleted rats. J. Nutr., 84: 173. 3 Fleck, A., J. Sheperd and H. N. Munro mind that the content of other amino acids 1965 Protein synthesis in rat liver: influ also may have been suboptimal. ence of amino acids in diet on microsomes The results of this investigation indi and polysomes. Science, 150: 6 2 8 . cate that components of cell-sap were re 4. Sidransky, H., T. Staehelin and E. Verney 1964 Protein synthesis enhanced in the sponsible for differences in levels of amino livers of rats force-fed a threonine-devoid acid uptake between liver cell prepara diet. Science, 146: 7 6 6 . tions from different dietary groups. It ap 5. Frost, D. V., and H. R. Sandy 1949 Assay pears that an initial step of protein bio of dry proteins by rat repletion method. J. synthesis was more active when there was Nutr., 39: 427. 6. Rippon, W. P. 1959 A comparison of sev a relative methionine deficiency in the eral methods for estimating the nutritive diet. This step requires the activation of value of proteins. Brit. J. Nutr., 13: 2 4 3 . amino acids by enzymes of narrow speci 7. Stone, D., and S. Joshi 1962 Some evi ficity; amino acyl adenylates are formed dence for a pathway of amino acid incor poration, in rat-liver microsomes, which does and linked to appropriate acceptor chains not require transfer ribonucleic acid. Bio- of soluble RNA (sR N A ). The necessary chim. Biophys. Acta, 55: 335. 9 6 E. A. KEAN 8. Korner, A. 1961 Amino acid incorporation 12. Hoagland, M. B., E. B. Keller and P. C. into ribosomes. Biochem. J., 81: 1 6 8 . Zamecnik 1956 Enzymatic carboxyl ac 9. Gomall, A. G., C. J. Bardawill and M. M. tivation of amino acids. J. Biol. Chem., 2 18: David 1949 Determination of serum pro 3 4 5 . tein by means of the biuret reaction. J. Biol. Chem., 177: 7 5 1 . 13. Allen, E. H., E. Glassman and R. S. Schweet 10. Fleck, A., and H. N. Munro 1962 The 1960 Incorporation of amino acids into ribo precision of ultraviolet absorption measure nucleic acid. 1. The role of activating en m ents in the Schmidt-Thannhauser procedure zymes. J. Biol. Chem., 235: 1061. for nucleic acid determination. Biochim. Biophys. Acta, 55: 571. 14. Allen, E. H., E. Glassman and R. S. Schweet 11. Hoagland, M. B. 1955 An enzymatic mech 1960 Incorporation of amino acids into anism for amino acid activation in animal ribonucleic acid. II. Amino acid transfer tissues. Biochim. Biophys. Acta, 16: 2 8 8 . ribonucleic acid. J. Biol. Chem., 235; 1068. Bioassay of Vitamin K in Chicks * 1 JOHN T. MATSCH1NER and E. A. DOISY, JR. Edward A. Doisy Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri ABSTRACT A convenient, precise bioassay for vitam in K is reported based on the analysis of chick plasma with Russell’s viper venom. Data obtained by this proce dure were normally distributed for statistical evaluation. The relative molar activities of several forms of vitamin K and the concentration of vitamin K in a number of animal tissues are given. Bioassay remains an indispensible tech venom according to Hjort et al. ( 2 ) . In nique in studies on the occurrence and mammalian plasma, this procedure de metabolism of vitamin K. Despite a num tects changes in prothrombin and Factor ber of available procedures, assay in chicks X ( 3 ) ; however in chicks the assay may has been used most often because these be specific for prothrombin since the ac animals are sensitive to deprivation of the tivity of Factor X is not present. The vitamin and respond in a reliable manner reference standard for this assay was a to prophylactic or curvative treatment. pool of plasma obtained from chicks fed During recent years we have adopted a for 2 weeks a commercial ration 3 contain modified chick assay which appears to have ing an added 0.5 ag of phylloquinone/g.4 considerable advantage over published The addition of vitamin K was necessary methods. to ensure uniform maximum prothrombin activity. Aliquots of the reference pool MATERIALS AND METHODS diluted to various concentrations were Chicks. Day-old White Rock chicks of analyzed with each group of experimental mixed sex were otbained from a local samples. Finally, clotting times were used hatchery. Preliminary studies with sexed to calculate the activity of the experi chicks indicated no difference between mental samples as a percentage of the male and female in response to deficient activity of the reference standard. To diets or to repletion with the vitamin. The evaluate the statistical aspects of this pro chicks were housed in wire-floor brooders cedure, individual samples of reference with contact heat.2 Room temperature was plasma were compared with the reference maintained at 24° and a roosting period pool. For 78 chicks the average pro was provided between midnight and 6 a m thrombin was 102 ± 1% 5 and individual by timed overhead lights. Fresh food and values closely approximated a normal dis water were provided daily. tribution. Diet. Chicks were grown for 10 days Received for publication April 21, 1966. with the vitamin K-deficient diet shown in 1 These studies were supported in part under grant no. DA-MO-49-193-62-G41 from the Office of the Sur table 1. After 10 days they were separated geon General, Department of the Army and Public into groups of ten and fed the same diet Health Service Research grant no. AM09909-01 from the National Institute of Arthritis and Metabolic Dis containing controlled amounts of vitamin eases. The opinions expressed are those of the authors K for 4 days. Lipids were conveniently as and not necessarily those of the Department of the Army. _ . . sayed by mixing them in place of an equal 2 James Manufacturing Company, Fort Atkinson, Wisconsin. amount of corn oil in the deficient diet. 3 Purina Startena, Ralston Purina Company, St. Prothrombin After the chicks had Louis. . . , . 4 Reference to specific forms of vitamin K is made m been fed the experimental diet for 4 days, accordance with the recommendation of the Nomen clature Commission of the IUPAC (4). The K 2 vita they were anesthetized lightly with ether mins are menaquinones (abbreviated MK-n) and vitamin Ki becomes phylloquinone (abbreviated K). and samples of blood were taken by car The length of the side chain is designated on the basis diac puncture. Plasma was analyzed for of the number of isoprene units (n). s Throughout this report means are accompanied coagulation activity using Russell’s viper by their standard error. J. N u t r it io n , 90: ’66 97 98 JOHN T. MATSCHINER AND E. A. DOISY, JR. TABLE 1 Vitamin K-deficient diet for chicks Soy protein 1 3 5 .0 0 DL-Methionine 0 .7 5 G ly c in e 0 .3 0 Glucose monohydrate 2 4 8 .9 5 Vitaminized glucose monohydrate 3 5 .0 0 S a lts 4 4 6 4 5 .0 0 C e llu lo s e 5 3 .0 0 Fat-soluble vitam in mix 6 0.10 C o rn o i l 7 1.90 1 ADM C-l Assay Protein, Archer-Daniels-Midland, Minneapolis. 2 Cerelose, Corn Products Company, N e w York. 3 Vitaminized glucose monohydrate supplied the fol lowing vitamins in m g / 1 0 0 g of diet: thiamine, 1 ; riboflavin, 1; Ca pantothenate, 5; pyridoxine, 0.5; niacin, 2; folic acid, 0.1; vitamin B 12, 0.005; biotin, 0.01; p-aminobenzoic acid, 1; inositol, 25; and choline chloride, 250. 4 Mameesh and Johnson (1). 5 Alphacel, Nutritional Biochemicals Corporation, Cleveland. 6 The fat-soluble vitamin mixture supplied the fol lowing vitamins in f i g /g of diet: vitamin A acetate, 5; vitamin D 3, 0.035; and vitamin E acetate, 60. 7 Mazola, Corn Products Company, N ew York. RESULTS AND DISCUSSIONS The response of chicks to vitamin K is shown in figure 1. Each point is the mean prothrombin activity of at least 20 chicks. PHYLLOQUINONE (pg/g diet) In the upper region of the curve, average Fig. 1 Response of vitamin K-deficient chicks normal prothrombin activity was observed to diets containing controlled amounts of phyl- in chicks that received the diet containing loquinone. Each point is accompanied by the approximately 0.4 pg of phylloquinone/g. standard error of the mean. The lowest detectable concentration of vitamin K was less than 0.05 pg/g. phylloquinone/g. Body weights as low as Chicks used in these studies generally 80 g at 10 days and a weight gain as low weighed about 130 g at 10 days and gained as 30 g during the assay did not alter pre approximately 50 g during the 4 days of dictable prothrombin activities. the assay; however, during the winter As shown in table 1, the deficient diet chicks were often stunted by exposure to contains corn oil; however, several other inclement temperatures during shipment. oils may also be fed without providing sig This prompted an examination of the ef nificant amounts of vitamin K. The bioas fect of weight gain on the assay. In table say of several oils at 2 % of the diet is 2 prothrombin levels are shown for nor shown in table 3. Only peanut oil and one mal and underweight chicks fed the defi brand of soybean oil contained detectable cient diet or a diet containing 0.2 pg of amounts of vitamin K. TABLE 2 Comparison of prothrombin in normal and underweight chicks Body weight D iet Prothrombin 10 d ays 14 days 9 9 % D e fic ie n t 1 0 6 ± 3 ( 1 9 ) 1 14 9 ± 4 6 ± 0 . 3 1 3 2 ± 3 ( 3 1 ) 1 8 2 ± 5 5 ± 0 . 4 Supplemented 2 8 0 ± 1( 10) 1 1 4 ± 2 4 6 ± 7 12 9 ± 2 ( 2 0 ) 18 4 ± 5 4 5 ± 3 1 Mean + s e . Number of observations in parentheses. 2 0 . 2 f i g of phylloquinone/g of diet after 1 0 days. BIOASSAY OF VITAMIN K 99 TABLE 3 dione was less active than expected. MK-9 Bioassay of several dietary oils (H ) which differs from MK-9 by the pres Dietary oil Prothrombin ence of a single saturated isoprene unit in the side chain (7 ) was approximately as % active as MK-9. The remarkable inactivity C o rn 1 5 ± 0 .4 2 S o y 3 5 ± 1 of dietary 2-methyl-3-dimethylallyl-1,4- S o y 4 19 ± 1 naphthoquinone (M K -1) first observed by Cottonseed 5 5 ± 0 . 2 Wiss et al. (5 ) was also obvious in the S a fflo w e r 4 ± 1 present assay. P e a n u t 9 ± 1 O liv e 5 ± 0 . 4 In addition to studies with pure samples of vitamin K we have also analyzed the 1 Mazola, Corn Products Company, N e w York. 2 Mean ± se. vitamin K content of several animal tis 3 Crisco Oil, Procter and Gamble, Cincinnati. sues. After some preliminary studies, it 4 Durkee Famous Foods, Cleveland. 5 Wesson Oil Sales Company, Fullerton, California. was possible to obtain direct assays by mix ing 30% of homogenized raw tissue with the standard vitamin K-deficient diet. Tis TABLE 4 sues obtained from the packing house were Relative molar activities of several forms of vitamin K of the best grade available; tissues from laboratory animals came from healthy V ita m in 1 R e la tiv e specimens fed commercial chows. The data molar activity 2 recorded in table 5 were obtained by the as M K -l i say of tissue from at least 10 animals. There K-2 3 0 was a low level of vitamin K in bird liver M K -2 35 K -4 100 compared with mammalian species, but M K -4 15 6 among mammals there was no apparent M K -5 1 1 6 basis or predictability for the concentra M K -7 122 tion of hepatic vitamin K. The richest M K -9 78 M K -9 ( H ) 67 source we have observed is beef liver which M K -10 4 9 may contain as much as 1.2 ug/g of tissue. M enadiol diphosphate 3 164 Those tissues in which vitamin K was not M e n a d io n e 4 9 detected contained less than approximately 1 Most of the vitamins were donated by Hoffmann- 0.07 ug/g; i.e., vitamin K was barely de LaRoche, Inc., Nutley, Ne w Jersey, through the gen erosity of Dr. O. Isler. Menaquinone-7 (MK-7) was tectable in chick and rat liver. In confir donated by Dr. E. A. Doisy. Samples of vitamin K9(H) mation of the results of Dam ( 8 ) , Bou- (MK-9(H)) were donated by Dr. M. Weber and Dr. C. Coscia. 2 Phylloquinone (K-4) was arbitrarily assigned an activity of 100. TABLE 5 3 Synkavite, Hoffmann-LaRoche, Inc. Chick bioassay of animal tissue T issu e V ita m in K 1 In an earlier extensive study, Wiss et al. (5 ) determined the biological activity of M/9 Liver, beef 0 .9 2 several forms of vitamin K using the assay Liver, veal 0 .2 7 of Dam et al. ( 6 ). In the present study we Liver, lam b n d 2 examined the activity of some of these L iv e r, d o g 0 .5 3 vitamins and extended the assays to more L iv e r, p ig 0 .2 5 Liver, rabbit 0 .3 5 lipophilic forms of vitamin K which have Liver, monkey 0 .3 3 recently assumed biological interest. Each Liver, rat (M ) 0 .0 7 vitamin was compared with phylloquinone Liver, rat (F ) 0.10 which was arbitrarily assigned an activity Liver, guinea pig 0 .1 5 Liver, turkey n d of 100. All vitamins were assayed at 2 Liver, pigeon n d concentrations within the useful region of Liver, chicken 0 .0 8 the curve shown in figure 1. The tabulated Liver, duck n d values shown in table 4 are the average of Heart, beef n d Kidney, beef n d these assays. By comparison with earlier M uscle, beef n d studies, the menaquinones appear to have 1 Activity expressed as phylloquinone. exhibited greater activity, whereas mena 2 None detected. 10 0 JOHN T. MATSCHINER AND E. A. DOISY, JR. man and Slater ( 9 ) and others, extrahe- thesis of thiamine in the rat. J. Nutr., 65; patic tissues contained little if any vitamin 1 6 1 . 2. Hjort, P., S. I. Rapaport and P. A. Owren K. In a separate study with rats, com 1955 A simple specific one-stage prothrom parative assays of fresh and irradiated bin assay using Russell’s viper venom in beef indicated the presence of about 0.04 cephalin suspension. J. Lab. Clin. Med., 46. ug of vitamin K/g of fresh tissue; how 8 9 . 3. Hougie, C. 1956 Effect of Russell’s viper ever, this is below the level of detection venom (Stypven) on Stuart clotting defect. in the present assay. Proc. Soc. Exp. Biol. Med., 93; 570. The data presented here emphasize that 4. IUPAC Biochemical Nomenclature Commis remarkably small amounts of vitamin K sion 1965 Nomenclature of quinones with isoprenoid side-chains. Biochim. Biophys. are required for normal function. The Acta, 107; 5. concentration in liver, from less than 0.1 5. Wiss, O., F. Weber, R. Riiegg and O. Isler to 1 |ig/g, defines a potency and effective 1959 Über die biologische Activität der ness for vitamin K approximating that of Vitamin Ki und K 2 und ihrer Isoprenologen. Z. Physiol. Chem., 314: 2 4 5 . the notably trace vitamins such as biotin 6. Dam, H., I. Kruse and E. Spndergaard 1951 and vitamin Bi2. Determination of vitamin K by the curative technique in chicks. Acta Physiol. Scand., ACKNOWLEDGMENTS 2 2 ; 2 3 8 . 7. Gale, P. H. B. H. Arison, N. R. Trenner, A. C. The authors are indebted to Mrs. Jose Page, Jr., K. Folkers and A. F. Brodie 1963 phine Amelotti and Carol Kuhn for cap Characterization of vitamin K 9( H ) f r o m able technical assistance. Mycobacterium phlei. Biochemistry, 2; 200. 8. Dam, H. 1935 The antihemorrhagic vita min of the chick. Nature, 135; 652. LITERATURE CITED 9. Bouman, J., E. C. Slater 1956 Tocopherol 1. Mameesh, M. S., and B. C. Johnson 1958 content of heart-muscle preparations. Na The effect of penicillin on the intestinal syn t u r e , 17 7 ; 1 1 8 1 . Proceedings of the Thirtieth Annual Meeting of the American Institute of Nutrition SHELBURNE HOTEL, ATLANTIC CITY, NEW JERSEY APRIL 12-16, 1966 COUNCIL MEETINGS II. Elections The Council of the American Institute The 682 ballots were counted by Drs. of Nutrition met Sunday evening, April Dorothy Arata and Duane Benton. The 10, and Monday morning and evening, following were elected: April 11. The actions of the Council were President-elect: presented at the Institute business meet George M. Briggs ings and are included in the report of Councilor: those meetings. Alfred E. Harper Nominating Committee: SCIENTIFIC SESSIONS M. L. Scott, Chairman A total of 348 abstracts of papers was C. O. Chichester accepted by the Institute; 70 of them were D. B. Coursin transferred to other societies; 43 were ac B. C. Johnson cepted from other societies, making a total R. M. Leverton of 321 papers programmed by the Instit- tute. These were arranged into 26 regu III. Membership Status lar AIN and 3 intersociety (atherosclero As of April 1, 1966, there were 1,034 sis) sessions. In addition, two informal members of the Institute: 914 active, 102 conferences were held, Poultry Nutrition retired and 18 honorary members, this be and Ruminant Nutrition, and the following ing a net increase of 35 members since half-day symposia were presented: last year. Fifteen members retired during 1. The Relationship of Nutrition to the year. The Clinical Division reports a Central Nervous System Devel total membership of 171. opment and Function Notice of the deaths of these members 2. Trace Elements in Nutrition was received this year: 3. Nutrition Education Bertha A. Bisbey (Charter M ember), April 3, 1 9 6 5 4. Improving the Nutritional Value C. W. Carrick, February 17, 1966 of Cereal Grains by the Use of Amy L. Daniels (Charter Member), January Gene Mutations and Selective 3 1 , 1 9 6 5 Breeding Richard H. Follis, Jr., December 5, 1965. 5. Interactions of Nutrition and In Allen R. Hennes, November 12, 1965 fection Josiah S. Hughes, August 12, 1965 H. H. Mitchell (Charter M ember), March 28, BUSINESS MEETINGS 1 9 6 6 Business meetings were held on Tues Elsie Z. Moyer, July 1, 1965 day, April 12 and Friday, April 15. Dr. Lydia J. Roberts (Charter Member), May O. L. Kline presided at both meetings. 2 8 , 1 9 6 5 W. D. Salmon (Charter Member), February I. Proceedings of 1965 meeting 5 , 1 9 6 6 Harry H. Sobotka (Charter Member), De The Proceedings as published in the cember 24, 1965 Journal of Nutrition, 8 6 : 439-450, 1965, Hertha H. Taussky, November 7, 1965 were approved. Robert R. W illiams, October 2, 1965 J. N u t r it io n , 90: ’66 101 10 2 AMERICAN INSTITUTE OF NUTRITION The following resolution in honor of Dr. Failing sight forced her retirement in 1941 and precluded further scientific Bisbey was read: study. Dr. Daniels’ wide range of inter RESOLVED: That the American Insti ests and her keen wit made her a de tute of Nutrition assembled at Atlantic lightful companion. Her high standards City, New Jersey, at its annual meeting, and unfailing honesty set an example April 12, 1966, place in its minutes for that will long be remembered by her perm anent record this statement of deep students and co-workers. regret and sorrow at the loss by death of Charter Member, Dr. Bertha A. Bisbey, The following resolution in honor of on April 3, 1965, and further, that spe Dr. Mitchell was read: cial recognition be given to her unique qualities that: enabled her to translate RESOLVED: That the American Insti her knowledge of chemistry and her tute of Nutrition, assembled at Atlantic appreciation of the limiting nutrients of City, New Jersey, at its annular meet of flour into practical recommendations ing, April 12, 1966, wishing to express to obtain the “nutritious loaf,” which its deep regret and sorrow at the passing proved useful for the socially under of one of its most distinguished Charter privileged; perm itted her to organize and Members, Harold Hanson M itchell, place administer a nutritional research and this statement in its minutes for perma training program during the early years nent record. of our Society; afforded her students Educated at the University of Illinois, the hum an understanding and encourage Dr. Mitchell spent his entire scientific ment needed to set and obtain high career at that institution, joining the scholastic standards and sound research staff in 1911 and continuing until his principles; and gave her the courage retirement in 1955. Few investigators and strength to achieve a successful have had an influence on nutritional career and to inspire others to make research and concepts equal to that of nutrition their life’s work. Dr. Mitchell. His research program was characterized by logical planning and The following resolution in honor of m inute attention to experim ental details. He was an early advocate of the statis Dr. Daniels was read: tical treatment of experimental data. RESOLVED: That the American Insti Among his many research contributions tute recognize the loss by death of Dr. the following are outstanding: the de Amy Louise Daniels on January 31, velopment of the Thomas-Mitchell 1965, in her 90th year. Dr. Daniels was method of protein evaluation and dem truly a pioneer among women scientists. onstration of the correlation between pro She was a Charter Member of the Amer tein value and amino acid content; the ican Home Economics Association espousal of controlled feed intake in (1909), was the first woman to receive nutrition studies; the importance of nu a Ph.D. in Physiological Chemistry from trient balance in rations; energy and Yale (1912), and was a Charter Mem mineral metabolism studies of farm ani ber of the AIN. mals and of m an; the factorial approach After teaching in the universities of to determination of nutrient require Missouri and W isconsin and taking grad ments. Together with these research uate study at Harvard Medical School, accomplishments Dr. Mitchell is known Dr. Daniels came to the University of for his meticulous and astute approach Iowa in 1918 as Professor of Nutrition to the scientific literature and for his in the new Child W elfare Station. Un great ability to summarize and to criti til 1925 she was also affiliated with the cally evaluate the issues of his day. He Department of Pediatrics, and managed was firm in his opinions, but fair and a well-baby feeding clinic. Her research always a gentleman in their defense. was directed toward establishing nutri Attendance in his classes was a re tional requirements for infants and chil warding experience. His course outlines dren. She was the first to show that were prepared and presented with great infants given cow’s milk feedings needed clarity and detail, and he encouraged additional vitamins. The effect of vita open discussion and evaluation of the min D on calcium and phosphorus reten data and conclusions. As a culmination tion of infants, and the magnesium re of his long teaching and research ex quirements of young children are among perience he devoted his retirem ent years her many contributions to nutritional to writing a 2-volume treatise on Com needs during growth. parative Nutrition of Man and Domestic In 1930, Dr. Daniels was appointed to Animals which will long serve as an in the first White House Conference on valuable reference. Child Development and in 1937 she Dr. Mitchell served on the editorial received the first Borden Award given by board of the Journal of Nutrition f o r 19 the American Home Economics Associ of the first 25 years of its existence. ation for her work in child nutrition. He received the AIN Borden Award in PROCEEDINGS 1 0 3 1945 and was elected a Fellow in 1958. The following resolution in honor of At the time of his death he was the Dr. Salmon was read: nominee for the 1966 Osborne and Mendel Award which was presented post RESOLVED: That The American Insti humously at the annual banquet. He tute of Nutrition, assembled at Atlantic received the Morrison Award of the City, New Jersey, at its annual meeting, American Society of Animal Production April 12, 1966, place in its minutes for permanent record this statement of in 1950 and was elected a Fellow of sincere regret and sorrow at the loss by that society in 1960. death of one of its distinguished Charter The circle of his scientific admirers Members, W illiam D. Salmon, on Febru was wide and he had a close circle of ary 5, 1966. intim ate friends. His was a full life and Dr. Salmon pioneered in nutritional a rich one. We who are his beneficiaries research in the South. Among his many realize a great loss but recognize how contributions were his work in demon greatly we have been privileged. strating the nature of vitamin B as a complex of vitam ins; his observations on The following resolution in honor of the influence of dietary fat on the body Dr. Roberts was read: fat composition; his discovery that zinc deficiency was the primary cause of RESOLVED: That the American Insti parakeratosis in swine; his showing that tute of Nutrition, assembled at Atlantic dietary deficiency, especially of choline, City, New Jersey, at its annual meeting, can produce cancer in experimental ani April 12, 1966, place in its minutes for mals; and providing the evidence that perm anent record this statem ent of deep influenced legislation in Alabama requir regret at the loss by death of one of its ing the enrichm ent of white flour, white distinguished Charter Members, Lydia corn meal, and grits. Jane Roberts, May 28, 1965 in Puerto He served his institution, Auburn Uni R ic o . versity, with distinction as professor, And further that high tribute be paid department head, and professor emeri to Dr. Roberts for her versatility in ex tus, and he had a marked influence on cellence including research, teaching and the quality of both its teaching and re action programs in nutrition; for her search. His alma mater, the University two distinct careers, one at the Univer of Kentucky, awarded him an honorary Doctor of Science degree in 1958 and sity of Chicago until retirement in re named him a Centennial Distinguished search and teaching, and the other at the Alumnus in 1965. He was one of the University of Puerto Rico in teaching most loyal AIN members, serving for and in community demonstrations of the twelve years on the editorial board of the value of nutrition in improving the living Journal of Nutrition, and as member or standards of low-income fam ilies; for di chairman of numerous committees. He rection of graduate students in research was elected a Fellow of the Institute in on nutrient and food needs, especially of 1962 and was a nominee for president children and the effects of supplement elect at the time of his death. ing diets of children with different food Dr. Salmon had an unfailing sense of stuffs; for her contributions to profes humor and a sharp wit to match his sional and nutrition policy-making keen intellect. His death is a great loss groups such as the Food and Nutrition to our Society and to his countless Board of the National Research Council, f r ie n d s . the Council on Foods and Nutrition of the American Medical Association, the The following resolution in honor of Dr. White House Conferences on Children Sobotka was read: and Youth and the American Dietetic RESOLVED: That the American Insti Association; for her contribution and tute of Nutrition, assembled at Atlantic aid to nutrition programs in develop City, New Jersey, April 12, 1966, at its ing countries and training for the Peace annual meeting wishing to express re Corps; for her writings, not only re gret and sorrow at the loss by death of search papers but interpretive books, one of its distinguished Charter Mem such as Nutrition Work with Children, bers, Harry H. Sobotka, place this state Patterns of Living for Puerto Rican Fam ment in its minutes for permanent rec ilies, the Dona Elena Project, and the o rd . text book on nutrition planned primarily Dr. Harry Sobotka was awarded a for use in the Caribbean area; for her Ph.D. in chemistry under Professor dynamic and friendly leadership, her in Richard W illstátter, a Nobel laureate in spiration to students and her ability to enzyme chemistry. His early appoint win support for nutrition programs not ments were those of Research Assistant only from other professionals but from at the Rockefeller Institute, and Re legislators and governors. search Associate at the New York Uni- 1 0 4 AMERICAN INSTITUTE OF NUTRITION versity-Bellevue M edical Center. In 1928 terprise could be applied to the better he was appointed Director of the De ment of humanity. partment of Chemistry of the Mount The bestowal of honors was accom Sinai Hospital in New York City, a post panied by calls to service from many he held until 1965, when he retired. quarters, and Dr. Williams gave of Dr. Sobotka was well known and himself unstintingly in positions which honored throughout the scientific world. demanded judgment, responsibility, More than 300 scientific communica broad knowledge and a high order of tions and a number of books and mono competence in furthering the cause to graphs attest to the prolific output of which he was devoted. Of particular the group which he headed; his mono note were his valuable contributions graph on the Physiology of the Bile still as a member of the Food and Nutrition remains a classic. At the time of his Board of the National Research Council death he was editing a work on alka and as Chairman of its Committee on loids and awaiting the eighth volume of Cereals from its founding in 1940 until Advances in Clinical Chemistry which his retirement in 1959. he edited in collaboration with C. P. In 1957 his colleagues in the American Stewart of the university of Edinburgh. Institute of Nutrition elected him as By his interest in clinical chemistry, their President. In this capacity, as in Harry Sobotka was instrumental in the all others he filled, he served with the founding of the American Association greatest distinction, regarding it, char of Clinical Chemists and served as its acteristically, not as a well-deserved first president. He received the first Van honor but as an opportunity for addi Slyke Award in 1958 and the Ames tional service. Award from the Association in 1962. Robert R. Williams, one of the fore In 1964 he was awarded an honorary most pioneers of nutrition, architect of doctorate by the University of Perugia, cereal enrichment programs throughout Italy, and also received the medal of the the world, outstanding scientist, warm Société de Chemie Biologique. humanitarian, has an abiding place in Harry Sobotka was a busy man, ever history and in the hearts of those who in search of knowledge. His high stand were so fortunate as to know him. Be ards led him through a long and sucess- cause of what he was, the purpose is ful career as a scientist. His death clearer, the road straighter, the goal leaves a void in our scientific commun nearer, and those who follow will ever ity which will be difficult to fill. receive inspiration from his life’s work. The following resolution in honor of IV. New Members Dr. Williams was read: The membership committee considered RESOLVED: That the American Insti the qualifications of 92 nominees. The tute of Nutrition, assembled at its an nual meeting in Atlantic City, New following 77 nominees, recommended by Jersey, on April 12, 1966, wishes to the Council, were elected to membership express its deep sorrow and profound at the buisiness meeting: sense of loss at the passing of one of its most distinguished members and be N E W M E M BERS — 1966* loved colleagues, Robert R. Williams, Abelson, Denis (C) Herman, Robert H. (C) on October 2, 1965. Abernathy, Richard Paul Hopkins, Leon L., Jr. As a scientist, philanthropist, hum an Allred, John B. Jacobs, Francis A. itarian and man, Dr. Williams won re Baldwin, R. L. Jansen, G. Richard Brown, Elmer B. (C) Jones, Don Paul (C) spect, admiration and affection. His Brown, William Duane Kalbfleisch, J. McDowell life was, and will continue to be, an Campagnoli, Mario (C) Kaufman, Nathan (C) inspirational example — a life marked Carpenter, Lawrence E. Kies, Constance V. by intense dedication to the high pur Carpenter, Mary P. Klain, George J. Carroll, Catherine Klavins, Janis V. (C) pose he set for himself, the eradica Chalupa, William Knittle, Jerome L. (C) tion of dietary diseases which have so Christakis, George J. (C) Kornegay, Ervin T. long plagued mankind. Christensen, Halvor Niels Kronfeld, David S. Cornwell, David G. MacDonald, Ian (C) His monumental scientific achieve Coulson, Walter F. Majaj, Amin S. m ent was the identification and synthe Dempsey, Hugh (C) Marion, James E. sis of thiamine, the culmination of an Donald, Elizabeth A. McGandy, Robert B. (C) arduous quest spanning a quarter cen Doyle, Margaret D. McKigney, John I. Ellenbogen, Leon McRoberts, Milton R. tury. While recognition came in the Evans, Joseph L. Meade, Robert J. form of many honors from a host of Fougere, William Mistry, Sorab Pirozshah nations, he spurned personal financial Gevirtz, Norman (C) Mookerjea, Sailendu Sekhar Gortner, Willis A. Motzok, I. reward in order to create the Williams- Hackler, L. Ross Munro, Hamish N. (C) Waterman Fund for the Combat of Hall, Charles A. (C) Navia, Juan M. Dietary Diseases as a mechanism Hanna, Fikri M. (C) Peifer, James J. Hanson, Lester R. Raun, Ned Smith through which the fruits of his labors Harrill, Inez Sandstead, Harold H. (C) and those of his co-workers in this en- Hawkins, George E. Savage, J. E. PROCEEDINGS 1 0 5 Schroeder, Henry Alfred Vaughan, David A. Schulert, Arthur R. Wang, Hwa. Lih their efforts in making the newsletter such Seward, Coleman R. Ward, Gerald M. a success. Sipple, Horace L. Welch, James Graham Smith,, John T. Wheby Munsey Stephen (C) AIN and the Nutrition Society of Canada Snook, Jean Twombly Yang, Modesto G. will plan for a joint meeting in 1970, Srebnik, Herbert H. Yeh, Samuel D. J. Tinoco, Joan W. H. Zalusky, Ralph (C) hopefully to be expanded to include the Turk, Donald E. Ziffer, Herman (C) Vanderveen, John E. recently established Latin American Nu trition Society. * For institutional affiliations and addresses of new members, see the Federation Directory of Members to The Council has initiated the procedure be published in the fall of 1966. (C) Also elected to membership in the Clinical that the President will poll the members Division on April 30, 1966. of the newly elected Nominating Com mittee as a means for their selecting their Drs. Anne M. Briscoe and Fred Kern, own chairman. Jr., already members of the American In It has been a long-standing custom to stitute of Nutrition, were elected to mem hold in confidence the identity of the re bership in the American Society for Clin cipients of the annual AIN Awards and ical Nutrition. also those to be named Fellows until the HONORARY MEMBERS actual presentation of these honors at the Society banquet. Dr. Kline announced The following scientists were elected to that the AIN Council had taken the im Honorary Membership: portant decision to alter this arrange H. A. P. C. Oomen, Director, ment to permit pre-meeting announce Institute for Tropical Hygiene, Amsterdam, Holland ment of Award winners and Fellows. This B. S. Platt, change was made to allow for more effec National Institute for Medical Re tive publicity concerning the individuals to search, M.R.C. Human Nutrition be honored, their specific scientific ac Research Unit, Mill Hill, London, complishments which won them recogni N.W. England tion, and the nature of the Awards and Jean Tremolieres, Institut National d’Hygiene, their sponsors. Under the present ar Paris, France rangement our news releases were always competing with all the others resulting V. President’s Report from the Federation meetings. President Kline advised the member A joint committee representing both AIN ship that AIN will continue to pursue its and ASCN is being established to under efforts to obtain support for developing a take a thorough revision of the constitu word list or guide to nutrition terminology tion and by-laws of both societies. The that would serve as a basis for a documen purpose is to improve the precision of both tation system for nutrition science litera documents and to resolve certain incon ture. sistencies between them. Since the initiation of the Sustaining As The American Board of Nutrition has sociates program last year, 19 organiza requested the Councils of both AIN and tions have become Sustaining Associates ASCN to accept the responsibility of mak of the AIN. Contributions ranged from ing a nomination to fill any vacancy that $100 to $1000, making a total of $5,500. occurs on the Board. The latter will elect Sustaining Associates are listed in the one of these nominees to maintain a bal Journal of Nutrition each month. ance between those working in human The Council has approved a budget of nutrition and those in clinical nutrition. up to $5,000 for the expert advice, con The AIN Council has accepted this assign sultation and conferences required for re ment and the ASCN Council will act upon vising the leaflet, “Careers in Nutrition.” the request at its next meeting. President Kline advised that, due to the VI. Executive Secretary’s Report pressures of other duties, the AIN Nutri tion Notes editors, M. R. S. Fox and W il Dr. Waddell reported that the total liam Boehne, have asked to be relieved of funds available to the AIN Committee for this assignment. A vote of appreciation the support of travel grants to the Vllth and thanks was made to the editors for International Congress of Nutrition in 1 0 6 AMERICAN INSTITUTE OF NUTRITION EXHIBIT A Balance Sheet — December 31, 1965 A s s e t s C a s h ...... $ 4 6 ,0 9 7 .7 5 A c c o u n ts R e c e iv a b le ...... 5 ,9 3 8 .7 5 I n v e s t m e n t s ...... 2 1 ,8 3 2 .6 8 F u r n i t u r e a n d E q u ip m e n t ...... $ 1 ,6 4 5 .5 7 L e ss A c c u m u la te d D e p r e c ia tio n ...... ( 8 2 .2 8 ) 1 ,5 6 3 .2 9 P r e p a id E x p e n s e s ...... 8 4 .0 0 T o ta l A s s e ts ...... $ 7 5 ,5 1 6 .4 7 L i a b i l i t i e s a n d F u n d C a p i t a l A c c o u n ts P a y a b le ...... $ 1 5 ,6 9 6 .5 0 F u n d C a p ita l ( s e e E x h ib i t B ) ...... 5 9 ,8 1 9 .9 7 T o ta l L ia b ilitie s a n d F u n d C a p ita l ...... $ 7 5 ,5 1 6 .4 7 EXHIBIT B Statement of Income and Expense and Fund Capital For the Period April 1, 1965 through December 31, 1965 F u n d C a p ita l F o rw a rd e d , 4 / 1 / 6 5 ...... $ 4 0 ,5 1 5 .9 6 I n c o m e ; P a g e C h a r g e s ...... 1 1 ,9 0 0 .0 0 Interest Income 1,368.20 M e m b e rs h ip D u e s ( 8 9 7 @ $ 7 .0 0 ) ...... 6 ,2 7 9 .0 0 S u p p o rtin g M e m b e rs C o n tr ib u tio n ...... 5 0 .0 0 A n n u a l M e e tin g R e g is tr a tio n s ...... 5 ,5 5 2 .7 0 S a le o f S p e c ia l P u b lic a tio n s ...... 1 1 2 .0 0 A d d itio n a l N e t S u b s c r ip tio n In c o m e — W is ta r I n s t i t u t e ...... 3 ,7 2 3 .7 5 E d ito r ia l A llo w a n c e — W is ta r I n s t i t u t e ...... 7 ,2 7 5 .0 0 A w a rd W in n e r s R e c e p tio n R e c e ip ts ...... 4 6 0 .6 5 P ro c e e d s f r o m S a le o f S e rie s K B o n d ...... 4 9 3 .5 0 O th e r I n c o m e ...... 7 .0 0 T o ta l I n c o m e ...... $ 7 7 ,7 3 7 .7 6 E x p e n s e s : S a la r ie s a n d W a g e s ...... $ 5 ,3 4 1 .4 2 P a y r o ll T a x e s ...... 1 4 4 .7 0 P e n s io n a n d G ro u p I n s u r a n c e C o n tr ib u tio n s ...... 2 9 2 .2 7 E d ito r ia l O ffice E x p e n d i tu r e s ...... 3 ,7 8 5 .0 0 O th e r P e r s o n a l S e rv ic e ...... 1 2 .3 6 H o te l a n d T ra v e l ...... 4 ,3 1 9 .7 6 A d d r e s s in g , M a ilin g a n d S h ip p in g ...... 3 0 8 .6 6 T e le p h o n e a n d T e le g r a p h ...... 2 1 5 .3 1 P r i n t i n g a n d E n g r a v in g ...... 6 8 1 .2 9 S u p p lie s a n d D u p lic a tin g ...... 7 5 4 .9 4 R e n ta l o f E q u ip m e n t a n d S p a c e ...... 1 1 6 .0 0 E q u ip m e n t R e p a irs a n d M a in te n a n c e ...... 2 5 .7 8 D e p r e c ia t io n E x p e n s e ...... 8 2 .2 8 D u e s to A A A S ...... 4 0 .0 0 A w a rd W in n e r s R e c e p tio n ...... 4 6 0 .6 5 C o ffee L o u n g e — A n n u a l M e e tin g ...... 1 3 4 .8 1 O th e r E x p e n s e ...... 2 6 1 .6 0 $ 1 6 ,9 7 6 .8 3 F A S E B B u s in e s s S e rv ic e C h a rg e ...... 9 4 0 .9 6 T o ta l E x p e n s e ...... $ 1 7 ,9 1 7 .9 7 Fund Capital at 12/31/65 $ 5 9 ,8 1 9 .9 7 PROCEEDINGS 107 Hamburg was $45,000 (National Institute of one or more AIN members residing in of Arthritis and Metabolic Diseases, the Bethesda area, and that they be in $15,000; National Science Foundation, structed to review the financial records of $10,000; Nutrition Foundation, Inc., the Institute in the Federation office. AIN’s $5,000; and AIN $15,000). This was ap present business arrangement with the preciably less than had been anticipated, Federation includes a professional audit while the number of applications was of the AIN accounts within the Federa greater than expected. The Committee tion audit. awarded 121 travel grants out of a net VIII. Dues total of 180 applications. Motion was made, seconded and passed Two additional group flights to Ham to accept the recommendation of the AIN burg and return were announced, supple Council that membership dues remain the menting those previously described in the same as for last year ($ 7 .0 0 ). travel folder sent to all AIN members and to applicants for travel grants. These were IX. Editor’s Report —- for trans-Atlantic travel only to suit those Journal of Nutrition who wished to spend more than 21 days The editor of the Journal of Nutrition, abroad but wished to arrange for their Dr. Richard H. Barnes, submitted his re own European travel. port for the calendar year 1965 with a comparison to 1963 and 1964. He called VII. Treasurer’s Report particular attention to the current guide Dr. W . A. Krehl distributed copies of for authors and asked that it be followed the financial statement, reproduced here, for manuscripts submitted to the Journal. and introduced to the membership the It was reported that the AIN Committee AIN business manager, Mr. John Rice. on Publication Management has been The Auditing Committee reported that, on asked to investigate the costs and proce their behalf, Dr. John Bieri of the Bethesda dures for moving the redactory services of area had examined the records of the AIN the journal to Beaumont and submit a finances and found them in order. The recommendation to the Council. Auditing Committee recommended that in The report and proposed budget were the future the auditing committee consist approved and are summarized below: Editing and Publication Operations (Calendar year) 1 9 6 3 1 9 6 4 1 9 6 5 Volumes published . 8 0 , 81 8 2 , 8 3 , 84 8 5 , 8 6 ,8 7 Pages published (including papers, biographies, announcements, and proceedings) 1 4 3 6 1 3 3 9 1 3 8 4 (Scientific papers only) 1 3 8 3 ) ( 1 2 6 7 ) ( 1 3 1 3 ) Papers published (including 3 biographies) 2 0 4 191 189 Papers submitted 3 1 2 2 9 3 3 0 3 Papers rejected 113 112 98 Rejection rate (based on no. papers submitted) 3 6 % 3 8 % 3 3 % Supplements published Letters to the Editor 1 2 Operating Schedule Avg time for date of receipt of manuscript to mailing to Wistar Press: Avg no. days with reviewer 21.2 2 0 .7 2 1 .5 Avg no. days out for revision 2 4 .8 22.6 22.2 Avg no. days in office, in mail or in unavoidable delay 3 4 .9 2 9 .2 3 0 .6 A v g to t a l days 8 0 .9 7 2 .5 7 4 .3 Avg time from date of receipt of manuscript to mailing by Wistar P re s s : Avg no. months with Editorial Office 2.6 2 .4 2 .5 Avg no. months with W istar Press 3 .2 3 .2 3 .0 A v g to t a l months from date of receipt to mailing of Journal 5 .8 5 .6 5 .5 1 0 8 AMERICAN INSTITUTE OF NUTRITION Summary of Finances in the Operation of the of research workers in basic and applied experi Editor’s Office, Journal of Nutrition mental animal sciences in the functions and ac July 1, 1965 — December 31, 1965 tivities of the American Institute of Nutrition. A symposium on “Nutrition Education” was Balance brought forward ...... $ 9 5 9 .2 7 organized for the annual meeting by a sub R e c e ip ts , A IN ...... 6 ,0 0 0 .0 0 committee on Nutrition and Education, chaired Total receipts and by J. K. Loosli. Participants in the symposia in balance available ...... 6 ,9 5 9 .2 7 cluded John B. Youmans and George V. Mann, Co-chairmen, and the following speakers: W. H. E x p e n d i t u r e s ...... 4 ,7 5 1 .2 9 Griffith, A. E. Harper, F. W. Hill, John F. M ueller, and Dena C. Cederquist. B a la n c e ...... $ 2 ,2 0 7 .9 8 In addition, two conferences were organized (This statem ent covers a period of only 6 m o n th s for the annual meeting as follows: (a) 31st because the beginning of the fiscal year was Annual Poultry Nutrition Conference, chaired by changed by the AIN business office from July 1 F. H. Kratzer. A major part of this program to January 1.) dealt with “Effect of Nutrition on Connective The Council approved the budget proposed by Tissue and Cartilage Formation in Chickens.” Dr. Barnes for the Editor’s Office for the year (b) 7th Annual Ruminant Nutrition Conference, starting January 1, 1966. chaired by R. L. Reid. The topic of this con ference was “Food Intake by Rum inant Animals.” X. Report of the Clinical Division: Efforts to collect a series of slides depicting nutritional deficiency disease in experimental J. F. Mueller animals have been initiated (see Proceedings of Dr. Mueller advised the membership of the Twenty-ninth Annual Meeting, Journal of Nutrition, 86: 445, 1965). The response by AIN the coming ASCN annual meeting in members to requests for appropriate slides has Atlantic City and invited all members to been limited as yet, but this project will be given attend and participate in the Second Mc additional emphasis by the committee during Collum Award banquet. the coming year. Even now the number of Dr. Mueller reminded the members of slides available to the committee is very en couraging. Members are asked to send slides to the Norman Jolliffe Fellowship Award. the committee chairman or to the Excutive The purpose of the award is to stimulate S e c re ta ry . interest in clinical nutrition among medi B. Joint Committee on Biochemical cal students in the U. S. and to give finan Nomenclature: S. R. Ames cial support to those active in the teaching of clinical nutrition in American medical The Committee confined its activities princi pally to problems of vitamin nomenclature and schools. to the establishment of working liaisons with other nomenclature groups. XI. Reports of Committees and The nomenclature of compounds of interest to Representatives biochemists is continually under review by com mittees of the IUPAC-IUB Commission on Bio A. Standing Committee on Experichemical Nomenclature (CBN). Results of their mental Animal Nutrition: G. F. Combs deliberations are published and become accepted terminology for scientific journals. The Inter The purpose and function of the Committee on national Union of Nutritional Sciences (IUNS) Experimental Animal Nutrition as stated in the occasionally has had representatives at CBN de operating procedure approved by the Council are liberations. In this country, the Office of Bio as follows: Purpose: (a) To emphasize the role chemical Nomenclature, NAS-NRC (OBN) was that research workers in basic and applied ex formally launched on July 1, 1965, with the ap perimental animal nutrition have played and will pointment of Waldo E. Cohn as director. The continue to play in the development of the sci objectives of OBN are defined “to coordinate in ence of nutrition, (b) To arouse greater interest formation concerning biochemical nomenclature on the part of these scientists in the functions of efforts of national and international organizations th e A IN . Functions: (a) To provide representa and to take appropriate steps for the stimulation tion of the experimental animal nutritionists in of efforts and the dissemination of this inform a the affairs of the AIN Council, (b) To be re tion in appropriate journals and by other m eans.” sponsible to the Council on m atters pertaining to Nutritionists both here and abroad appear to experimental animal nutritionists, (c) To work have taken a relatively passive role in the estab with the Ruminant Nutrition and Poultry Nutri lishment of biochemical nomenclature and more tion Conferences in behalf of the Council, (d) active participation is desirable. There is neces To initiate steps necessary for the conduct of sity for immediate action in some areas, espe specific activities and meetings, including sym cially the vitamins and related compounds. posia, considered desirable in the accomplish The Committee considers that generic descrip ments of the purposes set forth above, (e) To tors for the vitamins are useful and necessary. develop and initiate other approaches designed to Because clarification of the IUPAC-IUB-CBN posi stimulate the general interest and participation tion in this area was essential, CBN was asked PROCEEDINGS 1 0 9 if the designation of suitable generic descriptors 2L-a-T ocopherol: of the vitamins was within its province. CBN 2L,4'D,8'D-a-Tocopherol (2S,4'R,8'R-a-tocopherol) advised that it is not now nor does it propose to is the i-epimer of a-tocopherol. It has been pre become in the forseeable future concerned with pared in pure form by resolution of 2DL-a-tocoph- the nomenclature of vitamins as vitamins. It erol (see above) and its biochemical and nutri considered the problem of generic descriptors of tional properties are being extensively investi vitamins as lying outside its province and within the province of IUNS. W here CBN has concerned gated. In view of the importance of this stereo itself with substances with vitamin activities, it isomer in research on the biochemistry of a-to has done so on a chemical basis not on an ac copherol, the name 2L-a-tocopherol is proposed tivity basis. Our Committee is thus free to work for the substance hitherto known as the ¡-epimer with IUNS to establish suitable generic descrip of a-tocopherol. tors for the vitamins without conflict of interest Tocopheryl esters: w i t h C B N . The following system of nomenclature for the In view of historical precedent and since no stereoisomers of tocopherols and their esters was change seems justified, the retention of the -yl proposed by the Committee and accepted by the suffix to refer to tocopheryl esters is proposed C o u n c il. (e.g., a-tocopheryl acetate). a-Tocopherol: (3-, 7- a n d S -T o c o p h e ro ls: The above proposals can be directly applied to Following its isolation from natural sources, th e c o r r e s p o n d in g s te re o is o m e rs o f j3,- y- a n d 5- naturally occurring a-tocopherol was termed a- tocopherols. tocopherol. Later, to distinguish it from syn thetic a-tocopherol, naturally occurring a-tocoph With the formation of OBN, the Joint Com erol was referred to as d-a-tocopherol. Recent mittee on Biochemical Nomenclature becomes evidence has shown it to be 2D,4'D,8'D-a-tocoph- superfluous and will be discontinued. The Com erol (2R,4'R,8'R-a-tocopherol). In view of its mittee recommends the formation of a perm anent natural occurrence and in agreement with IUPAC Nomenclature Committee of AIN. This committee practices for other vitamins having asymetric would serve to evaluate and make recommenda centers, the name a-tocopherol is proposed for tions in areas not being reveiwed by CBN and the pure subtance hitherto known as d-a-tocoph- OBN and in cooperation with IUNS would re e ro l. solve nomenclature problems that are peculiar to nutritionists. Racemic-a-tocopherol: C. Ad hoc Committee on International Racemic-a-tocopherol, synthesized from racemic isophytol, is a mixture of the eight possible Nutrition: W . N. Pearson stereoisomers of a-tocopherol. It was incorrectly This Committee recently carried out a survey termed di-a-tocopherol (ra.-a-tocopherol) imply of the AIN membership to determine the extent ing identity with the previously synthesized to which individual members were involved in 2DL,4'D,8'D-a-tocopherol derived from natural phy- international nutrition programs. The results of tol (see below). To resolve the present confu this survey were the basis of a report, prepared sion and in recognition of its being racemic at by the Committee last year, showing the num three asymetric centers, the trivial nam e racemic- ber of members who had actually worked in a a-tocopherol is proposed for the mixture of eight foreign country, those members who expected to stereoisomers hitherto known as di-a-tocopherol. participate in foreign nutrition programs in the immediate future, and the names and addresses 2DL-a-tocopherol: of 625 foreign students trained in the U. S. by The synthetic a-tocopherol derived from natural members of the AIN. Copies of this report are phytol was the first substance to be named dl-a- available from the office of the Executive Secre tocopherol and has been uniformly confused with ta r y . racemic-a-tocopherol (see above) following syn Dr. Pearson reported to the Council that the thesis of the later substance from racemic iso Committee, having completed the above report, phytol. The synthetic a-tocopherol derived from had not embarked on any further programs dur natural phytol has been shown to be 2 d l ,4 'd ,8'd- ing the past year, but he recommended the con a-tocopherol (2RS,4'R,8'R-a-tocopherol), a m ixture tinuance of the Committee and the work which o f o n ly 2 o f th e 8 possible stereoisomers of a- had been started. tocopherol. As the acetate ester, it was termed President-elect Schaefer, in his informal com di-a-tocopheryl acetate which as a 1% solution in ments to the members, reported that the Council oil was the former International Standard. Syn had taken positive action to continue this work thetic a-tocophervl acetate derived from natural and that he would appoint a strong committee phytol (2DL,4'D,8'D-a-tocopheryl acetate) is pres to carry it on. Specifically, the latter was ently being specified for the Animal Nutrition charged with developing a contract between the Research Council Vitamin E Reference Standard. Agency for International Development and AIN To resolve the present confusion and in recogni in support of the AID “Food for Peace” program. tion of its being racemic at only the 2 position, This would include, among other things, the de the name 2DL-a-tocopherol is proposed for the velopment of a roster of nutrition scientists work mixture of 2 stereoisomers hitherto known as ing, or available for work, on foreign assign synthetic a-tocopherol from natural phytol. ments; foreign students being trained in the 11 0 AMERICAN INSTITUTE OF NUTRITION U. S. and the location and availability of those The Committee on Marine Protein Resources who have completed their training; and a review Development, with the help of Mr. George K. of ongoing research associated with foreign in Parman, Executive Secretary, has continued to s titu te s . be active in its advisory service on the research and production of marine protein concentrates D. Representative to the Food and Agri for human consumption and the effective utili culture Organization: B. S. Schweigert zation of such products. This committee has FAO has been active in a number of fields of stated that the fish protein concentrate, from interest to nutritionists during the past year and whole hake, as prepared by the Bureau of Com of particular significance is the summary in the mercial Fisheries process, is safe, nutritious, November-December issue in 1965 of FREEDOM wholesome and fit for human consumption. FROM HUNGER for the 20 years of program The Agricultural Board is now printing re activities in this field. It is appropriate to point vised publications on nutrient requirements of out that while extensive progress has been dairy cattle, poultry and rabbits. A new publica achieved in a number of countries in improving tion on nutrient composition of feedstuffs was food production and health, expanding world released. A special steering committee of the population in relation to food supplies has re Agricultural Board arranged and conducted a sulted in an even greater concern to achieve public symposium on the scientific aspects of “freedom from hunger” in 1966. pest control, (January 31 — February 3, 1966) Three other reports of interest published by a Proceedings which will appear around May 1, FAO or jointly with WHO are appropriate for 1 9 6 6 . mention: (1) Protein Requirement, Report no. The affiliated Agricultural Research Institute 37; (2) Specifications for the Identity and Purity held its annual meeting on October 18-19, of Food Additives and Their Toxicological Evalu 1965, on the theme of World Food Needs and ation: Food Color and some Antimicrobials and Production — Present and Future, Proceedings Antioxidants, Report no. 38; and (3) Symposium of which are published. The 1966 program will on Industrial Feeding and Canteen Management d e a l w i t h The Role of Animal Agriculture in in Europe, Report no. 36. Meeting World Food Needs. E. Representative to National Research A Symposium on Research in Agriculture a lso was sponsored jointly by USDA and the National Council Division and Boards: G. F. Combs Academy of Sciences on February 23-25, 1966. The Division of Biology, NRC-NAS, under the The Agricultural Research Institute is cooperat able leadership of Dr. A. G. Norman, through its ing with USDA and state agricultural experi Food and Nutrition Board, Argicultural Board, ment stations in preparing a national inventory Agricultural Research Institute, Institute of Lab of agriculture. Publication is expected by mid oratory Animal Resources, U. S. National Com summer, 1966, after it has been analyzed. mittee of International Union of Nutritional The Institute of Laboratory Resources is cur Sciences, U. S. National Committee for the Inter rently updating and revising Laboratory Ani national Biological Program, U. S. National Com mals II, Animals for Research; Standards f o r th e mittee for the International Union of Biological care, breeding and management of mice, rats, Sciences and the U. S. National Union for Pure hamsters, guinea pigs, cats, dogs, rabbits, pri and Applied Biophysics, has had another most mates (Macaca m ulatta); the shipping of dogs, productive year. the utilization of test animals; and the shipment The Food and Nutrition Board, which com of laboratory primates. In preparation are poul pleted its 25th anniversary with its 68t h m e e t try standards, genetic standards, laboratory ani ing, has published an historical brochure, Twenty- mal quality standards, laboratory animal pro five Years in Retrospect. curement standards, health standards for Other publications of the Food and Nutrition international shipment of laboratory animals, Board during the year include: Pub. 1282, Pre- and primate standards (other than rhesus). School Child Malnutrition — Primary Deterrent The U.S. National Committee for the Inter to Human Progress, and Pub. 1270, Some Con national Biological Program has progressed siderations in the Use of Human Subjects in nicely in planning the basis for participation of Safety Evaluation of Pesticides and Food Chem U.S. scientists, including nutrition scientists, in icals. Also, the first edition of the Foods Chem IBP. The preliminary framework of the U.S. will be available in the summer of icals Codex program of IBP is presented in USNC, IBP Pub. 1966 with specifications of identity and purity no. 1, NRC-NAS, August 1, 1965. for about 500 food chemicals. Other new reports Members of the AIN should be fully aware of progressing include: The Role of Cereals in World Nutrition, Nutritional Diseases Around the the nature of this program and unique oppor tunities which it can provide for nutrition re World — Geographic Nutrition, a n d Dietary Fat and Human Health. search scientists interested in international nu tr itio n . Other activities include a program in food microbiology which deals with: 1 ) improving F. Representative to the Federation: the reporting of food-borne diseases; 2) s t a n d ard methods of microbiological examination of A. E. Schaefer foods, and; 3) microbiological control measures The Federation Board acted to approve the under conditions of good manufacturing prac addition of another wing to the new building at tic e s . Beaumont (Milton O. Lee Building). PROCEEDINGS 111 The Federation has established a patent policy water supplies which are deficient in concerning inventions by employees which will fluoride, has been shown to improve be voted on by the Board after review by legal the durability of dental enamel and to c o u n s e l. decrease the rate of dental decay, and The Federation bylaws have been revised to Whereas this well-tested public health define functions and duties of Executive Di measure has the support of the U.S. rector, Comptroller, Treasurer. Affairs of the Public Health Service, the American Federation are in good order; the new Executive Medical Association, the American Den Director, Dr. J. F. A. McManus is continuing the tal Association, and many other sci leadership of the Federation in a commendable ence-oriented groups, therefore m a n n e r . BE IT RESOLVED that the American Institute of Nutrition at its annual G. U.S. National Committee, IUNS: meeting April 15, 1966 recognizes flu G. F. Combs oridation as a safe, effective and low- The U.S. National Committee of the Inter cost means of improving dental health national Union of Nutritional Sciences held two by improving nutrition. meetings during the year and continues to sup port the admission of IUNS into the Interna B. Future Federation meetings; tional Council of Scientific Unions. To this end, 1967: April 16—21, Chicago the Committee has encouraged strengthening the 1968: April 15—20, Atlantic City present program of the IUNS to include partici 1969: April 13-18, Atlantic City pation in the International Biological Program, to develop a documentation of nutrition science C. The next Council meeting will be at the international level, to encourage a com held on October 29, 1966, in Washington, mon nomenclature for international use, and to D.C. maintain a list of nutrition scientists by coun tries throughout the world. ANNUAL DINNER AND A revised Constitution and By-laws for IUNS PRESENTATION OF FELLOWS has been prepared for presentation to the dele A N D A W A R D S gates at the forthcoming Congress. The Com The annual banquet was held Thurs mittee has encouraged the appointment of a nutrition scientist to act as coordinator of nu day, April 14, 1966 at the Shelburne trition science pertinent to the International Hotel, with 377 individuals attending. Biological Program. Dr. Kline presided. The Committee continues to encourage the Dr. Charlotte Young, as chairman of development of stronger nutrition societies in other countries as a means of strengthening the the Fellows Committee, introduced the IUNS. newly appointed Fellows, whose citations H. Ad hoc Committee on Meeting follow: M a x K l e i b e r — i n r e c Evaluation: Carl D. Douglass ognition of his distin Dr. Douglass advised the membership that guished career as an AIN had formed a committee to assess the inspiring teacher, au effectiveness of the annual meeting in serving thor and research the needs of the membership. To provide the scientist; for his at basis for sound recommendations, the committee tempt to make nutri asks that members give diligent care to the tion a quantitative sci AIN questionnaire prepared by this committee ence by expressing which will be sent to all members. biological concepts in mathematical and uni I. Affiliation with AIBS: R. W . Engel versal terms; for his The AIN committee considering the feasibility unraveling of the con of AIN affiliation with AIBS advised that they ditions which influence had no recommendation to make at this time. MAX KLEIBER the metabolic rate of the body; for his dis J. International Biological Program. covery of the relationship between body size and See U.S. National Committee, IUNS, XI, metabolic rate; for his contributions concerning the evaluation of diets for animals, particularly G, of these Proceedings. his “replacement equivalent” method of investi gating the utilization of dietary energy; for his XII. New Business pioneering use of isotopes in the clarification of A. Resolution on Fluoridation. The the biogenesis of the constituents of milk. His success as a teacher and lecturer is due in part following resolution was adopted by the to his independence of thought but in large membership at its meeting on April 15, measure to his great sense of humor. He re 1966: ceived the AIN Borden Award in 1952 and the WHEREAS the addition of appropriate Morrison Award of the American Society of amounts of fluoride, to those municipal Animal Science in 1953. 11 2 AMERICAN INSTITUTE OF NUTRITION S a m u e l L e p k o v s k y — fatty acids. He has determined the dietary re for his distinguished quirements for polyunsaturated fatty acids in career of research in several species, including man, and the factors both fundamental and which influence these requirements. He has de applied nutrition; for veloped several methods for the analysis of fatty his contributions to the acids and has carried out extensive investiga early discoveries of the tions of the interrelationships of fatty acids with B vitamins including: other lipids. He has made estimates of the the relationship of vi dietary linoleate intake of men and of infants t a m i n B 6 to xanthu and assessed the requirements of the latter from renic acid excretion serum fatty acid analyses. and to the level of dietary protein, the THE CONRAD A. ELVEHJEM AWARD identity and structure A FOR PUBLIC SERVICE of riboflavin, the rela SAMUEL LEPKOVSKY IN NUTRITION tionship of thiamine The first Conrad A. need to the fat and carbohydrate of the diet, Elvehjem Award of and the metabolism of pantothenic acid; for his $1000 and a scroll contributions to our understanding of the mech was presented to Dr. anisms of digestion, hunger and satiety. His Charles Glen King, modest unself-seeking and generous attitude to Associate Director, In ward his work and his fellow scientists is well- stitute of Nutrition Sci known throughout the world of nutrition. His ences, Columbia Uni quiet voice has cleared up many a controversy versity. The award and his chuckle has smoothed out many argu- was made in recogni m e n ts . tion of Dr. King’s long J o h n B . Y o u m a n s — and distinguished ser for a distinguished ca vice to the public as reer as physician and exemplified by the fol medical educator whose CHARLES GLEN KING lowing: his isolation leadership in clinical of vitamin C perma- nutrition has profound nently benefits man; he has been unsparing in ly affected nutritional his efforts to further the cause of good nutrition science; for his design through professional societies and national and and application of the international agencies; as teacher and counselor technic of nutritional he has infected students and associates with his survey in the United spirit of public service. Also as the first Director, States and abroad prior and later President, of the Nutrition Foundation to World War II and he established a pattern for the support by in his major contributions dustry of nutritional research and education JOHN B. YOUMANS to ICNND; for his ef which has been widely imitated. This Founda fective guidance in tion has had a profound impact upon the devel clinical nutrition and research for the U. S. opment of nutritional science and the application Army through the Office of the Surgeon General of research findings to the benefit of mankind and for the American Medical Association through long membership on the Council on everywhere. Dr. King has indelibly imprinted the importance of nutrition on the institutions Foods and Nutrition and as its Scientific Di rector. As a teacher he has exerted a telling of government, of industry, and of education. effect upon the careers of many members of MEAD JOHNSON AWARD FOR AIN. RESEARCH IN NUTRITION Dr. Kline, as toastmaster, presided at The 1966 Mead John the presentation of the following awards: son and Company Award of $1000 and a BORDEN AWARD IN NUTRITION scroll was awarded to Dr. M. Daniel Lane, As The 1966 Borden sociate Professor of Bio Award of $1000 and a chemistry, New York gold medal was pre University School of sented to Dr. Ralph Medicine. Dr. Lane was Theodore Holman, Pro selected in considera fessor of Biochemistry, tion of his fundamen Hormel Institute, Uni tal contributions to our versity of Minnesota. understanding of the The award was made mode of action of bio in recognition of Dr. tin in enzymatic car M. DANIEL LANE Holman’s outstanding boxylation reactions, contributions to knowl and of his pioneering demonstration that in edge of the metabolism biotin enzymes the vitamin is covalently bound and the requirements in an amide linkage involving its carboxyl group of those im portant con- ralph t. holman and the epsilon amino group of lysyl residues of stituents of milk, the the protein. PROCEEDINGS 113 OSBORNE AND MENDEL AWARD The 1966 Osborne Man and Domestic Animals.” His many publi and Mendel Award of cations on the biological evaluation of proteins $1000 and a scroll was had a profound influence on methodology and awarded posthumously understanding of this important field and illus to Dr. Harold H. Mit trate the thoroughness of his approach to scien chell, Professor Emeri tific matters. His interpretive reviews on cal tus of Animal Nutri cium requirements, fluorine toxicity, and the tion, University of effect of environmental stress on nutrient metab Illinois. It was pre olism revealed his unusual capability as a sented in recognition meticulous critic. His influence was felt also by of his pre-eminent stud the excellence of his teaching and the accom ies in mammalian pro plishments of many students. A charter member tein, mineral and and Fellow of the Institute, he served for 24 energy metabolism, cul years on the editorial board of the Journal of HAROLD H. MITCHELL minating in the au Nutrition. He received the AIN Borden Award thorship of a two- vol in 1945, and the Morrison Award from the ume compendium, “Comparative Nutrition of American Society of Animal Production in 1950. AMERICAN INSTITUTE OF NUTRITION Founded April 11, 1933; Incorporated November 16, 1934; Member of Federation 1940 OFFICERS, 1966-1967 Fellows Committee: S. L. Hansard (1967), chair man; P. E. Johnson (1967), W. H. Griffith President: A. E. Schaefer, Nutrition Section, Of (1968), Agnes F. Morgan (1969), R. M. fice of International Research, National In Forbes (1969). stitutes of Health, Bethesda, Maryland. Committee on Honorary Memberships: P a u l President-Elect: G. M. Briggs, Department of Gybrgy, chairman; Grace A. Goldsmith, R. Nutritional Sciences, University of Cali W . E n g e l. fornia, Berkeley, California. Auditing Committee: W. A. Gortner, chairman; Past President: O. L. Kline, 3509 Rodman G. V. Vahouny. Street, N.W., W ashington, D. C. Finance Committee: W. A. Krehl, chairman; Secretary: W. N. Pearson, Department of Bio H. W. Howard, D. V. Frost, Grace A. Gold chemistry, Vanderbilt University School of smith, James Waddell (ex officio). Medicine. Nashville, Tennessee (1969). Symposia Committee: E. L. R. Stokstad, chair Treasurer: W. A. Krehl, Clinical Research Cen m an; F. J. Ingelfinger, S. M. Garn, Lucille ter, University of Iowa, Iowa City, Iowa S. Hurley, Doris H. Calloway, W. S. Hartroft, ( 1 9 6 8 ) . C. G. King. Councilors: N. S. Scrimshaw (1967), E. L. R. Committee on Publication Management: W . J. Stokstad (1968), A. E. Harper (1969), A. Darby (1968), chairman; F. L. Iber (1967), B. Eisenstein (1969). D. M. Hegsted (1967), R. W. Engel (1968), Executive Secretary: James Waddell, 9650 Rock J. F. Mueller (1968), James Waddell (con ville Pike, Bethesda, Maryland 20014. tinuing) secretary. Ex officio: W. N. Pear son. A. B. Eisenstein COMMITTEES Committee on Nomenclature: S. R. Ames, chair Nominating Committee: M. L. Scott, chairman; man; P. L. Harris, H. H. Williams, H. E. D. B. Coursin, B. C. Johnson, Ruth Leverton, Sauberlich, M. E. Shils. C. O. Chichester. Public Information Committee: M. S. Read Membership Committee: K. W. King (1968), (1968) , chairman; A. L. Forbes (1967), chairm an; C. H. Hill (1967), Dena C. Ceder- C. J. Ackerman (1967), Ruth L. Pike (1967), quist (1969), G. V. Mann (1970), B. L. Mary A. W illiams (1967), P. L. W hite (1968), O'Dell (1971). J. T. Sime (1968), R. S. Goodhart (1968). Nominating Committee for Mead Johnson Aivard: Committee on Experimental Animal Nutrition: P. L. Day (1967), chairman; A. R. Kem- G. F. Combs (1969), chairman; W. H. merer (1968), L. M. Henderson (1969). Pfander (1967), vice-chairman; R. W. Luecke Nominating Committee for Borden Aivard: P . H . (1969) , E. W. Crampton (1968), F. W. Hill Weswig (1968), chairman; E. L. Hove (1968), J. K. Loosli (1967). Ex officio: (1967), K. E. Harshbarger (1968). Hans Fisher (1967), D. S. Kronfeld (1967), Nominating Committee for Osborne-Mendel A. E. Schaefer (1967). Aivard: M. O. Schultze (1967), chairman; Ad hoc Committee on Science Fairs: C. W. Carl Alex Black (1968), L. E. Holt, Jr. (1969). son, chairm an; E. L. W isman, Helen M. Dyer. Nominating Committee for Conrad A. Elvehjem Ad hoc Committee on Sustaining Associates: Award: E. E. Howe (1967), chairman; F. C. H. Krieger, chairman; W. R. Graham, W. Quackenbush (1968), R. E. Shank (1969). H. L. Sipple, James W addell (ex officio). 114 AMERICAN INSTITUTE OF NUTRITION Ad hoc Committee on International Nutrition: Federation Proceedings Editorial Board: P . L. R. W. Engel, chairm an; Guillermo Arroyave, Harris (1967). D. B. Hand, W. G. Unglaub, L. J. Teply, Federation Proceedings Translation Supplement: J. B. Youmans, W. J. McGanity, G. F. Combs, J. A. Stekol (1968). W. N, Pearson (ex officio). Ad hoc Committee on Nutrition Education: A. Editorial Board E. Harper, chairman; A. L. Black, Doris H. The Joitrnal of Nutrition Calloway, G. F. Combs, K. W. King, H. A. R. H. Barnes, Editor (1969); H. PI. Williams, Schneider, T. B. Van Itallie, C. W. Woodruff. Ad hoc Committee on Recommended Constitu Associate Editor; E. Neige Todhunter, Bio graphical Editor; Roslyn B. Alfin-Slater tional Changes: J. C. Fritz (chairm an), W. (1970), George H. Beaton (1970), George H. Griffith, L. M. Henderson, R. S. Goodhart, M. Briggs (1967), Samuel J. Fornon (1969), J. F. Mueller, I. C. Plough, G. M. Briggs, R. M. Forbes (1967), L. M. Henderson James W addell (ex officio). Ad hoc Committee on Evaluation of Meetings: (1968) , F. W. Hill (1968), Jules Hirsch (1967) , E. E. Howe (1967), B. Connor C. D. Douglass, chairman; S. R. Ames, Johnson (1970), Gennard Matrone (1968), Myron Brin, Mary A. Morrison. C. H. Hill, Paul M. Newberne (1969), Boyd L. O'Dell K. W. King, J. M. Iacono, James Waddell (1969) , William N. Pearson (1970), H. E. (ex officio). Sauberlich (1969), Clara A. Storvick (1968). U. S. National Committee, IUNS: O. L. Kline (1967), chairman; C. G. King (1967), W. J. Darby (1967), W. A. Krehl (1967), W. M. Officers, American Society for Clinical Nutrition Beeson (1968), A. L. Forbes (1968), R. W. (A division of the American Institute of Nutrition) Engel (1968), G. F. Combs (1989), Char President, R. E. Hodges; President-Elect, R. E. lotte Young (1969). Ex officio (voting) A. Shank; Past President, C. S. Davidson; Sec E. Schaefer. Ex officio (non-voting): A. G. retary Treasurer, A. B. Eisenstein, 818 So. Norman, R. K. Cannan, Harrison Brown, Meramec Avenue, Clayton, Missouri; Coun E. C. Rowan. cilors: R. M. Kark (1967), W. S. Hartroft (1968) , J. J. Will (1969). W. A. Krehl REPRESENTATIVES (ex officio). National Research Council Boards and Divisions: Editorial Board G. F. Combs (1969). American Journal of Clinical Nutrition American Association for the Advancement of Science: R. L. Lyman (delegate), Ruth M. W. A. Krehl, Editor-in-Chief; R. E. Hodges, Leverton (alternate). Associate Editor; M argaret J. Albrink, Ernest National Society for Medical Research: H . C. Beutler, G. J. Gabuzda, Francisco Grande, Spruth, H. A. Schneider. S. A. Hashirn, Victor Herbert, Robert Jack- Food and Agriculture Organization: H . E. son, C. M. Leevy, M. E. Shils, D. M. W atkin, Säuberlich (1968). George M. Owen, H. E. Sauberlich. Federation Pziblic Information Committee: M . S. J a m e s W a d d e l l , Executive Secretary Read (1968). American Institute of Nutrition