PYRIDOXIN DEEIGIENOY - AN EXPERIMENTAL STUDY a thesis for the degree of doctor of Medicine(M.I).) in the U niversity of Glasgow L.R.G. AGNEW,M.B. ,Ch.B. Rowett Research In stitu te , Buckshurn,Aberdeenshire * March,1950 * Now McGhie Gancer Research Scholar, Department of Medicine,Royal Infirmary,Glasgow ProQuest Number: 13870154 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13870154 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 I am deeply indebted to Dr D.P.Guthbertson and Dr J.W.Howie for th eir helpful advice and encouragement during the period(l947- 1949) when the work reported here was in progress CONTENTS In tro d u c tio n Rage 1» Scope of Thesis Rage 3. Rart 1 Part 1 (Methods) Page 4. 11 (Results) ...........................Rage 11. M (Discussion) ...................Rage 19. u (Summary) ...........................Page 32. Rarl ,.„g Part 2 (Introduction) .......P a g e 34. ** (Methods) ...........................Page 35. 11 (Results) ...........................Rage 38. 11 (Discussion) ....................Rage 88. '* (Summary) .........................Page 96. References (Parts 1 and 2) ..Page 99. 1. INTRODUCTION The story of pyridoxin began in 1926 when Goldberger and -Lillie reported that rats fed a diet deficient in what was regarded as the pellagra-preventing(P-P) factor developed a ch aracteristic derm atitis. Once the m ultiple nature of the vitamin B complex was realised ,th e separate nature of Goldberger and L illie* s factor - which by th is time had been variously called "factor Y!l (Chick and Copping, 1930) , the "antiderm atitis factor"(Hogan and Richardson,1936),"vitamin H" (Booher,1937),and "factor l"(Lepkovsky,Jukes and Krause,1936)- was recognised,and Gyorgy' s(l934) suggestion that it be called "vitamin BgM was generally accepted. Vitamin Bg was isolated as a pure c ry stallin e organic compound in 1938,and the following year its structure was known and it was synthesized by H arris and Rolkers(l939) in the United S tates, and by Kuhn,-Westphal,Wendt and W estphal(l939) in Germany. A shorter name than 2-methy1-3-hydroxy 4,5 dihydroxymet- hylpyridine was needed and b iolo gists welcomed the suggestion of Gyorgy and Eckhardt(1939) that th is new substance be called "pyridoxin". "Vitamin Bg" and "pyridoxin" are often regarded loosely as synonyms,but as two other substances - pyridoxal and pyridoxamine,an aldehyde and an amine derivative respect­ ively of pyridoxin(Snell,1944) - possess the same biological activ ity in higher animals,Rabinowitz and Snell(l948) have suggested that the name "pyridoxin" be reserved for the specific substance mentioned above,and that "vitamin Bg" should be used as a group term fo r pyridoxin,pyridoxal and pyridoxamine. The structure of these compounds is shown in F ig .1. GHO HO OHoOH HO ,0H ch3 CH3 CH Pyridoxin Pyridoxal F ig .1. Structure of Pyridoxin,Pyridoxal and Pyridoxamine Pyridoxin is essen tial for m ice,rats,chicks,dogs,sw ine and,pos8ibly,monkeys*aad growth failure,ow ing to anorexia, has been reported in deficiency states in these animals. A lso,in rats,pyridoxin deficiency causes derm atitis - the so-called "rat acrodynia”^Birch,Gyorgy and H arris,1935) - and, in long-standing cases,epileptiform fits(C hick,fil Sadr and Worden,1940);w hilst in dogs(Fouts et a l. .1938) and swine (Wintrobe et a l. .1943) hypochromic m icrocytic anaemia has been described. Pyridoxin is important in protein metabolism. Normal tryptophan metabolism is in terfered with in pyridoxin-defic­ ient rats and m etabolites such as xanthurenic acid are excreted in the urine(Lepkovsky,Koboz and Haagen-Smit,1943). Bacteria capable of u tilisin g members of the vitamin-Bg complex(pyridoxin,pyridoxal and pyridoxamine) can convert them into amino acid decarboxylases. For example,pyridoxal can act as a coenzyme and catalyse the decarboxylation of amino acids such as tyrosine(Gunsalus and Bellamy,1944). SCOPS OF THESIS PART 1. Antibody Production in Pyridoxin-deficient Rats. Stoerk and Sisen(1946) and Stoerk,Eisen and John(l947) reported that pyridoxin-deficient rats immunized with sheep erythrocytes developed serum-antibody(haemagglutinin) levelb far below those of inanition control rats and of rats fed a complete d iet ad libitum . They also reported a strik in g reduction in the number of fixed and circu latin g lymphocytes in pyridoxin-deficient ra ts. I t seemed of in te rest to attem pt to confirm and extend these findings,and the f ir s t part of th is thesis is devoted to th is work. PART, g. Haem aturia^in-Pyridoxin-def icien t R ats. During these experim ents(to be described in Part 1) on antibody production in pyridoxin-deficient hooded ra ts, gross macroscopic haematuria was noted in several animals fed the pyridoxin-deficient diet but not in corresponding control ra ts. The second p art of th is thesis is concerned with a detailed study of th is phenomenon. 4. PART 1, ANTIBODY PRODUCTION IN PYRIDOXIN-DEFICIENT RATS As mentioned above,Stoerk and Eisen(l946) and Stoerk, Eisen and John(l947) reported that pyridoxin-deficient rate immunized with sheep erythrocytes developed serum-antibody (haemagglutinin) levels far below those of inanition control rats and of ra ts fed a complete diet ad libitum . Axelrod, Garter,McCoy and G eisinger(l947),using human erythrocytes as an antigenic stim ulus,obtained sim ilar resu lts. It seemed of in te rest to attempt to confirm these findings and to use not only sheep erythrocytes but also a k ille d culture of a pathogenic organismfB acterium typhosum. Further,Dimick and Schreffler(l939) observed "complete atrophy" of the thymus in pyridoxin-deficient rats;and as Stoerk et a l. (1947) reported a strik in g reduction in the number of fixed and circulating lymphocytes in pyridoxin-deficient r a ts ,it seemed worth studying the effect of pyridoxin deficiency on lymphoid tissue in the ra t,p a rtic u la rly as the work of White and Dougherty(l946) indicated the importance of the lymphocyte in antibody production by virtue of the elaboration of gamma-globulin. METHODS Hooded L ister In stitu te rats(Rowett In stitu te strain ) of both sexes were used. They were housed in individual cages with wide wire-screened bases to prevent coprophagy. Unlimited access to water was allowed,and they were weighed daily. DIET. The animals were weaned and maintained on the synthetic diet shown in Table 1. The B-complex vitam ins (Table l),except choline,were made up in a ‘master mix* which was stored at 0-4°C. An appropriate amount of th is powder was thoroughly mixed with the casein of the d iet. // Table i. Composition of the experimental diet y Vitamin supplements added to casein Other vitamin supplements v Content mg./ioo g. Amount and Foodstuff (%) Vitamin diet Supplement method of feeding Sucrose 73'° (-Inositol IO'O Choline chloride 15 mg./rat/day. 30 mg. Vitamin-free casein i8-o p-Aminobenzoic acid IO'O in o* 1 ml. distilled (Glaxo Lab. Ltd.) Nicotinic acid 4-0 , water fed by pipette . Margarine 5-o Ca-pantothenate 2-0 jn * every alternate day Salt mixture (Mc­ 4-o Riboflavin 0-3 Radiostoleum (^yn- 5 mL/250 g. margarine Collum 185 +0'22I Aneurin o-3 thetic vitamiitsD, g. potassium iodide/ B.D.H. Ltd.) kg. Pyridoxin-HCl* o-3 a-Tocopheryl acetate 2 mg./rat/week. Fed in arachis oil by pipette * Pyridoxin-HCl was fed to control animals only. DESIGN OF EXPERIMENT, Prelim inary studies indicated that weanling rats fed the pyridoxin-deficient diet(Table l) failed to grow as rapidly,owing to anorexia,as litter-m ates receiving the same diet with pyridoxin. Any differences,e.g, in antibody titre s or thymus weights,between a pyridoxin- deficient rat and a fu lly supplemented rat might thus be attrib u tab le not only to pyridoxin deficiency but also to inanition. Failure to make allowance for th is in the design of the experiment makes i t impossible o r,a t b e st,d iffic u lt to d ifferen tiate effects resu ltin g from simple failu re of the rat to grow,from effects sp ecifically referable to lack 6. of the p articu lar vitam in in question. The prelim inary experiments mentioned above indicated that "inanition controls" were e sse n tia l,end two methods - paired-feeding and paired-weighing - were considered. Before discussing these methods it should be pointed out that inanition is not,of course,a specific feature of pyridoxin deficiency and occurs,for example,in deficiency of riboflavin,aneurin and pantothenic acid. Paired-feeding consists in making another rat isocaloric with the rat fed the deficient diet by feeding this control rat the same amount of food,plus the specific vitamin,consumed by the animal fed the deficient diet. Paired-weighing consists in mimicking in another rat the weight curve of the rat fed the deficient d iet, and th is is readily done by simple underfeeding. As in paired-feeding,the control rat receives the specific vitam in. This is the most exacting - and hence the most accurate - control available for n u tritio n al experiments. Paired-feeding is not such a reliab le control as it does not necessarily resu lt in the weight curve of the paired-fed rat p arallelin g that of the rat fed the deficient d iet. This only occurs in short-term experiments. At the end of a long-term experiment (P ig.2) with a quartet of litter-m ate rats of the same sex