BE IHflttBBCa Of (IBM B3RIYATIV ' Mf THE HS?A90U 3K 0? KQHMaL AND TCJIOB TI33UJ53 37 Wilson 0 ^ Grant M f %%% & 1 -Jilt float* autatfcfcad to tl® Facmlty of thm Ciradttato iohool of tl* Wnlvtralfcy of tfarylaad in partial fulfillaent of th® raqttiraaa&ta for the dagr*® of Bootor of Jfelidaoph? 1942 UMI Number: DP70368 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. Dissertation Publishing UMI DP70368 Published by ProQuest LLC (2015). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 for her generous assistance throughout this investigation, I would like to aeknowledge a debt of gratitude to ay wife, I wish also to thank Dr, iohn 0, Krants, ^r.» and the Internation­ al Osoeer Research Foundation for providing the opportunity and necessary facilities, and Miss Dorothy Klhler and Dr. Sylvan S* Forman for shenieal syntheses* w ,c *a . 96898 T1BXJS 01* OOMTSHT-1 Page Introduction 1 Experimental 7 Oxygen Oonaumptioa IS Anaerobic Uyeolysls 17 Methylene Blue 'Reduction 18 n~8utyl Urea m Sample Ore a Derivative 2£ Inhibition of Xueoiaie Acid Oxidation 32 Comparison of VIeasea 32 Gluoose Concentration 34 !ea~apedfi®ity of Reagent 34 Comparison of Carbohydrate* 36 Order of iddltlon 30 Brain Brel 42 Methylene Bine Reduction Timeo f Sueelaata 44 Glucose and p-Phanylene Diamine 46 .Gydrogendon Concentration 47 Influence of Calcium 49 Various Media 51 Aerobic Glycolysis 54 Dlaeusaion 56 Conclusions 61 Literature Cited 62 LIST OF TABLK3 Table Pag® lo * I !xs»pi® of hydrogen transportation 6 II Relationship of the urea derivatives 11 III Th# effsot of an homologous series of urea derivatives on oxygen consumption 13 I? The offset of aortain urea derivatives on oxygon oonsumption ie F The influence of urea derivatives on aaaoro* bis glycolysis 18 FI Xnfluonoo of urea derivatives on anaerobic dehydrogaaation. SO VII Effect of varying the concentration of n*butyl urea on oxygon consumption rats 88 Till Influent® of dlmltrophenol on tissue treated and untreated with it-fentyl urea 86 IX Addition of p^phenylen* diamine to brain slices with and without n«butyl urea 88 I Addition of succinate to tissue slloss treated vith n~butyl urea 31 II The sffsot of glucose on the oxidation of suc­ cinic said in the presence of n-butyl urea 38 XII Iff®si of glucose on succinic oxidation in the absesos of n-butyl urea 33 ..XIII .The iuflug&o* of carbohydrates and derivative® oh the oxidation of aueoial® acid 38 XIV Effect of elapsed time between the additions of glucose and succinate on the oxidation of the latter 40 XV tfttooluls -acid oxidation a® influenced by- the addition of glucose subsequent to that of the substrate and n*butyl urea 41 xn Succinic acid oxidation as influenced by the addition of ti^butyl urea subsequent to that of the substrate "ml glucose 42 The influence of glucose on succinic cell m i oxidation of brain brel 43 Methylene blue reduction time of succinic acid m u a® influenced by glucose 45 XXX The effect of glucose on the oxidation of jKpheaylene 4lamina 45 XX Influence of calcium an! pH of media. on eueclaate inhibition by glucose 4@ XXI The effect of eaXelum ©a euoolalc noil oxi* dotlea In the absence of n*butyl urea 51 XXXI The effeot of Him lent an! trlvalent ion® on succinate laglbition by glucose 52 XXXII The Influence of calcium on the inhibition of succinic acid by glucose in various media 53 JOCIT Lactic acid production and resultant pH of respiratory media 55 w i m m B Mo. mg® The effect of an homologous series of urea 1 derivatives on Xo*> 14 The effect of adlel urea derivatives on a methylene blue reduction time 21 The Influence of varying the concentration of 3 n-butyl urea on oxygen consumption 23 4 The influence of varying the concentrations of glucose on the oxidation of succinic acid 35 I mrnomctim 9Mlsi A study of the inf luma®, of various derivatives of urea on oellular metabolism h*e been undertaken with m twofold purpos® s 1* to aampere the ram®tIona of various normal and tu n e r lis iM s * E~ to determine the mod® and alto of tot ion of these compounds« A discussion of the history and voluminous literature of Biological oxidations la f r beyond tha seope of this paper. Extensive treatment of this 'subject la presented la tli© review* by 01 xoa 11938)» Oppenhelmsr and iSbajpt (1939}f 3xent.«’&yorgyl ! 1930) f Elvehjem r>nd Wilson , and .1111 ott (1941)9 and many other®. it will thus s^flac to outline a few of tha systems examined la tha a oofs a of study • 1. Bcanlretioa. True respiration h--a its 'site in tha ©all and is concerned with tha oxidation of foodstaffs with the .V;:acfniripllad xalaasa of energy* Cellular. mmynm /..activate'. tha ultimate union of tha hydrogen of the, stbst state wiihgeatygcn. It Is from this combination of hydrogen and oxygen that -the vital energy of life is drewn. 2H 4- |0 g = HgO + E nergy Tha liberated energy may be employed a imply as a *cures of % fee-fit or a a the driving foree In one of the a inter one body f fume 11 o " s, The foregoing eosab inatlas ia a result of many sseondary relations dealing mainly with the release 'ad transportation of hydrogen of the foodetuff to oxygen * Typleal examples of the types of mnzymrn system® involved will fee deeerlbed. *• Pehydro&eaaeo* Hydrogen oeeurrlag ae & constituent of foodstuff® Is relatively inert »t body temperatnre« In order that hydrogen may fee released* p^rtle.lpation of a de- hydrogenating eazyaa or dehydrogenase la essential* Tfeeee substances are known to fee protein in nature feet their eacaot mode of *eetiVatina* hydrogen for release ia as yet unsolved* The known dehydrogenases are specific ss re^'-rde their substrates, a.g*, laetie aoid, sueeiaio sold, glnaoae=de- hydro?emse*9 feut set in cod aeration v.lth eert in other non- specifIn oellular enzymes* ?*■•' "Coen%yaiea« It we® early discovered In the; «•*«'# of most physiological substrates or foodstuffs tfe : t the specific dehydrogenase required the activity of a second enzyme in removing hydrogen from the substrate* The latter substance wee termed e eo-defeydrogeaaee, or more commonly, a eo-enzyme and ia re©exalted In two forma, aoentym.es 1 tad II, Chem­ ically, I is a di-phoephopyrldiaa nucleotide, and II is a tri-phopbopyridiae nucleotide* 3pon hydrolysis* Coenzyme 1 yields phosphoric acid, rifeoae, adenine, and nicotinic acid amide* '*heA, for eifiple, leetie aoid is added to a mixture 5 of its dehydrogenase and Oeensyiae lf pyruvic acid ia pro* dueed and the nicotinic act# amide of the eoenxya* la re- due@<9: by addition of hydrogen* tactic 2 lactic acid + Coen^yme ------------— > dehydrogenase I Pyruvic acid + Comnzfmm•BH 4* fellow Siwmaa* The d*hydregen--'tioia or oxidation of the •w>ww*««Jv^rs*m6f!»ww ^tiv^wM^TWWw1 ■ fHwuw * reduced ooenzysse is effect®# through the mediation of mr<* burg#s Yellow Snsyme* This substanae9 flavoprotein9 consists of m high moleemlsr weight protein and a prosthetic group* The latter9 called riboflavin* Is composed of phosphoric acid, rthos* and lso^alloxezine* The 2 atoms of hydrogen arc transferred from the nicotinic acid amide of reduced Coeazyme X to the ico~allox*slae ring of the flavoprot«iafa prosthetic group* In this manner reduced OocsEymc 1 is re~ oxidized ^-ttd the flavoprotetn reduced.* Ooenzyme•IH + Vlavoprofceia ----- > doemaym© +■ flsvoprotalii* ZB 5. four (forboo foqexftoxrUo acids* mother "hydrogen trans­ port^ system Interposed between tbs primary foodstuff and oxygen is the succinic •>©!# scries of SgeatoOyorgyl (1939). Hegleoting moMnt&rlly the enzymes involved, the following is an outline of the reactions which occur when labile hydrogen is added to oxaloacetic and fuaarlo acids* Oxclo* acetic acid is reduced to malic by the addition of two atoms of hydrogen* These -atoms are then transferred to fumsric -old which becomes sueaInto on hydrogenation* 4 By virtu® of defeydrogenationf malic *soid reverts to the original oxaloacetic m id* COOH 000!! I I HQfl SOI zb i m i m Done tor * 00' > H001 --- I * ------- I oooh omu Oxaloacetic Mail© 00011 COOH I I OH BOH | PII | OB' — BOH > &n I 4— I 0003 000.13 Fusaart© lueolai© «» the system© described have s©n#«ra#a with lb) *aotivati<mf'l (b) r«§wl, tail, C o} transport of substrate hydrogen, th« final stag© ia Sydregen tmbsfer la effected specifically in mltml tissues by succinic dehydrogenase * This enzyme removes H atoms of hydrogen from succinic ©old, thus oxidizing it to fuaarie; and the hydrogen passes to dytoohram© which Is thereby re­ duced. Cytochrome is a beautin containing protein-which functions as the specific lin k between the hydrogen transport system® and oxygen, iron in oxidized oytooh yom© :.is; In the ferric condition nxid is readily reduced by electrons from added hydrogen to th© ferrous state, An eazym®, cytochrome oxidase* natnungsferment5* of Marburg, lndophenol oxlia«©f ©t©,t re-