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1952 Biotin and Oleic Acid in the Nutrition and Metabolism of Certain Microorganisms. Emilie Anne Andrews Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Andrews, Emilie Anne, "Biotin and Oleic Acid in the Nutrition and Metabolism of Certain Microorganisms." (1952). LSU Historical Dissertations and Theses. 8018. https://digitalcommons.lsu.edu/gradschool_disstheses/8018

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IN tm NUTRITION AND METABOLISM OF CERTAIN KICBGGRGANISMS

A Thesis

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doetor of Philosophy in The Department of Agricultural Chemistry and Biochemistry

by Erailie Anne Andrews B. S., Southeastern Louisiana College, 1946 M* S#, Louisiana State University, 1949 February, 1952 UMI Number: DP69396

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Unpublished theses submitted for the masterfs and doctor!s degrees and deposited in the Louisiana State University Library are available for inspection. Use of any thesis is limited by the rights of the author. Bibliographical references my be noted* but passages may not be copied unless the author has given permission. Credit must be given in subsequent -written or published work.

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LOUISIANA. STATE UNIVERSITY LIBRARY m & m L zm m n

The author wishes to express her sppreeietlon to iir* Virginia lu ftliii&m for her $m&r ou» share of the mrk included In th is rnlnm md for her consistent eaeoeregaaeat in the deeelogfiaent of the problem*' «’*% John f . Christaan has ale© isindly eootrfkmted vaiusfeke direction sodl •xp^rinektsl eeaist&ae«« the aether wishes to thank or. t. A* H %#r for hie constaat interest throughout this writ*

i i

355410 TABLE OF CONTENTS

CHAPTER FACE I« INTRODUCTION AND SELECTED LITERATURE SURVEY...... X I I. EXPESIMBNTAL METHODS...... 22 A* Materials used...... •••••* ...... •...... 22 B. Microbiological assays for biotin or biotia substitutes, *. * ...... 23

G. Isolation of hexokinase...... 30

0. Ensyme assays...... 3 2

£. Glucose determinations ...... 33

F. Removal of interferences in glucose analysis...... 35

XIX. EXPERIMENTS AND DISCUSSION OF R E S U L T S ...... 3? A* The turbidiaetric-titrimetric disparity in oleic

acid stimulation of Lactobacillus Casei ...... 37 B. Oleic acid in the biotin metabolism of

Saccharomyces Cerevisiae. 49

C* A linkage for biotin in carbohydrate metabolism. 56

IV. SUMMARY...... 66 SELECTED BIBLIOGRAPHY ...... 69

VITA...... 76

i l l LIST o r TABLES

TAM PAGE I . Composition of a Basal Medium for Lactobacillus Cased* 25

XI. An A ssa y Medium for Biotin Being Saccharoasyces ...... 29 I I I . Turbidimetric-Titrimetrie Ratios Obtained with Various Concentrations of Oleic Acid, Oleic Acid Flue Albumin,

and Nopaicol 6- 0 . 4 2 * * - XV. Effect of pH on Turbidimetric-Titrimetrie Eatios Obtained with Oleic Acid and wopalcol 6*0 ...... 44 V. Effect of Glucose Concentration on Turbidim etric-Titri- metric Eatios from Tubes Containing 1,000 Mieromicro- grams of Biotin and Bo Oleic Acid* ...... 44 VI. Ratios of Turbidimetric to Titrimetric Assay Values Obtained with L. Arabinosus...... 46 VII. Utilisation of Various Source® of Carbohydrate by S. Cererislae Java Grown Upon Basal Media Containing 1 Ml.

Egg White Per 100 Ml...... 57 VIII* Biotin Content of Cells, Supernatant, and Uninoculated Medium for L. Casei Grown on a Biotin-Deficient Medium*.* 57

if U ST OF FIOURfiS

FIGUHE PAGE 1. tjrpie#X StMdMd Cfjrwto RwpwiB. of JUetoto.lU*# egstik 24

2. Typical Standard Growth Response of Saccharoamses IgMdllB...... 26 3* Apparent Biotin Content toy Titrim etric and Turtoidlmetrlc Methods with Varying Oleic Acid Concentrations.*10 4* Cooperative Growth Stimulation of £» cerevisiae Java by Biotin and by Oleic Acid on Basel Media to Which the Indicated Additions Were Rads,...... $2

5* Growth of Saceharomyces cerevlglae Java on Biotin* Deficient Media in the Presence and Absence of Igg White. • • 53 6* Synthesis of Biotin by Celle of jl* eereviflae Java Grown on Biotin~Deficieut Medium# Assays Performed

by L. easel and ft* fragilis as shorn.. * ...... 54

7 k 6. The Relative Stimulatory Affects of Biotin and Biotin plus Adenylic Acid Upon Glucose U tilisation by k* oasel Cells Grown on low Glucose Biotir^Beficient

M edia...... * ...... 42 9# Relative Mates of Glucose Utilisation by Cells of ft.

cerevigiae 139 Grown on High* and iow-Biotin M edia#.#...,.. 63

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three hitherto unexplored phase® of the relationships of biotln and oleic aeid to the metabolism of certain microorganlems have been studied# These phases ares (1) the anomaly between titrim etric and turbidimetric microbiological assays using media containing oleic aeid; ( 2) the phenomenon of oleic acid stimulation of yeast in biotia-free systems; and ( 3) the relationship of biotln to carbohydrate metabolism in microorganism©. The various functions of biotin in cellular metabolism have beer, the source of much interest and quite intensive research for the last ten years. Various workers have shown this vitamin to be necessary for the function of the deaminases of aspartic acid, threonine, and serine, and the decarboxylases of oxalacetic and succinic acids; to function in various tissue decarboxylations and to be related in some manner to oleic acid# The methods used in phase® (1) and (2) were largely those of microbiological assay, utilising various strain® of yeast or Lacto­ bacilli. Phase (3) involved the us© of resting cell suspensions and cell-free extract®. The extent of reaction was measured In a ll cases by the disappearance of glucose# Several methods of glucose analysis were employed# The turbldimetric-titrimetric disparity which exists in Lactobacillus case! assays of oleic acid was examined under various

v i condition*! (a) varying the concent ration of oleic acid* (b) vary­ ing the length of incubation time, (c) including serum albumin or supplying oleic acid in an ©sterified fora* (d) making various substitutions inohd additions to the medium, (e) varying the pH of the medium, (f) varying the concentration of glucose per tube, and (g) comparing the effect observed tilth |»* arablnoaus. Under most of the conditions tested the apparent biotln contents as measured by turbidities were approximately twice the values as measured fey titration, the phenomenon showed some pH sensitivity, the turbidimetric titrim etric ratios increasing as the pH was raised to 7*0 because of the probable increased toxicity of oleic acid with decreasing acidity. An Increase of turbidities as compared with acidities was obtained upon lowering the glucose content of the basal medium in the presence of biotin, not oleic acid. Only the substitution of a high molecular weight ester of oleic acid for the latter compound in the assay medium completely eliminated the anomaly. On the basis of these findings several explanations were offered for the disparity. Upon consideration of the current theories of surface adsorption andltpo-protein effects, it seems most likely that oleic acid is adsorbed upon the bacterial cell membrane and by means of steric effects prevents the utilization of the jtettHmmn amount of glucose by the bacterial c e ll• That the phenomenon of oleic acid stimulation of microorganisms in biotin-free systems is applicable to yeast was demonstrated with

v ti Saccharomyces cereviaiae Java* Oleio acid was found to b© capable of stimulating growth of the organism on a biotin-deficient sucrose medium in the presence of aspartic acid* Synthesis of biotln by cells of the same organism grown in a medium deficient In biotln was shown to occur* Cells of 4* easel grown on a biotin deficient medium were also found to contain low concentrations of biotln or its nutritional equivalent* Growth of £• cereviaiae Java did not occur in the absence of biotln if glucose! hydrolysed sucrose, or fructose were substituted for sucrose as a carbohydrate source* From the latter observation the third line of research was developed t the relationship of bio tin to carbohydrate metabolism* Stimulation of glucose utilisation by cells of S. cerevtsiae 139 grown on media containing 2 mierograms of biotln per lit e r was found to be considerably higher than that of cells grown in the presence of 2 x 10 «*2 mierograms of biotln per liter* A linkage of biotin to hexokinase function was postulated, but could not be demonstrated with cell-free enzyme preparations * A continuation of the biotin studies described above should be made on hexokinase resolution* If the enzyme can be resolved by dialysis or the addition of biotin-binding reagents so that its biotin relationship becomes demonstrable, a further contribution will be made to existing knowledge of vitamin function in metabolic pathways*

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For way investigators, biochemistry has become the study ©f ©/©lie processes. These eiM&ymtieaXlyMsetaXyaed cycles, interwoven fey d irest or indirect link ago* when viewed as a whole become the dyna&ie framework of life# individual reactions of each system are enzymatically governed and are constantly in a atate of equilibrium with other related reactions* 3ince m individual efsype is capable of catalysing a series of reactions fey itself > it beeomes necessary to study snsymatie reactions both individually and ©elleotively as a means of understanding the processes of life« la reseat years it has beeetes quite clear that we say Justify ourselves ia detailed study of the metabolic schemes and aeohanisiss of microorganisms through tne concept of ©pmparativ© bloc heads try . This concept, introduced fey fcluyver £l), intimates that the fuada~ mental easyaatie reactions ©f all cells are the ea m regardless of cell origin. Again and again metabolic reaetions which have been demonstrated to oeeur in certain microorganisms have la te r been shown to be applicable, with slight modifications in swe eases, to higher plants and animals# This approach to the study of metabolism i& by far the easiest Since the single cell organism presents a living system conveniently simple for study, as well as being rapidly

1 2 and inexpensive to maintain* Originally the interests of the bacteriologist lay simply in the cultivation of organisms, usually pathogens, for the purpose of a tody, The actual growth requirements were cosisldered to be of little importance, though it was frequently quite difficult to maintain the bacteria on the crude media available* Until the advantage of chemically defined media became more ©bvioua, the media were usually changed mere or less empirically to make them suitable for the maintenance of growth. Turning to the use of chemically defined media required that a careful study of the nutritional requirements of microorganisms fee made. As the nutritional requirements of more microorganisms were determined i t began to fee obvious that a eomple- mentary interrelation must codLst between the nutritional requirements of a particular microorganism and Its individual synthetic abilities* With the passage of time then, a more general concept ©f nutrition versus metabolism has developed* This Is best conveyed by quoting directly from a review fey &• C. J* G. Knight (2}V

When the nutritional question is viewed from the stand* point of metabolism, many of the apparently difficult questions of definltioa and nomenclature disappear* The substances which an organism takes from it s nutrients are used as material for building up the new cells* These cello daisy but a eos$>leoc Interwoven series of processes, which is the life of those cells, and consists in taking compounds from the environment and synthesising other compounds to make new cells* The cxtent and rate of railtiplieation of now cells will depend on the efficiency with which the processes of construction are carried out* This efficiency (here 3 used in the general and net only the thdiwodynamie sense) will dearly depend partly cm the availability of the materials for construction#' This 'wSXX in tan* depend upon the rates of u tilisa tio n and synthesis of the various materials of the easyme systems whose continued funetiouiBg is the life of the cells* The fundamental biochemical processes of adl~life*~*the essential metabolism of the ceils—form the cardinal feature* end certain of these processes are common to the widest variety of cell* Where organises may d iffe r, however* le in the mean* whereby the amterlale for these processes are acquired» hut here a sharp metaphysical distinction Into "acquired from the environment" end "synthesised by the cell" Is not possible* for it ie clear that a certain rate of synthesis night be too dew to yield a required sub­ stance at the required rate# Effectively then the cell would depend upon an external source of supply to a degree which would be relative to the rate of synthesis of this substance# Hence a given substance* required as a component of one of the essential metabolic processes* might appear in three different roles as a component of the nutrients* It might appears (1) as ass * essential 9 nutrient, when its rate of synthesis by the cell was so slew as to be insignificant; ( 2) as a growth stimulant* when its rate of synthesis was somewhat faster but still alow enough to be a limiting factor; or (3) as a substance not required at all for nutrition, because the cell could synthesise it so fast that it was not a limiting factor in growth* It Is the metabolic process which is the essential thing and the compounds used in eariying it out are essential metabolites* i»e** the substrates used for the process* or the substances which form parts (prosthetic groups* etc*j of the enayme systems which carry out these essential reactions#****** What matters Is to show how any given substance which affects growth plays its part# And very often It will be found that i t has a relation to seme essential metabolic processes, the role it plays in nutrition reflecting the mode fey which the cell acquired a sufficient quantity of it at a sufficient rate*

As the development of bacterial physiology has proceeded from the use of mixed cultures of organisms and unidentified media to the utilisation of pure cultures and chemically defined media* it has been 4 possible to identify many metabolic products and to create easily reproducible schemes of assay for essential vitamin and amino aeid components of the basal medium, the use of these more or less routine procedures has led to many important observations in bacterial metabolism. from investigations which used proliferating microorganisms to determine metabolic products, bacteriologists turned to the use of incubated washed cell suspensions* These ‘resting 1 sells served as the source of active ensyme systems, stable over a period of hours, which were uncomplicated by the demands of reproduction. This technique was introduced by Quests! and tohetham ( 3) and, In combination with other standard biochemical methods, has been extensively used* The chief limitations of the technique are the impermeability of the cell m il to certain substrates and the interfering action of other ensymss which may be present. Within the last fifteen years It has become possible to define the properties of the individual ensyste through the use of cell-free extracts. The wet-crushing m ill was developed by Booth and Green In 193$ (4) • Cell-free extracts may also be obtained by lysis of cells with toluene or acetone, by crushing them with ground glass, or by alternately freusing mod thawing them. Ultrasonic waves are sometimes applied to produce cellular disintegration. Bo method is applicable to all organisms. Certain easymatic systems cannot be isolated under say of these conditions since their action apparently Is linked in some maimer to the intact cellular structure* It is necessary for the «\ I 1 1 1 I

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15$ p?*» •VVMdtai 9ir?*t®*ttT »*M» *«artxo«l»%*w J» »ft*fti(rf *«artxo«l»%*w »*M» 9ir?*t®*ttT p?*» •VVMdtai ti I I f t «a*wn •<«% «? «l MRf* «a*wn •<«% P»ATOA«t *«W PAAftTWl i V § 8 resolution which they designated as "aging" consisted of treating the washed tells with molar phosphate buffer for a short period of time* Llehstein and Christman (16), having demonstrated the occurence of "aging" in cells of several types, concluded that not only biotln but also adenylic aeid was involved in aspartic acid deamination* Al~ though many biological materials would cause stimulation of aged cell preparations at pH 7, at pH 4 the effect was specific for biotln and adenylic acid, the stimulation being independent and sometimes additive. Llehstein (1?) made the observation that certain systems, both "aged" and cell-free, which could be stimulated before refrigeration by biotln and adenylic acid together or by yeast extract alone, after refrigeration responded only to the yeast extract* He was led to believe that a preformed biotin-containing coenssym® for aspartic acid deaminase eclated in yeast extract and that adenylic acid was concerned in its formation. Further evidence for the existence of such a coenzyme was presented by Llehstein and Christman (18). By paper strip chromato­ graphy they were able to isolate a substance, or substances, from yeast extract which activated the deaminases of aspartic acid, threonine, and serine* the material was shown to be neither biotln nor adenylic add as such.

bright, (19) was able to duplicate the "aging" process in Escherichia coli of Lichstein and Itobreit (15) and to reactivate the aspartic deaminase system by the addition of biotln specifically* 9

Pilgrim* Axelrod* and Uvefeje® (20) ia 1942 observed that liv e r homogeaate from bietln~defieient rats had a greatly decreased rate of pyruvate oxidation compared with controls* That bietia ia concerned la oxalaeetie acid decarboxylase was demonstrated ia four separate l&boratori©® by four different tech­ niques almost simultaneously* lardy, j^** ( 21) using a medium defleiaat ia both Motin and aspartic aeid demonstrated that tbs growth of L* arabiaosue could be stimulated by the addition of osaiaeefcie aeid or bicarbonate lea* Shiv* sad Rogers (22) obtained results indicating that biotln funct ions for £* coli in the earboxylation of pyruvic is eaalacetle aeid by mesas of inhibition analysis* Similar data for such a Metis function for yeast collected ia the same laboratory by Garrison sad Babies has sever been published* lbs resting sell approach was used by iiehstein and Umbreit 121) • The se lls were grows os complex. Matin-containing medium* harvested* sad “aged" ia molar phosphate buffer* After “aging’1, the cells lost their ability to produce CQg* sad restoration of activity occurred apes the addition of bio tin* the actios of biotln was specific* It was found possible to substitute oxalaeetie or malic aeid for aspartic acid* The conclusion was dram that biotin must be linked in seme manner to the eoensy^e of oxalacetic acid decarboxylase* Ochoa, et al«f (24) concluded that biotln is concerned in the action of ox&i&cstie aeid decarboxylase either as a structural exponent S I S. 1 A 43 •H i 1 1 3 2 •Hi 49 3 1 * 2 8 a 2 33 « I 3 8 ^ 5 4* 1 I i | I s 1 3 I 1« %* ^ * 1 89 3 § 1 £ ° 40 I I 2 I ! 8 .*» I 8 *» I -S3 89 4 § * i tt 4 4* £• « s i 3 t S. 3 S 4» 11 2 8 4 I +»S 40© Ih 4 » 4 4 sj I * a 3 s sr 2 4 ! 3 a 1 8 1 i » 3 : a © i « ?» 2 * : 3 $ I <1 I 1 ! ! I 3 a 3 I i 0 g I o 1 « 1 «* i <9 Oc ■3 0 4 © ! < e I O a -P *S *\ £ 1 g ^ M *ri a 1 o t laI i S i £ % ■*» <9 1 I *-g • *5 1 *5 ® +» -0 TJ 1 9 i a i a T3 III’ i 4 a ? *» i !I 8 £ fire fire jj| aiidifcioo* vitro* of a be&teg rasitfcre of normal rat a I JB & ^ I " £ » I •HU i § 1 3I 43 9 1 1

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? i ves« that biotia i ia for aaea©***^ produsfcioa of fcfca tha usalio I u 1 9 *» * 4»^3 i $ I S fe 9. 33A i i $ i 1! c4 31 jS i 9 3! £ 'S 9 1 3 O o <*% frt § u9 2? 9 +» -*J 1 * st £ 8 jf I I « 9 t 3 « >* 3 I % r+ 4* * £ S 1 1

a s 1 1 strains of diphthorlao C« would not ^row in tho presence of S1 Hi 14 a l l known growth factors unless additional m aterials from serum, milk, or coiaaiercial casein mere added, One of the active materials was isolated, purified, and shown to he oleic acid* Oleic aeid in concentrations of 0*3 to 0.6 mg./lO ml, medium gave good growth in the presence of saponin for Ervainelothrix rhusiosathlae (39). If saponin were omitted, as much as 0.2 mg./lO ml. was inhibitory. Both the fungus Fityrosoorum ovale (40) and the anaerobic bacterium C. tetan i (41, 42) require oleic acid, the optimal concen­ tra tio n , however, varies from 100 mg./lU ml. medium with the former to 0.01 rag./lO ml. for the la tte r. Bauemfeind, et g l., (43) in 1942 first discovered the stimu­ lation by lipoidal substances which may occur in the course of assays for pantothenic aeid and riboflavin. In the same year Strong and Carpenter (44) discovered the effect of fatty acids on the growth of L. casei and worked out a method for eliminating this effect in ribo­ flavin assays. In 1945 I t was noted by Williams and Fieger (45) th at lipoidal stimulation of L. easel also occurs in microbiological assays for biotin. Tvey were unable to demonstrate eynthesie of biotln in tubes showing high acid and cell production in the presence of basal medium and rice oil without additions of pure biotin* In the same year Williams (46) noted a similar effect of lipoidal substances upon the L. arablnosus assay for biotin. I f Hedaoa U T Jrep o rted th at ©Xeie and aspartic acids would m% eeapietely replace bietla in biotln assay fey the choliaeless ipore frasga %mder the conditions used ia his laboratory. The addition, however, of oleic aeid and Tween 90 alone or la ee&btaaitoa with aspartic aeid predated * alight growth response la the absence of fciotin and sees stimulation ia Ita presence. Stadias of the phenomenon of oleic aeid stimulation of L* cssej were began by Williams and fieger (4®) la 1944* Their findings were as fellewst 1) The pH of the medium, Xsagth of iaoubation period, temperature, and concentration of eleic aeid added were found to affeet growth* Optimal Oeaditleas for growth were a t pH f«f, incubation period of ft to 190 hours, incubation temperature of fT0, and concentration of 400 mieregr&as of olele aeid per tube. 2) Tarbidiaetrlc assay showed a much higher biotin equivalence than did titr la s tr io meaeureaeate* 3) A number of fatty aeids were tested, of which iim oleie, la u rle , and myristde aside were found to be strongly inhibiting. Elaidle aeid, the treat Isomer of olele, was found to show greater stimulation than oleic acid* lb

4} Stimulation ia th® absence of one vitamin moo obtained only in the ease of biotin« Further studies of lipid stimulation of &• oasol by Willisa*® and Finger (49) lod to the foUewihg results*

1) |g« easel stimulation by lipids occurred whom essoin hydrelysats woo replaced h?r either ondoo acids, or peroxide treated ®c»ipQfi

Had 1 0 0 0 ffiicremierograas of biotin. 2) Of synthetic detergents exandned for stiau** la ter? off eat non-ionic detergents proved generally stimulatory* the most stiswlatoxy of all detergents tested were th» cleates*

3 ) Oxidatl*#t~reduotioa potentials detezttiaed on cultures containing standard bio tin, ©isle acid, or a non-ionic detergent corroborated acidiaetric and tiiriaetri© data*

4 ) It was postulated on the basis of experimental data that biotin functions as a sell persnea- t i l i t y faetor and can be replaced by the proper lipids*

Kodieek and bordea ( 50) demonstrated the inhibitory action of oleie, lin o ie le , and linolenie aeids upon the growth of L. helvetieus and several other gram positive bacteria. The inhibition was reversed by eertaln surf see active agents* It was suggested that 17 evidence pointed to a phyeicochemical explanation for such behavior* In an extension of this work (51), the depression of acid production by certain uasaturaied fatty acids mu shown to increase with im~ saturation* It could be reversed toy other surface-active agents which were capable of forming molecular associations with the acids. A physicochemical mechanism for the variable effect of various acids on different microorganisms was farther elaborated upon* Williams, Broquist, and Snell (52) studied the phenomenon of dele acid stimulation in connection with various Lactobacilli. They learned thati 1) Oleic, linoleie, or a combined source of these materials was required by several of the strain s. The even-numbered, saturated fatty acid® Cg to C-^g were completely in­ active* 2) For i* bulgaricus oleic acid, although essential, was exceptionally toxic. The addition of certain inactive, water soluble esters of oleic add served as an excellent non-toxic source, 3) Although most of the lactic organisms do not require oleic acid on a complete medium, i t becomes essential in the absence of biotin, 4) Avidia does not nullify the action of oleic acid for the organisms tested. € I 1 & I £ I i sol I r -i © e ©si © •H 9 % v 4 -rS S © 0 * ? col <0 $« g 'S o l 4>1 4-1 -al 3o 2 g. © +9 I O *A E 1 I 2 U sp 3 t\ r 5

i the ehsndeal netur* of the fat-solubia substances with i i ■ g i Ii a St

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i s than the ei» fo m the usual caeei sodium was found ta b * unproductive for ft strain of e«eal kaciofaatoilli isolated from rats fed a hi$ily jwp£f£«NS diet * Uleie acid piov«A to be necessary* $ho organisms would

»ot grow1 a the &ba«nce of cleats when a ll viiasdaa wore supplied*

Guir&rd, Snell, and Wiliiaaa (64) acted the offset of acoiate

at a atimLaai for early growth la lastie acid bacteria# A probable

role for acetate la the syathoolo of various lipid® was suggested V

the a b ility of numerous compounds of title class to duplicate la seme

degree tito of foot of acetate*

fcponty^eigbt strains of Lactobacilli previously reported not

to grow in nodi* of known eowposlUon studied by Sibfty and

Snell (65)* All of these osaopt two repaired ololo acid or other

uftsetttxated fofcty aside for growth*

Broqoist and Snell (46) noted that elolo9 liB cleie, and

liaolonie acids when detoxified wore equally effective in promoting

growth of £• butyrleua and £* faeealis# Saturated fatty aside wore

shown to maintain the name synergism toward unsaturated fa tty acids

when the la tte r had been detoxified •

the offset of positional* and atetvivomrim, on the biotic

lik e activity of octadeeenoie soldo was studied by Oreonburg, j>£ aj.#t {67 j, using arahinoous* Wide variations in activity of the trans

foias were obtained though only minor differences occurred among the

ois forme.

Carlson* Whiteside-Carlson, and ftoepetos ( 68 ) studied the ability of various fatty acids to substitute for hiotln In the 2 1

growth-produeiion of Ieuconoatoc> With all strains tested oleic and

lauric acids in the form of their ester®, Tween 80 and 20 respectively, produced a higher level of growth in sucrose medium than that obtained with biotin. Half-maximal to maximal growth was obtained for these

strains when the esters were substituted for biotin in glucose and

fructose media. The stearate and palmitate esters were less active

than oleate and la urate. In sucrose media their ability to sub-*

stitute for biotic paralleled the dextran-synthesiaing capacities of

the organism, They could not substitute for biotin in media containing

glucose and fructose.

Thus i t becomes c le a r th a t o leic acid has been demonstrated to be an essential growth factor for various microorganisms, It

possesses a definite interrelationship with biotin and the hypothesis

has beam advanced that biotin functions in the synthesis of oleic

acid. The inhibitory action of the acid at higher concentrations is

believed to be either a atari® or lipoprotein effect. chaptjsk 11

KXPmMMTAL METHODS

A* Materials Uo#d«

1* Microorganisms.

The organises used were Lactobacillus casei 7469, Laeto* bacillus arablaoaua 17-5, Sageharomyoea cerevisiae Java 4125, and

Saccharoayces fragills 8644, all obtained from the American Type

Culture Collection, and Saccharoiaycea cerevisiae 139 obtained from

Hoffaan-Lakoche• For the isolation of hexokin&se, compressed brewer's y east was obtained from Anheuser Busch Incorporated .

2* Natural Products«

O leic acid, obtained from the Hormel Foundation, had an iodine value of 90.83, as compared with the theoretical of 89.87» The adenosine triphosphate (di-barim salt) bought from either Nutritional

Biochemicals Corporation or Sigma Biochemical® was claimed to be 90 per cent ATP. Vitamin-free casein hydrolysate was obtained from

Nutritional Biochemicals Corporation.

The adenylic acid (adenosine-5~pho sphoric acid) was kindly furnished by the Ernst Bisohoff Company.

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I I

? i . 3 3 3 •3 I * preafcur*) Alter £ 1 1 pmmxto

i sterile sterile vat®r fimt, ©1 titia 0.1 ial« me^l was* l * at 121° (15 jBl*a£bc» CAL SANDARD GRWT RESPONSE E S N O P S E R TH ROW G D R A D N STA L A IC P Y T

ML. O.i N ALKALI OCNRTO O BOI I MICROCRAMS IN BIOTIN OF CONCENTRATION O F L A C T O B A C IL L U S C A S E I E S A C S U L IL C A B O T C A L F O 200 400 0 lRMTl VALUES TlTRlMETRlC 0 O IUE I FIGURE UB IERC VALUES TURBI DIMETRIC 600 600 1000 0 u 40 30 60 0 < 50 70 60 90 too 25 TitfHuB I c o m m or a mm. tm m for c ms.

Cae«ia, h^rely»«5(100 a&8 ******* XQ*Q $« jB**Glt50©### 0B&yd8,0ti||*«**«##«&«»««*&«*#»«*#* ft*#*##a♦ **.« «»**#»40 #0 $» Satina a##4ai* trife^rat©«•ft***************#«***#«#«#•«*«««* 66*0 $*

L - C y s t i a * * 9 ««*•»«« «#*##« a «ft ftft ft a » »»## $440-4 » ft#a *«aft» o a a s a a #» * ft 400

J^TrgpUt&hein*******0« 0**0* *****000 *000009 00**0*00 200 qfr .400 ntga

c&i*yarog«i0 »a«»««a #.*.«* «*»«#«« <«««*»«*IftO a«« fa f’ti&MSitta fefdlDU® jpil§0j|il0&0# « «.# # # .# ?#.«## a «## * # * # $ * # » a#1*0 **•##* g* tim&iestom hmgW&temgm*«»**#«»*******®»«»#**«»*«.«a*# 400 mg* Satina cbloild^ftftftftft.ftft ft ft ft ft # ft# ft * ft ft ft ft ft ft ft * 0 ft-ft * # ft ft ft ft 4 ft ft # «• ft-ft ft ft ft # 20 mg* Ferrous suUfafee ftajiitai^dgattOft •««#«*«* # #* $*###*### ##■■*# * * * ####20 mg* tSaftgBROt StiUfofe# 20 sag,

M m im *ulf&t«e ft ft* ft ft ft*# ft® ## **#« ft# ft ftftftft#ft#ftft##ft.ft ft#*#####ftft20 ft# lag*

Guanine J$ydL$0$^orl40#ft«******«#* * #**a#«#*#* # *«#•«##.«#*.###»#20 mg* #**##« » ft ft ft ft a #« * ft * ft ft ft# ft# » ft «#«»*• #4# ft# «#.«###*#*20 m* a «-» ft u ra c il a« # ## ## «• •■« • *#« a «#«a«#*»a# «# a * •«? ## a # # •#«## «eft«o» #2® a«» m* a

7hl& X iA & ]9SFCijP0ehlOrl

0 a l « l u s f t t f t t o t f e f t E i a t t t f t ft « « » a « « # # ft » a » » * ft ft# .» * a a a # a « « #mm ft « * jig * lito tin io 0014## e »# * a ## a#«# <»*•«»*»♦&«*• a*ftft*»#«ftft#Bftft*ftftft»»*2000 J*g o

•#*'•*«»**•*»♦«»»* •»««#**•*•*«««#*«•2000 jug* PjrUoxkn* kftiroeklwrid***#* *#**■#*«*«* **»*»*** ******* ******* MtiMjug* 2 6 TAILS I ■ (Continued) w w m n o u of a basal kkdigm rot uctqbacillus^asei

para-An&no bensoio a c i d ..,,...... ZOOj/g»

Folic acid...... m o ...... SOjut* \ Water* ...... *to 1000 ml*

inoculated and incubated at 37° for 48 to 96 hours. Upon completion of the incubation period the mixtures were diluted with 10 ml* of distilled water and equal volumes of each solution were taken for analysis by titration and turbidimetry# titrations to pH 7 against

0.1 N. alkali were made with a Beckman pH meter* Turbidities were determined h? reading against a distilled water blank in a Beckman spectrophotometer a t wave length 6000 f* and s l i t width 0.3 mm*

When only the acidimetrie values were desired titration© were made on the undiluted samples.

2. Saccharomvces fraglljs. The following assay procedure is based on that of Snell, et al.

(70). Stock cultures were carried on agar slants (1 per cent glucose,

1 per cent yeast extract, 2 per cent agar, pH 5.0) and subcultured in tubes containing approximately $ ml. of glucose medium (1 per cent yeast extract, 1 per cent trypton®, 0.5 per cent monopotassium phos­ phate, and 1 per cent glucose) or of a similar medium to which one m irtp tit #*#*&• j* r MUt M fc#«&m&*& In Ut* «f Us# glue©##* After U iw « tatuMA#* «t Jfl!% mAmMmmin* m m &&%*& tkr##% Sm m n it# 1# *&» «ff 4 1 # tU ie i *mfc»r *n#m m & m sto 4 , In « #p#$. *#*««# #f v#t#r* ia&# Wit& «f * i&UAiit#*« l» w l(iM dilution *f tn# •#»¥* *mpm*lm wm miiim$Lm% tar ife# im m fa M m of ««ftfe Jj&? &U «ff latteas* fiie- ®m>*

©eatratimm m*A wmm 0 * Ilf* , Uf***9 i # " \ m T ^ $ 1%*^# m& l$T® #f MeUn $er t#i*« a «itii&*jr*l #*ar## 1#

£#* #M##*4ti.«& #1 t&# fa##* s&##As» i t | i w In f#H # £1* for ©isiinai gtevtb* #i£m#$9Mt to §&$ mm9*$ f m m iirt ®£ mAizm mm #t#*ilA*H®A. m i %km iMtatoMU fli# selnttaMl in m t*#t#d m i %m wbm&mi $&#f£n mMAmimmm plntt4 In %fwm k f t tm bm i l l & i>0 m + i mmI Mi# fc© # 1m#1 «b3m# #ff 2 #&* ft# t%fe*» «n» *#£ ®t®ryj.s©4 for fift*** $4.m.%m #1 i2l0« $## I*r#wU##X/ intnsdftlni w « «$&## im I ni* It- e ta i tot# an* Hi# tvtom mm k&mh*nM *t jflP* M m Mill f n U t i # #f grq#in in### i# appear in ito# list## no M#tl% i&# ln#n» Mil## «## #t#rp«i* After iaMMtattafc it# Ante#* «w# ##H w*«k*Kk t» Mipis# #11 tn# «#LUf ##i to* t^rM iiti*# «#r# reti m etast & tiit&iXMm % m b im k i# t#»& m m m %% ##v# im'Aglte &w& I* # u t#141^ &»i #n« 28 too

90

70

50

ui

30

20

-6 -5 -4 -3 -2 0 LOG CONCENTRATION OF BIOTIN IN MICROGRAMS

FIGURE 2

TYPICAL STANDARD GROWTH RESPONSE OF SACCHAROMYCES FRAGILIS * * * * * * ♦ $ m m m n & * * £ f £ £ t £ O***** €> O -4 r- es «n €4 ^ o ct o o o * * * * "i * o o O o o

» * * * * « * * * * * *

a

1& ■&sa

* I

I ttt 3

acid*.* 0*0 mg 0*0 acid*.* 1to ! o ■2 li ! Ill Boric 30 TABLE II (Continued) ASSAY MBOItM FOE BIQTO b$XM

Pyridoxins hydrochloride* 400>g. Riboflavin* ...... 200 >«-g. Thiamine hydrochloride ...... 400j+g. Water*****.********.*...... to 1000 ml*

C* Isolation o£ h^okinase*

Hexokinase was isolated according to the general procedure of Berger, eb §1* (71), although purfication was not carried to the final point ©f crystallisation. In detail the method used was as follows s

Five pounds of brewer 1 s yeast were warmed to 37® over a two hour period* One hundred and sixty m illilite rs of toluene were well mixed with the cells, and the mixture was permitted to liquefy p artially at 37° for forty-five minutes* At the end of this period,

104 ml* of 50 per cent glucose were added and the mixture stirred for an additional forty minutes* Then 2*3 kg* of crushed ice and 52 ml* of 50 per cent glucose were added* The mixture stood for IB hours at 6 * The suspension was then centrifuged in 250 ml* bottles at 3000 rpm. A second centrifugation was generally necessary to produce a sufficiently clear supernatant. The supernatant stood overnight at 0 to 6 °* 3 1 .•w„r foe ja^tain of iae by the m w m k ’ s©X»»* p i ff pe^oaut aleohol* fturlng ibis addition tto* b * w m RlgMft jo * n& m lafowed.fo *$rn *%&** f * After standing fo r mm hour* tom suspension m * %}m ®°Xd* ^ resulting precipitate m * suspended la 480 aX% of X per dint glucoae stirred •ViMgft.M-tte «eld* ■ i l l ffMstainiiaE auanendod om tain. •*«■»« seat,!1!.fluked down* awl th e p& oX the #»|H«PS^titat aijuated .fo i#f, bf ttei ..aOdttloa of appr&xiroiely

Xt « U i f 0*1 H aestip add* . 4 alenty preeipib&te wm removed fey centrifugation* 1feo,gi&ti&io& .©£$ bo 3 : *X« of X»3 i eodfom f a m i n e raised the gH to §*&, end .Hi. s&* of ;«X«efeoX.iMir* «MM* (m id brought toe eXeefcolie eeabeub to sXMfctly less then the 2f ,f#y rent tfeeified by dexger end ooworkore for the original 20 pounds of yeast* Xt appears, however, to he necessary to lower the concentration, slightly for « smaller quantity of yeast*) x rather large gray preoipitate ms discarded# flie addition of 343 ml* sore of alcohol caused precipi­ tatio n 0$ a tetter small end seceftieaaXXy gMqr mbsrinX* the latter me diaselead in 00 el# of X get seat glucose* fomtgH&vt nUli* lite r s i f $«X M ee«fcab*> buffer, pH 3*4* were added, to the glucose solution and the protein again fraetionaied between 23 and 4$ per seat alcohol# the resulting fraction ms. dissolved in 40 »su of 1 per seat glucose and aa equal quantity © 1 0 * 0 2 M acetate buffer, pi 5*4 # containing 1 per seat glucose ess added*

The enayme me then adsorbed at 0® os 40 ml* of alumimiM hydroxide gel# The mixture was stirred mid left to stand for 30 was then aentf&fegsi ©at, washed with threeiOOHsl. perUe&s *£ 0*01 l ft««tii« buffer* j>H 5*4 * load aatraeied with three 1 M » portions ®£%m U phoephat* buffer, pH 7>l$* Item* extracts ware combined sad used fa r assay* Aa active aluaioufli hydroxide gel m$ obtained by dissolving

33*1 s* af alaaimai sulfate ©eiadeeahydmte la 600 wt* of water and adding 14© «&* of esm rttswtad «AM©aiua fhla was dilate*toeae U ler, centrifuged* mat the supernatant discard* ftweasidaw w waehsdwltitaisUlladwnteriaatil l^Ci air of tep&t* aataot titrated ©*2 ml* of leas of ©*1 tt eaifurie aeid to the aeihyi rad end *®int* ’'thus precipitate was than suspended i& 250 a&* o f water aa* activated further bgr heating at X© 0* over steam for four hours*

!B*a£

- tfsjnh&fta* assays were carried m.% a t 3# l h @*1 $ phosphate buffer, pH 7*15* Although sode variation M the eonceniratioa ©f a& ted tls was wade firm time to tine, assay tubes generally soatalaed the fellow iaglaiiw dleatss .....

0*2 « l. of Q*4 A magneeiim chloride ©»dal*ef f *1 M phosphate buffer, pi' 7*15 ' 1*0 Si* of glucose, 1 iag*M* 0 .1 el* of bexokinaae preparation 0*2 3sl« of additions*

Kith the e x c e p t io n of the easyme preparation, ail materials were sob** blued and equilibrated la the water bath for tea minutes* After c*\ e l l

wi& $ wi& I

•fii 5 i i I 8

*

** f

into * t@»& into tufe* * t@»& if ®| aasu *<3* I I 1 ! I I #tfi 01 iboijip* trO’HWiTTJ

o«X» stm j#i®t twiawfej 1BW«TP»« -to port ‘p»inTTJ «** I u t 5 i i

jmgt ***** jr® a**Tt »»° «f S*»BJ»

m « n w » JO io ■%%%*& wa jt» wswS £*w**-»i51? IrSKJ ■ 35 Saaas S s s m k I*--a 15 per cent solution of cupric sulfate, pentahydrate was prepared* To this was added 1 or 2 drops of concen­ trated sulfuric acid per liter.

flgaiiml—Twenty-flv. great. of m um lm raolybdate war. dissolTad. fa 450 ml. of distilled w ater, and 21 ml. of concentrated su lfu ric acid were combined with i t . Three grams of disodium acid arsenate heptahydrate dissolved in 25 ml* of water were added* The entire solution was placed in toe incubator at 37° for 4® hr s . , and then stored in a glass-stoppered brown bottle#

Procedure: One m illiliter of supernatant from toe sine sulfate-barium hydroxide precipitation described in section f was pipetted into a

Folin-bu blood sugar tube, graduated at 25 ml* One m illiliter of a mixture of .25 parte copper reagent A to 1 part' copper reagent B was added to each. Solutions were mixed and heated for 20 minutes in a boiling water bath. At the end of this period the tubes were cooled In cold water and 1 ml. of Arseno-molybdat© reagent was added to each. After mixing and the evolution of C02 were complete, the tubes were diluted to

25 ml* and read In the Evelyn photo electric colorimeter at 515

Suitable standards and a blank were included with each assay* The method appeared to be most sensitive for the range 20 to 100 micro grams of glucose.

F. HaaovaJ. o£ Interferences i£t glucose toOyaie ■

Sooogyi in 1945 (75) suggested a method for remoral of the reducing substances which give interference in toe determination of blood sugar. I 8 ! 5 '•* a ! *r* |*8 * I i ? m fu tn* '** o *•1 s, © i s £2a % £ g 4* % S % i I

m m im ts, m mmumw qf . hesueb

The problem under consideration has three separate, yet Inter* related phases* The first of these is the result of observations made during the study of the nutritional effects of oleic acid stimu­ latio n on 1 * easel* The second is concerned with the application of the phenomenon of olele acid stimulation to a different microorganism, Saccharomarces cerevisi&e. The third section consists of m attempt to demonstrate more clearly the connection of biotin with certain phases of carbohydrate metabolism* Each of these divisions will be discussed separately*

a « 3M in Oleic Acid 3ti*auIation of X^c^obacillus Casei*

It has been generally assumed that equal validity may be assigned to the two micro biological assay procedures most widely em­ ployed i the titration of acid and the measurement of turbidity of c e ll suspensions* In 1946, however, i t was reported by Williams and

Fleger (4 &) that there was lack of agreement between titra tio n and turbidity in 1 . easel assays of biotin-free media containing oleic acid* The point seemed of sufficient importance to require further investigation* 37 9* The amemaly has been examineeI from several stai^points : (1) varying Urn c©iicentration of oleic acid, {%) varying the length of incubation time, (3) including serum albumin, or supplying oleic acid in an esterified form, (4) making various substitutions in add additions to tiie medium, (5) varying the pH of the medium,

(6 ) varying the concentration of glucose per tube, and

(7) comparing the effect observed with, arablnoeug « Quaere! Procedure*

la all eases triplicate tubes were analysed for each dilution of te st substancej turbidity, titra tio n measurements, and where necessary, glucose determinations were made on aliquots from the same tube. Except where otherwise stated the pH of the biotin-free medium was adjusted to 5*8 and the tubes were incubated for 72 hours. That the high turbidities observed throughout most of this investi­ gation were due to high cell production is supported by (a) the agreement of turbidities with cell Volume measurements (40), (b) the agreement of turbidities with plate counts of viable cells, and (c) the normal appearance of cells on microscopic examination. 39 Sine© turbidimetri© and iiirim etrte values obtained by mlcn>~ biological assay have been assumed to be directly comparable in value, the ratio of the apparent biotin content lay turbidimetric assay to the apparent Matin content by titrimetjie assay theoretically should be 1*0. The turbidimetri c-titrimet ric ratios so frequently referred to in the following pages are based on the above concept. The method of calculation becomes obvious through examination of the data in Table V. !• VMtetipn la th . CQnc*otistioB of p l.l* aeM- The oleic acid eontent per tube was varied from 10 to 4,000 mierograms, and biotin-fre© tubes were compared with those containing 300 mieramierograms of biotin in addition to oleic acid. The results are illustrated in Figure 3# where apparent biotin content as determined from the growth in the oleic acid tubes is plotted against the logarithm of the concentration of oleic acid per tube. From these curves it may be seen that both acid and cell production were stimulated by oleic acid with and without biotin, but cell production much more so* This disparity is obvious over a wide range of concen­ tratio n , agreement of titra tio n and turbidity data being approached only at very lew concentrations of oleic acid in biotia-free tubes* 2. Variation 1b th. Imgth oi Incubation tlac.

lubes containing 25, 50, and 100 micrograms of oleic acid were inoculated and incubated for 24, 4&, 72, and 120 hours. To a duplicate set of tubes 400 adcromicrograas of biotin were added per AND T U R B ID IM ETR IC M ETH O D S WITH VARYING VARYING WITH S D O ETH M IC ETR IM ID B R U T AND A PPA REN T BIOTIN C O N T E N T BY TITRIM ETRIC ETRIC TITRIM BY T N E T N O C BIOTIN T REN PPA A

APPARENT BIOTIN CONTENT ( m ILL I M IC ROG R AMS PER TUBE) —O UBDMTI, O ITN ADDED BIOTIN NO TURBIDIMETRIC, O 0— ADDED . BIOTIN NO TITRIMETRIC; O—O — UBDMTI; 0 MCOIRGA BOI PR TUBE PER BIOTIN MICROMICROGRAM 500 TURBIDIMETRIC; 0—0 0.2 0.8 0 0

A . TR(ERC; 0 M1 O CR6RM ITN E TUBE M PER 1C T BIOTINITROM 500 ICR• (METRIC R06 RAM ; 6 ! LOG OLEIC OLEIC LOG LI AI CONCENTRATIONS ACID OLEIC 1 ACID IUE 3 FIGURE TUBE) PER (MICROGRAMS CONCENTRATION 1 40 tube. The highest ratios of turbid imetrio to titrimetric data were attained at hours* After that period of time, fairly constant ratios averaging about I .9 6 were found, the higher concentrations of oleic acid tending to produce higher ratios. The presence of biotin along with the oleic acid had very Util© effect on the disagreement between the two methods of assay. 3* MasUM? a£ asss ateste ataas a£ afMsMIM sisis. asM- It has been shown that the inhibiting effect of oleic acid on the early growth of £, easel can be counteracted by the addition of sterile biotin-free albumin to the medium or by adding oleic acid In the form of an ester (76). The effect of these two variations was consequently studied from the standpoint of turbidity and titration analyses. Tubes containing either 25 or 50 micrograms of oleic acid were inoculated with and with©ut albuMda* S«veral concentration® of hopalcol 6-0 gave values very dose to the theoretical 1.0. In the ease of the ester, the ratios of approximately 1.0 were achieved by high acid production paralleling the high turbidity, instead of a reduction in turbidity to match low acid production, as the case might have been* The acidity in the hopaicol 6~Q tubes reached the amount theoretically possible to obtain from the glucose content of the medium. The ratios obtained in the presence of albumin were brought about by high cell growth and low acidity. These data are shown in Table 111. 42 ■*>,v : ‘ tABS i l l BAtios m x m m w rn ¥mm®* wmmm&mu® OF Q4M0 AQAO, OkSMAeii) PLUg iMUM* AK& HQFAbGOt 6-0

Concentration Turbidimetric-Titrimetric Eatle Per Tube Oleic Acid Oleic Acid / 2$ mg* Albumin iopalcol 4-0

. *5 .. - 1 * 1 0 ..:.. 1.98 50 1.96 2.04

100 . 1 #08 ■, 200 0,95

600 1 .0 0

*•' Alteration^ in the medium. A great number of substances were tested in the a tte st to reduee the high ratios obtained to the theoretical value ef l.G. Al­ though certain inhibitions and stimulations were noted in some cases, the effects operated uniformly on both acid and cell production, and the usual abnormal ratios were obtained. The changes were as

follows s (1 ) eaeh of the following substances was substituted in turn for the glucose of the basal medium*-maltos©, fructose, galactose, lactose, mannose, sucrose, xylose, ara binose, and mannitols ( 2 ) small amount* of the folio wing amino acids were added singly to the casein hydrolyeate medim--al*nine, serlae, methionine, threonine, lysine, arginine, phenylalanine, histidine, proliae/lsoleueine, valine, leucine, tyrosine, cysteine, glutamic acid, aspartic acid, glycine, and tryptophane; and (i) the following miscellaneous substances were added—stearic acid, ascorbic acid, oxaiacetic acid, adenosine tri«* phosphate, glueoce-l-phosphate, haxose diphosphate, iodoacetic acid, and sodium fluoride* 5. Variation to pH of medium. The effect of variation in pH on the turbidity-titration ratios was studied in the following manners The usual medium was made and divided into seven portions* Each portion was adjusted to a different pH, the range being 4*0 to 7.0 by increments of 0.5 pH unit* Biotin standards and tubes containing several concentrations of oleic acid and ikopaicol 6*0 were set up for each pH. At pH values of 4*0 and 4*5 there was no measurable growth, and at pH 5*0 growth was so low xn the presence of oleic acid that the values obtained were not reliable* Data for the other pH values are given in Table IV* I t is obvious that for oleic acid there is a rise in the ratio with increase in pH* The off set however in the presence of h opal col 6-0is rela­ tively slight* The Increasing ratios obtained at the higher pH values for oleic acid probably reflect the increasing toxicity of oleic acid with decreasing acidity* The infinite values obtained at pH 7*0 are due to a complete lack of acid production in the presence of some cell growth* “*r44

TABLE IV mmm w p$ m mnm

ommm with oleic agio mu nopalcol 6~q

Turbidimetrie^Titrimetrio Hatio pH Go&esnfey&tlen per Tube '■ \ ■ , ' .v , $ .0 . 5,5 6 ,0 6,5 7*0

jU f hopaicoi 6 -0

59 0 .8 6 1 .0 $ 1.14 1*15 1.29

100 0.92 1 .1 2 1«16 , 1*56 1*54 200 0.87 1.15 u p . 1*4? 1.51 Oleic Acid 50 —— 1,72 2*2$ 4.56 . *sO 199 . ■■'•■ '* 1.72 . 2,0$ 5*6? CO

200 i *72 2 ,2 8 5*00 O0

The laties for ftepalcol 6-0 are seeewfeat higher at pH6.5 and

7 .0 , although a o tso extreme a* those observed with oleic acid* This

pH sensitivity with wepalcol 6*0 (and consequent disparity in acidity and turbidity) is especially interesting considering the widespread practice &£ adding sash noaienie pieate detergents to various micro- biologieal aseay media, An examination of the data from which the E © © i a & S I % I *»> H fl* £ f * a se I I I ? ? I I! 5- ? Vft % 3 $ I c*- 8 2 IS la , 8 t I t

fermentation neeess&ry to furnish energy for cell growth is less then 46 : 7ms T.-. wmm w mMmm mmmmm m Mtm mm mm mtmim i 9

Glucose 0*1 ft Apparent Per Cent Apparent Turbidimetrie- Acid Per :,V’ Biotin ..v Tsmmw . ■ BWtin v litrisaetric Per Tube Aliquot (Titrimetrie) mission (Turbidimetric) Batio

•; ml.

25 2.77 0 :‘-71*l ...v:. ",19ft oO : j-.» 3.95 120 63.1 370 . 3.03

n 5*22 •< 295- V: 54*0 371 ,... ,> 1*94 100 4.57 ■ 520 47.3 . ..320 1.53 125 7.42 46.1 . 900 . 1*23... 150 3.31 970 44 *1 .; 1,080 1 *1 1 . 175 3*09 .^360, u , ■ .44*5 : 1 * 1 9 •' 2QQ 3*34 1 ,0 0 0 44*7 lii000 1 *00

that involving the 20 grams of glucose per liter provided in the medium* Oleic acid thus may exert an effect of ©ierie hindrance because of its attraction for the lipo~proteln surface of -the bacterial cell and prevent access of the excess glucose which is necessary to make the products of fermentation equivalent, In terms of apparent biotin content, to those of cell production. The latter hypothesis seems the 47 most probable considering the present concepts of surface adsorption and the data involving pH effect®, differing olei© acid concentrations, and the contrast in the action of Hopaleol 6 -©# The fata of the glu­ cose that is not converted Into lactic acid is unknown except that it can be accounted for as a reducing substance, this fact was ascertained in two wan * U) fey bhaf fer-hartmann deieiwinabioa of glucose (48); and (b) by use of Dreyweod’a enthrone reagent( 42) •

7* ggmparlson of l# easel with.1 * srablnQSus# the turbidicwtric^titrimetrie disparity observed with jj,. casei was cheeked with £.# arablnoaus. and the data are given in fable VI« From two separate experiments conducted some weeks apart i t may be seen that the effect with Jt» arab^nosu® i® much lee® marked/ so much so that it is doubtful whether the disparity exists in this case# To summarise, one may then conclude that of the many substances and. variations in procedure tooted, not one was. completely effective . in removing the disparity observed in turbidimetric assay values ob­ tained with oleic acid# the inclusion of oleic acid in the foim of a high molecular weight ester, kopalcol 6-0, was effective at pH 5*0 to 6.0# In general, turbidity values were approximately twice titration values under all other conditions# Increasing the pH from

5 .5 to 7*0 resulted in an enormous increase in the turbidity-titration ratio because of the increased toxicity of oleic acid at higher pH values, particularly with respect to glycolysis# The only clue obtained with regard to the mechanism wnereby oleic acid selectively inhibits 40

eatxos of 'fuiTOiMimQ ,.,:0®TAIW WITH V* „■.,

Tube Contents Turbidlmetrlc-TItrimeirio Ratio

Qleie Acid Biotin .. Jjfrg$«1 , ..lafb* Z, .

'.-■■■■ ■" ■ , . >*«•

25 500 " : 0 .8 6 1: " 50 500 i.29 ioo 500 1 .0 6 25 600 v 1 .1 2

50 600 1.13 '

ioo 600 ' 1 .2 0 ;

acid production was found in the case of tubes containing biotin and decreased glucose, but no oleic acid. When compared with tubes eon- taiaing the usual amount of glucose and equal amounts of biotin, turbidities gave much higher apparent biotin values than did acidities • On this basis several possible explanations were offered for the strange disparity, the most probable of which is a physico-chemical one. B* SIMS MA M Me m atte a»t*boIlm of S»e«hero«ara*aG»reri»M». Since the discovery by Williams and Fieger (45) that oleic acid is capable of replacing biotin For certain mcroorganismB, a study of the inter relationships of biotin arid oleic acid in the metabolism of such microorganisms has been continuously underway in various laboratories* Although the original work, involved the use of , an extension of this phenomenon to yeast was attempted Of seise twenty»flve strains of yeast which were tested* g* cerevialae Java shoved the most pronounced response when oleic acid was substituted for biotin as a growth factor in the basal sucrose medium, and consequently i t was chosen for study

1. Big aMMta:

0— O NO ADDITION I ML. EGG WHITE PER 100 ML. MEDIUM 100

90 Id0 z < H 80| 1 z ce< h 70 i- z uio 60

50*—

10 20 30 40 50 60 70 80 TIME IN HOURS

FIGURE 5

GROWTH OF SACCHAROMYCES CEREVISIAF JAVA ON BIOTIN~DEF ICi ENT MEDIA IN THE PRESENCE AND ABSENCE OF EGG WriiTE 54 130

• 20

100 94

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8.3 5.7 4.4 12221 2.4 EZZ L ACTOBACILLU5 CASEl 3ACC H AROMYCES FRAG* LIS

KEY Iyeast MEDIUM YEAST SUPERNATE YEAST CELLS

FIGURE 6 SYNTHESIS OF BIOTIN BY CELLS OF S. CEREVISIAF. JAVA GROWN ON BIOTIN-DEFICIENT MEDIUM. ASSAYS PERFORMED BY L. CASEl AND S FRAGILIS AS SHOWN I I ®qq If &I8. 0 S (5 i r 2 3 1

69 3 p*%vintoQvwm r I S $ 0 *-% 3 s* I *~< I 1 1 £ § O I H» i or & £ H* St 0 V s- <+ e* 1 « e* O 3 1 C* tr £ £* © tra 3 D a P» £ ►J S' & I j @ j £ « q w j s p ? »I|^ JO (q) H> 0 H I i ea­ ts* I % 0 1 Vs*& I—1 I H* <5 1 fv paawft19^ «qow*x« &n*oq qqq w? qtwsojtd ssmi^eqns pajtoxoo I %** E fc* i S

«t I 40J Jftxtqooxioo «T ^T^OTJJTP «W («) J?q poermo »q Jtero jC*®s* T®iio « M £

o M JO

V* It *1 e« oqq qqp* pejr^dwoo IfffSH? •? wtvppon jfiq M t V» 56 substrates, m growth of $• cereviaiae could be obtained in the absence of biotin even on prolonged incubation • In conclusion, the growth of Baceharomygea csrevislae Java was stimulated by oleic acid on a biotin deficient medium in the presence of aspartate. Glutamate and succinate would not substitute for aspartate. When incubated for longer than three days, S. cereviaiae Java achieved fairly good growth in a sucrose medium lacking both oleic acid and biotin but containing aspartate. Analysis of hydrolysed cells from such cultures showed synthesis of biotin. Small quantities of biotin, or its nutritional equivalent, have also been shown to be present in L. easel cells grown in the presence of cleats*

C • A Linkage for Biotin in Carbohydrate Metabolism.

During studies of the ability of oleic acid to substitute for biotin in the fcrowth of S* Cerevlsiae Java, it was noted that data ob­ tained on the basal sucrose media could not be duplicated if glucose, fructose, or hydrolysed sucrose were substituted for sucrose as sources of carbohydrate. This appeared to indicate that biotin was essential for some phase of the monosaccharide glycolytic mechanism which is not involved in the utilization of sucrose. Data of similar implications nave been published by Carlson and Whiteside-Garlson (35) from their studies with Leuconostoc species. 0*0 ^ 0 1 x Z'Z ranfp©M p©q«T^30trpin 0*0 ifJUT * //

IfffR S J •? jEq itesav ow®®W?£ /q JlBSSV opt^diTFp^qJHJ

Hfiraw ii© ioiasa-raoia v w> m o n ot s s v o • ! n m HnicraM ojaTinaowrun aw 'jwxrwrajns 'srxso jo xsmsoo pra-vra IIIA ST8VX

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VAvr PfffX SW •? X9 axvsaiHoaavo i o s m n o senoraw jo sotxnrttxn HA STBTX S«0 I e the e o * J0& of § 30 I 0 •H8 4* O « 1 m 1 Su^ §• cereviaiae (a) (a) biotin and 3 of t

of of > If2 a 1 te t3 d 14 » a 3 3 S •H -p * ag % I «-4l ° 1 a • € I I I *3 • I « 8 s jo a I 4*

presence presence and absence © i P3 a 1 S O

the the i Q 2 o $ A so i w o i 1 4 *rl ** o 3 % i I a *3t* •s 3 4 © JKP I 8 I i i I I ! 1 p3 ©O •rtt 8« (b) (b) biotin / adenylic acid. of of the variation in the glucose utilisation of colls activity of hexokinase in 139 grown 139 grown on high- and low- biotin media, and (3) by studies 3 1 3 L. oassi were grown in the presence of Hopaleol 6-0 on a low glucose medium, The washed cells were then incubated in buffered glucose (a) with no additions* (b) with the addition of 1 H&erogramof biotin, and (c) with the addition of 1 ado regram of biotin / 1 microgram of adenylic acid, Details of the procedure follow, The glucoee content of the medium was reduced to 5 g* per liter, one eighth of its usual value, Nop*iCQl443,40 rag. per lite r of double strength medium, was substituted for biotin as a growth factor* Cells grown on two liters of this lot glucose medium containing hopaleol 6-0 were harvested afte r about 4# hours of incubation and washed once With d istille d water, The cells were then suspended in approximately 20 ml* of distilled water and added in 2 ail* aliquots to tubes containing the following materials at 35°*

5 ml. of phosphate buffer, pH 7*13 2 ml, of glucose (2 mg,/ml*) 1 ml. of additions.

Several series of three tubes were prepared, each series to be removed from the temperature bath afte r definite time intervals. The tubes contained (a) no additions, (b) 1 microgra®. of biotin, and (c)

1 microgram of blotln / 1 microgram of adenylic acid. At the end of the incubation period each tube was heated in boiling water for one minute to inactivate the enzyme systems. Suspended cells were then centrifuged out and 3 ml. aliquots of supernatant removed for precipi­ tation with zinc sulfate-barium hydroxide. 6 0

Figures y and B illustrate the rata of glucose utilisation from two euohwashed oell incubations, ' At incubation periods of 10 to 15 miautes it becomes obvious that some stimulation of glucose u tili­ zation i s produced by biotin alon©, end that an even more pronounced stimulation occurs in the presence of a combination of biotin and adenylic add.

2. tew w utm aatjoa tar X««a» CaU. (Haram ffltfe - mi, ifljfStw tte

2SSS8- Saechareaarees cereviaiae 139 was grown for 46 hours on two media differing only in biotin content. The high biotin medium con­ tained S miexograms of biotin per l it e r . The low biotin medium contained 2 at 10““^ micrograma per lite r . Approximately 29 g* of cells were obtained from 10 lite rs of the former medium as compared with 9 g. from 10 lite r s of the la tte r « Cells were suspended in 3 ml. of water per gram of cells. the following three incubation flasks were set up for cells from both high- and low-biotin mediat

(a) 50 ml. of to/ 20 phosphate buffer, pH 7, containing no glucose. (b) 50 ml. of U/ZO phosphate buffer, pH 7, containing 20 mg. glucose.

(c) 50 ml. M/2Q phosphate buffer, pH 7, containing 40 mg. glucose. 61 Nine m illiliters of cells were added to each flask and 10 ml. aliquots were removed from each at intervals of 0, 5, 10, and 15 minutes. These samples were immediately centrifuged to remove the cells, and the supernatants were analysed for glucose, Th® rates of glucose utilisation are shown in Figure 9 * During in itia l stages of glycolysis It is obvious that the rate of glucose utilization of the biotia-deficisnt cells is much slower than that of the cells which were grown on the high-biotin medium, 3* Effect of Biotin on Hexokinase. feast hexokinase was prepared several times, Enzyme assays were performed in the manner described in Chapter II and analysis made for the disappearance of glucose. The enzyme obtained showed either such a low level of activity that stimulations could not be observed or an exceedingly high degree of activity with no evidence of stimu­ lation in the presence of blotin or biotin with adenylic acid. The lack of a stimulative effect is not, however, conclusive evidence that biotin is not necessary for the function of hexokinase. It is entirely possible that the enzyme system as isolated is not sufficiently resolved. To summarise, a study of the linkage of biotin to the glycolytic cycle has been made. That biotin has a stimulative effect upon at least one of the enzyme systems involved has been demonstrated by the use of two different microorganisms. Washed cells of L, Casel which had been grown on a low glucose medium In the presence of Nopal col 6-0 showed an increased utilization of glucose upon the addition of biotin or blotin with adenylic acid to the glycolyzing suspension. A O 2 1— 5 < QC ADDITION OF BIOTIN PLUS 2 i _i ADENYLIC ACID, I MICROCRAM OF EACH PER TUBE

15 30 TIME IN MINUTES

O

ADDITION OF BIOTIN I MICROCRAM PER TUBE NO ADDITION _l i

30 TIME IN MINUTES

FIGURES 7 AND 8

THE RELATIVE STIMULATORY EFFECTS OF BIOTIN AND BIOTIN PLUS ADENYLIC ACID UPON GLUCOSE UTILIZATION BY JL CASE I CELLS GROWN ON LOW GLUCOSE BIOTIN-DEFICIENT MEDIA 400

360

BIOTIN CONCENTRATION OF oc 2 X I0"2 MICROGRAMS PER LITER u CL

BIOTIN CONCENTRATION OF 2 MICROGRAMS PER LITER

80

40

TIME IN MINUTES AFTER INOCULATION

FIGURE 9

RELATIVE R A T E S OF GLUCOSE UTILIZATION BY CELLS OF S. CEREVISIAE 139 GROWN ON HIGH- AND LOW-BIOTIN MEDIA $ similar increased rate of fermentation was shown by cells of S. cere- visiae 139 grown on a high-biotin medium when compared under sim ilar conditions with cells produced in the presence of a much lower con­ centration of biotin* Because of the differences observed in the utilisation by yeast of the d i- and the monosaccharides in the absence of biotin, and because of a somewhat sim ilar phenomenon noted in the metabolism of Leuconostoe specie® by Carlson and Whites id e*~Carlaon (35)* i t has been postulated that blotin must be linked to the action of on© of two enzymes x either hexokinase or phosphohexoisomerase, most probably the former. The preparations of hexokinase tested were not stimulated by the addition of biotin or of biotin with adenylic acid. Since, however, the enzyme system may have remained unresolved, th is was not regarded as a negation of the hypothesis. CHAPTLE IV

m m m

Previously unexplored phases of the relationships of biotin and oleic acid in the metabolism of certain microorganisms have been studied along three different lines: (a) the anomaly between turbidimetrlo and titrim etric microbiological assays of oleic acid-containing media has been examined under various conditions; (b) the phenomenon of oleic acid stimulation of microorganisms in biotin-free systems was studied with yeast; and (o) the relationship of biotin to some phase of carbohydrate metabolism has been demonstrated for several micro­ organisms* The turbidimetric-titrimetric disparity which exists in Lacto­ bacillus easel assays of oleic acld-containlng medium was examined under various conditions. Under most of the conditions tested the apparent biotin contents as measured by turbidities were approximately twice the values as measured by titra tio n . The phenomenon showed so u l s pH sensitivity, the turbidimetric-titrlmstrlo ratios increasing as the pH was raised to 7;0 probably because of the increased toxicity of oleic acid with decreasing acidity. An increase of turbidities as compared with acidities was obtained upon lowering the glucose content of the basal medium in the presence of biotin, not oleic acid. Only 66 67 the substitution of a high molecular weight ester of oleic acid for the latter compound in the assay medium completely eliminated the anomaly. On the basis of these findings several explanations were offered for the disparity, the most probable of which appears to be a physico­ chemical one.

The ability of oleic acid to stimulate growth of S&ccharoniyeeg cerevisiae Java ou a biotin-deficient sucrose medium in the presence of aspartic acid was demonstrated. Synthesis of biotin by this organism grown on a similar medium was shown to occur. Cells of J.. casei grown on a biotin-deficient medium were also found to contain low concentrations of biotin or its nutritional equivalent. Reproduction of S. cerevisiae

Java occurring upon sucrose medium in the absence of biotin, did not occur if glucose, hydrolyzed sucrose, or fructose were substituted for sucrose as carbohydrate sources.

From the latter observation the third line of research was developed: the relationship of biotin to carbohydrate metabolism*

Stimulation of glucose utilisation was observed when biotin or biotin with adenylic acid was added to cells of 1. casei grown on a low-glucose medium in the presence of i«opalcol 6-0. Hie rate of glucose utilisation by c e lls of 3,. cerevisiae 139 grown on media containing 2 miorograms of biotin per liter was found to be considerably higher than that of cells grown in the presence of 2 x 1CT2 microgram© of biotin per liter. A linkage of biotin to haxokinas© function was postulated, but could not be demonstrated in the purified ©nayue under the conditions used. 6S Several conclusions were drawn*

(1) The turbidimetric-titrimetrlc disparity observed in micro biological assays of oleic acid-containing media may b© removed by 'the substitution of a high molecular weight ester of the oleic acid for the l a t t e r compound in assay media* The anomaly is probably due, to an effect of sieric hindrance by oleic acid adsorbed on the cell membrane•

(2) Oleic acid is capable of stimulating the growth of S. cerevisiae Java on a biotin-deficient sucrose medium* Synthesis of bio tin or its nutritional equivalent occurs when the organism is grown for a prolonged period on a biotin-deficient sucrose medium* Cells of

L* easel grown on a biotin-deficient medium also synthesize small quantities of biotin* Growth of JJ. cerevisiae Java on medium lacking in biotin cannot be produced if the monosaccharides listed above are substituted for sucrose as sources of carbohydrate«

(3) Biotin exhibits a stimulative effect upon glucose utili­ zation by cells of L. easel and &* cerevisiae 139* A biotin linkage for hexokinaae has been postulated. SELECTED BIBLIOGRAPHY

Books. (1) Kluyver, A* J*, Chemical Activities c£ )&U reorganising, University ©f London Press, 1931* (2) Kaight, B-. G* J* G*, “Growth Factors In Microbiology", Vitamins and Hormones. Academic Press Incorporated, New York, 1945* 111* ppr pJ*'®®*

Journals. (3) Quastel, J , H. and Whetham, M* p« "The Equilibria Existing Between Succinic, Fmnaric, and Malic Acid in the Presence of Resting g* ife• w ,- w . y- (4) Booth, V# H# and Green, D. E, 11A Wet Crushing Mill for Micro­ organisms", Bioeheau jr. jg, 055, 1930.

(5) Wildier*s, E. «A Mew Substance Mecessary for the Growth of leasts", 1* £g, 313, 1901. (6) Kdgl, F. and Tennis, S* “Plant Growth Substances, Bios II Preblest—The Isolation of Crystalline Biotin from Egg Yolk" & jeSusM* cheg. 242, 4 3 , i?36.

(7) Bom , K. A. "gffeet of Qeeeiestion upon Nutritive Properties of Egg Mlite",gioctew. 712, 1927. (8) Gy#ray. P. "fiioftet* and Other Avitaminose*", Z. tr tz l. Fortbild. 28, 397, 4 1 7, 1931- (9) Allison, F. C., Hoover, S. R., and Burk, D,, "A Respiration Ceensrae". Selene. 78. 217. 1933. (10) du Vigneaud, V., Hofmann, K., iielville, D. B., and GyOrgy, P. "Isolation of Biotin (Vitamin H) from Liver", J. Biol. Chem. IM i, 643 , 1941.

69 70 (U) Eoaer, S. A,, Wright, M* M., and Borfiaan, A# "Aspartic Acid as a P artial Substitute for the Grovrfch-Dtimulating Effect of Biotin of Torula Cemoris". Proc. See* hxgtl* Biol, Med. JgL, ■. sm , im * • ■ ' ■■■ ■ ■ (12) Winaler* R* d ., Burk, B., anddu Vigneaud, V. "Biotin in the Fermentation, Respiration, Growth, and Nitrogen Assimilation by Iea»tV|rg|. Mg&S®* 25,m 4. (13) Stokes, J.L., Larsen, A*, and Gunness, M. "Biotin and the Synthesis of Aspartic Acid by Microorganisms", J. Bact. 54, S it, 1947. (14) Stokes, J* 1 ., Larsen, A#, and Cknness, M. "Biotin and the Synthesis of Aspartic Acid by Microorganisms", J. Biol. Chem. i& , 613, m f i ~ (13) Lichstein, H« ft* and Bmbreli, W. W. "Biotin Activation of Certain Deaminases", J . Biol* Cheat* 17Q, 423, 1947* (16) Lichstein, H. 6* and Christman, J, p* "The Role of Biotin and Adenylic Acid in Amino Acid D eam inasesifol* Chem. 175. 649, 1946. (17) Lichstein, H. C. "The Probable Existence of a Coensyme Form of Biotin", £. Biol. Chem. 177. 125, 1949* (16) Lichstein, M. C. and Christman, J. F. "The Nature of the Coensyme of Aspartic Acid, Berine, and Threonine Deaminases", J . Bafii* 5*> 1949. (19) Wright, L. D., C res son, E. L ., Skeggs, H. E, "Biotin In the Bacterial Deamination of Certain Amine Acids", Prof. Bod. Sxptl. Biol. Med. |2 , 556, 1949- (20) Pilgrim, F. J., Axelrod, A. E., and UveM-Jem, 0. A. "The Metabolism of pyruvate by Liver from Pantothenic Acid- and Biotin-Deficient Rats", J . Biol. Chem. 145. 237, 1942. (21) Lardy, H* A., Potter, B. L., and llvshjem, C. A. "The Bole ©f Biotin in Bicarbonate Utilisation by Bacteria", J . Biol. Chem. 169 . 451, 1947* (22) Shive, W. and Rogers, L. L. "Involvement of Biotin in the Bio­ synthesis of Oxalacetie and Alpha-Ketoglutaric Acids", J • Biol. Chem. l6£, 453, 1947- n (23)' Lichstein, H* C* and Umbreit, W, W. "A. Function for Biotin”, J . Me|* gggg, |Zg, ■ ■'■ (24) Ochoa, fV, Mehler, A., Blanchard, M. ,L.Jukes, T* H., Hofmann, G* E ., and Re&gaa,M. '’Biotin and Carbon Dioxide Fixation in Liver”, g. Biol, Chem, 170, 413, 1947*

(25) Buik, D, *nd Winsler, E. 0 , »HesVi*MA*»Ari^la-OoablmVla, Speeies-flpecifl*, end Other Hitamers of Biotin", Science 97. 57, 1943. (26) Suwiw*, W. H., toe. J. K., aiMl Partridge, W. H. "The Effect of Biotin on the Metabolism of Liver Slices from Blotiu- Deficlent Bats", Salens. 1 0 0 .250. 1944. (27) Lard/, H. A., Potter, B. L ., Burris, B. H. "Metabolic Functions of Biotin. 1. The Bole of Biotin in Bicarbonate Utilisation by L. Arabinosua Studied with cU ". 3. Biol. Chem. 179. 721, 1949, ' . ; (28) Delwiehe, E. A, "A Biotin Function in Succinic Acid Decarboxylation by Fropionibacterium Pentogaeeum”, J. Bact. 59* 439> 1949. (29) Lichstein, R, 0. "Further Studies on the Biotin Coensyme” J. Bact. 60. 45, 1950* (30) MacLeod, P. R., and Lardy, H. A* "Metabolic Functions of Biotin. I I . The Fixation of GO2 by Mental and Biotin Deficient Bats” J* Biol. Chem. 179, 733, 1949* (31) MacLeod, P. B., Grisolia, S ., Cohen, P. P ., and Lardy, M« A. "Metabolic Functions of Biotin« XII» The Synthesis of Citrulline fxom Ornithine in Liver Tissues from Normal and Biotin Deficient Rats”, J, Biol* Chem. 180, 1003, 1949. (32) Wessmaa, G. £• and Werkman, C* H. "Biotin in the Assimilation of Heavy Carbon in Oxalacstate”, Arch* Bioohem. 2&, 214, 1950. (33) broquist* H, p. and Snell, E* E. "Biotin and Bacterial Growth. I . Eolation to Aspartate, Oleate, and Carbon Dioxide", J. Biol. Chem. 188, 431, 1951. (34) Blanchard, M. L ., Koifces, S., del Campillo, A., and Ochoa* S. "Function of Biotin in the Metabolism of L. Arabinosus"» £. Biol. Chem, 18J, 875, 1950. (35) Carlson, W. W. and Whiteside-Carlson, V. "Biotin-Carbohydrate Interrelationship in the Metabolism of Leuconostoc” . Proc. Soc. Exptl. Biol. Med. 71. 416, 1949. 72 (36) AJX# 3. W** Hart, ty. and ;.Wex2eaahi •$*. M. "Mdbia in Sadeinic Aeid Oxidation", Ensymologia m . l. 1950* (37) Fleming, A. “On the etiology of Acne Vulgaris and Its treatment byVac&iBos", lancetl. (32) Cohen, $nj4or* 4* J^eU er# 4* H* ^Factors Concerned in the Growth of Corynebacterjum Diphtherias from Minute Inoculum" , £* Bact, 41, 5ai>' (39) Hutner, 3* $*,; “Some G*x>wth Requirements of Irysiaelothrtx and U jsterella", £. gae|. 2£, 629, 1942. (40) Bonham, ft* W. "Cultural Characteristics of . ,.,. „ ^ A Iip©pbyilc Fungus”, Proc. Soc. ixntl. Biol. Med. 4 6 ," W , 1941*

(41) Feeney, S. K*, t e l l e r , 4 1 M«, and Miller, P. A* "Growth Requirements of Clostridium Tetani. XI, Factors Exhausted by Growth of the Organism*, J. Bact. j£» ^59, 1943. (42) Feeney, E* E ., Mueller, J . E*> and M iller, P. A. “Growth Requirements of Clostridium Tetani, I I I . A Synthetic Medium1*, J. Bact. 46. 543. 1943* (43) Bauerafeiad, J* ©., Sotier, A* L ., and Boruff, C. S* “Growth Stimulants in the Microbiological Assay fo r Riboflavin and Pantothenic Add1*. Ind. and Eng, Chem, Anal. Ed* Ijt, 666, m (44) Strong, F. M. and Carpenter, L. E. "Preparation of Samples fo r Microbiological determination of Riboflavin", Ind. Eng. Chcm. Anal. M . 14, 909, 1942. (43) Williams, V. R. and Fieger, E. A., "Growth Stimulants fo r Micro biological Biotin Assay", Xnd. Eng, Chem*, Anal. Id, 12, 127, 1945* (46 ) Williams, V, R. “Growth Stimu .aat® in the Arabinosus Biotin Assay", J. Biol. Chem. 159, 237# 1945* (47) Hodeoa, A. 2* "Oleic Acid Interference in the ieurcspora Crases Assay for Biotin", £♦ Biol. Chem* 179* 49, 1949. (4E) Williams, V. R. and Fieger, I* A* "Oleic Acid as a Growth Stimulant for Lactobacillus Case!", £♦ Biol* Chem* 166, 233, 1946. 73 (49) William*, 9* R. and Fieger, Jfcu- A. "Further Studies of Lipide Stimulation of Laetpba^illiia Qassi". J* Biel* GhcBU 170* 399* 1947. (§0) Hodicek, S. and Worden* A* M. "The Effect of Unsaturated Fatty and Other Gram-Positive Microorganisms", Biocheia, J . ?8* 1945. (51) Kodicek, E. "the .Antagonism Between Unsaturated Fatty Acids and Other Active Agents on Gram-Positive Bacteria", Bulletin a* ia Sssiili Is £Ms&s ^ateBteB ip* * * 6 , i w . ($2) Williuie, W. L., Broquist, H. P., and Snell, E. Hi. “Oleio Acid and Related Compounds as Growth Factors for Lactic Acid Bacteria", J . Biol. Chem* 170, 619* 1947. (53) Middlebrook, Gl and Bubos, R.

(61) Potter, R* b* end KlvehJem, G. A. "Biotin and the Metabolism '«* m m iA p m w Biol, Chem. igg, 531, 1948. (62) Shull, G. M*, Thoma, K* W., and Peterson, W. H. "Amino Acid and Unsaturated Fatty Add Requirement® of Clostridium §mmmn» m m$$m * b > (63) Whitehill, A, E*, Qlesen, J* J*, and Subbarow, Y. "A Lacto­ bacillus of Cecal Origin Requiring Oleic Acid", Arch. m m r n * 15, 31, 1947* (64) Guirard, B. St., SaeU, £• I., and Williams, E* J. "The Nutritional Role of Acetate for Laetic Acid Bacteria. 1. The Response to Substances Related to Acetate", Arch, Biochem. £, 361 , 1946. (65) Litay, E. and Snell, E. £»• "Some Additional Requirements of Certain Lactic Acid Bacteria", £• Bact. |0 , 49, 1950. (66) Broquist, H. P. and Snell, £• £• "Biotin and Bacterial Growth. 1. Relation to Aspartate, Olsate, and Carbon Dioxide", J. Biol. Chem, 186, 431, 1951. (67) Greenburg, S. M., Cheng, A. L. S ., Melnick, D., and Deuel, H. J ., J r. "Biotin-Lik© Activity of Octadecenoic Acids as Affected by positional- and Stero-Isomerlaation", Fed. Proc. 10. 192, 1951. (66) Carlson, W. W., Whiteside-Carlson, V. and Kospetos, K. "Fatty Acids as Biotin Substitutes for Leuconostoc. Fed, Proc. £, 159, 1950. (69) Shull, G, M., Hutchings, B. L., and Peterson, W. H. "A Microbiological Assay for Biotin", J. Biol. Chem* 142. 913, 1942. (70) Snell, B. K., Eakin, R. E ., and Williams, E. J* ttA Quantitative Test for Biotin and Observation® Regarding Its Occurrence and Properties", J. Am. Chem. Soc. 62, 175, 1940. (71) Berger, L., S lein , M. W., Colowick, S» P ., and Cori, C. F. "Isolation of Hexokinase from Bakers Yeast", J . Gen. Physiol. 32, 379, 1946. (72) Morris, D* L. "Quantitative Betemination of Carbohydrates with Dreywood*s Anthrone Reagent", Science 107 . 254, 1948. 75

(73) Folia, 0. and Malmros, H. "An Improved Form of Folia1 s Micro Method fo r Blood Sugar Determination1*, J . B iol. Chem* S3.

(74) Kelson, M. “Photometric Adaptation of Somogyi Method for Determination of Glucose", g* ffool. Chem. 153. 375, X944-

(75) Somogyi, M. "Determination of Blood Sugar", B iol. Chem. 160. 69, 1945. (76) Williams, V. B., and Fieger, B. A. ^Further Studies on Lipide Stimulation of Lactobacillus Case!", J. Biol. Chem. 177, 739, 1949. VITA

ISmili© Anne Andrews was bora at Hickory, North Carolina, on

February 7, 192$* She was educated in the public schools of Morgan- to n , North C arolina, and graduated from Morganton High School in 1 942.

The same year she enrolled in Southeastern Louisiana College and graduated from that institution in June of 1946 with the Bachelor of

Science Degree in chemistry. In September, 1946, she entered Louisiana

State University as the recipient of an honor scholarship awarded at her graduation from Southeastern Louisiana College. She was a graduate assistant in the Department of Chemistry from 1947*1949* In June of

1949 she received the Master of Science Degree with a biochemistry major. Since th at time she has been employed as a research a ssista n t in the Department of Agricultural Chemistry arid Biochemistry of the

Louisiana Agricultural experiment Station, Louisiana State University, and has continued her studies toward the degree of Doctor of Philosophy in the field of biochemistry.

76 EXAMINATION AND THESIS REPORT

Candidate: Emilie Anne Andrews

Major Field: Biochemistry

Title of Thesis: Biotin and Oleic Acid in the Nutrition and Metabolism of Certain Microorganisms Approved:

r Major Professor and Chairman

n of nhe Graduate School

EXAMINING COMMITTEE:

tfrd 7^ J i -

Date of Examination:

November 19. 1951

PIKE BURDEN