Effect of certain polyenes, vitamin E, and related substances on the utilization of beta-carotene and vitamin A for storage of vitamin A in the kidneys and livers of rats

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EFFECT OF CERTAIN POLYENES, VITAMIN E, AND RELATED SUBSTANCES ON TM UTILIZATION OF /^CAROTENE AND VITAMIN A FOR STORAGE OF VITAMIN A IN THE KIDNEYS AND LIVERS OF RATS

BY EDWARD G. HIGH

Submitted to the Faculty of the Graduate School in partial fulfillm en t of the requirements for the degree, Doctor of Philosophy, in the Department of Chemistry Indiana University December, 1949

ProQuest Number: 10295206

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AGKNOVCLSDGEMENTS

The author's thusks are gives t© the followings the U. S. Department o f Public Health for a predoctorate research fellow ship in b io lo g iea l chemistry from August 2 , 1948 to August 1, 1949; to Dr. H. G-. Day for h is in sp iration , patience, and able guidance throughout the course o f th is research and the w riting o f the manuscript; and to my w ife for her encourage­ ment.

C O N T E N T S

I . In tro d u ctio n

Page 1

...........

IX* H is to ric a l and Plan o f Problem *............ A. Xanthophyll B* Lycopene G. Squalen© D. Phytol E. Vitamin E, P hytol, and Hydroquinone P. The E ffe c t o f Various Substances on th e D epletion o f Tissue Vitamin A

HI* Methods A* B* C* D. E* P. G* H*

......................................................................................... Source and P reparation o f Reagents D ietary Substances P reparation of Supplement M aterials Care o f Animals Method o f Preparing Vitamin A D eficien t Animals Method o f Supplementing P reparation o f Xanthophyll and Absorption Spectra P reparation o f Lycopene and Absorption Spectra Preparation o f Vitamin A and Lycopene Standards and C a lib ratio n Curves Procedure o f Analysis f o r Vitamin A i n th e Tissues

XV* Eaqperimental ................. A* R esults and Discussion of Carotene and Xanthophyll Data B. R esults and D iscussion of C arotene, Vitamin A and (X- and >£-Ionone Data C* R esults and Discussion o f Carotene, Vitamin A and Lycopene Data D. R esults and Discussion o f Carotene, Vitamin A and Squalen© Data E* R esults and D iscussion o f Carotene, Vitamin A and Phytol Data F* R esults and D iscussion o f Vitamin E, Phytol, Hydroquinone; Carotene Data G* R esults and Discussion of Data on D epletion of Tissue Vitamin A H* D iscussion o f th e E ffe ct o f Rat© of Growth on L iver and Kidneys Vitamin A Content I* A D iscussion o f Hepatic Deposition of Lycopene and Some F actors A ffecting t h i s Deposition V. General D iscussion and Conclusions VI* Summary VII# References

.........

..............

3

9

24

62 71 73

T A B L E S

AND

F I G U R E S

Tables

Page

I -E ffe c t of Carotene: XanthophyU Ratio on Tissue D eposition of Vitamin A from Carotene Fed

.........

IX-Effoct o f o(~and (S~Ionon© on Tissue D eposition o f Vitamin A from Carotene and Vitamin A F e d

29-31 32-33

H I-E ffe c i of Lycopene on Tissue Deposition of Vitamin A from Carotene and Vitamin A Fed . . . . . . . . . . . . . . . . . . . . . . . . . . . IV -Effeot o f Squalene on Tissue D eposition o f Vitamin A from Carotene and Vitamin A Fed . . . . . . . . . . . . . . . . . . . . . . ............

38 41

V-Effect o f Phytol on Tissue D eposition o f Vitamin A from Carotene and Vitamin A Fed ...................................... VI-Effect of V itanin E, Phytol, and Hydroquinone on Tissue D eposition o f Vitamin A from Carotene Fed

.

45 48

VIX-Effect o f Various substances on th e D epletion o f Tissue Vitamin A ........................................................ VH I-Bata I l lu s tr a ti n g Hepatic Deposition of Lycopene and Some Factors A ffecting t h i s D eposition ..........................

61

Figures 1.

Absorption Spectra of XanthophyU ............................ (Employed in th e f i r s t and second se rie s )

2.

Absorption Spectra o f X anthophyU (Employed in th e second s e rie s )

3.

Absorption Spectra of "Yellow" X anthophyU (Employed in th e second s e rie s )

17*1

4.

Absorption Spectra o f XanthophyU (Employed in th e t h ir d s e rie s )

17*1

5*

Absorption Spectra o f XanthophyU (Employed in th e fo u rth s e rie s )

6.

Absorption Spectra of XanthophyU (Employed in th e f i f t h s e rie s )

7.

Absorption Spectra o f "Oxidized" XanthophyU

8.

Absorption Spectra of Lycopene ....................... (S olvents 2 Skelly Solve B and chloroform)

...............

17.1 17*1

..................... ..........................* . . . . .............

17*2 17.2

..........

17*2 20,3

Figures ............. . . . . ...........................

21,4

9*

Vitamin A C a lib ra tio n Curve

10.

Lycopene C a lib ratio n C u rv e

11.

Lycopene C a lib ra tio n Curve w ith Reagent A dded.............

21.4

12.

E ffect o f Bate o f Growth on th e L iver and Kidneys Vitamin A Content when 30 Micrograms o f Carotene i s Fed D a i l y .........................

56.5

E ffect o f Bat© o f Growth on th© L iver and Kidneys Vitamin A Content when both 60 and 15 Micrograms o f Carotene are Fed D a i ly ............................

56.5

E ffect o f Bate of Growth on th e L iver and Kidneys Vitamin A Content when 13 Micrograras o f Vitamin A ............. are Fed Daily

56.5

13.

14.

15.

...............................

Absorption Spectra o f Saponified L iver E x tra ct . . . . . . . . (Comparison o f animals fed carotene only w ith those fed c both carotene plus lycopene)

21.4

61.6

16.

......... 61.6 Absorption Spectra o f Saponified L iver E x tra ct (Comparison o f animals fed vitam in A only w ith those fed both vitam in A plus lycopene)

17*

R elationship between th e Amount o f lycopene In g ested Daily and th e Quantity Deposited in the L iver o f Rats . . . . . . . 61.7

IHTHODUGTIOH

1 During the past decade rapid str id e s have been made in the f i e l d of vitamin research due in part to the discovery o f the antagon istic e f fe c ts of certain structural analogs.

To mention a few there i s 3~Fyridin®

su lfon ic acid for n ic o tin ic acid* pantoyltaurine fo r pantothenic add* pyrithiamine and oaythiamine for thiamine* and d esth iob iotin fo r b io tin .

All

of these antagonists are stru ctu rally sim ilar to the e sse n tia l m etabolite which they in h ib it - probably by competing for a sp e c ific p rotein .

Became ©

no true biochemical antagonist has been demonstrated conclusively fo r e ith er vitamin A or the provitamins* t h is in v e stig a tio n was undertaken to study the e ffe c t o f various stru ctu rally related compounds on both carotene and vitamin A as possible biochemical antagonist for the vitamin and provitamins, A comparison of the structures of so m o f the substances in vestigated as possible biochemical antagonists to both vitamin A and carotene w ill ju sti­ fy th e ir s e le c tio n . HoC CR~ KoC CRo ^.CN 1J H IJ ^ IJHH gf V" ^ HoC C-C=C'C=«-C=C-C*C-C=C-C=C-C=C-C=C'C’'C-C XCH„ I (| iHo CH3 CHo CHo I I HoC. ^C-CH, J CKo CH2 ^-Carotene 3 N ct^ 6-dibenzanthracene was

apparently th e most e ffe c tiv e in m obilizing sto red vitam in A.

I t has been

suggested th a t diben&anthracene may a c t by competing with vitam in A f o r some substance* possibly a protein* which serves as an anchor fo r the vitam in i n the l i v e r (2* 5 ).

le a s t adm inistered along w ith dibenzanthracene tre a te d r a t s

caused a fu rth e r decrease in hepatic sto re s of vitam in A and i t was suggested th a t y east ©lone may e x h ib it an antagonism fo r hepatic vitam in A (2)* the experiments with yeast were not well c o n tro lle d w ith resp ect

However*

to th© in ­

take of provitamins A. p-Dimethyl-airdno-azobenBene* dicumerol* carvone (16)* bromobensene and anthracene (5) have been found to be without e ff e c t upon depletio n of hepatic

vitamin A storage* The close s im ila rity in s tru c tu re s between various terp en es and t h e i r d e riv a tiv e s and vitam in A suggests th a t a biochemical an ta g o n ist f o r th e v i­ tamin may e x is t in t h i s la rg e group of compounds.

Although a number of

these substances have been te s te d fo r vitam in A a c tiv ity * only a few* such as carvone, have been stu d ied as possible a n ta g o n istic agents fo r vitam in A. Geraniol, phytol* ^ -io n o n e and several o th e r re la te d and u n satu rated com­ pounds have been te s te d in connection with b io lo g ic a l hydrogenation.

They

are oxidized to acid s and hydrogenated in varying degrees when in je c te d o r fed o ra lly to ra b b its (19).

I t ha® been claimed th a t menthol poisoning*

in te rfe rin g with uronie acid metabolism, cause changes sim ila r to those o f vitam in A deficiency (4 9 ). Although no tru e biochemical antagonist has been demonstrated fo r v i t a ­ min A, th ere i s one o r more known fo r each of the w ater soluble vitam ins (52) • The discovery ©f an an tim etab o lite f o r vitam in A would be indeed o f s ig n if i­ cant value in attem pting to gain an in sig h t in to the possible mode of action of the vitam in aside from i t s ro le in the v isu a l cycle.

A system atic study

o f rea d ily a v aila b le s tru c tu ra lly re la te d compounds seemed to be th e most lo g ic a l approach to t h is problem.

In view of t h i s , experiments were arranged

fo r th e purpose of te s tin g g e ra n io l, menthol, methyl ionone as w ell as saualene and phytol. was included a ls o .

Due to the rep o rt o f G hevallier e i a l. (13) d ieth y l e th e r

METHODS

9

A,

Source and Preparation of Reagents

Benzene- Commercial thiophen© fre e benzene was mixed thoroughly with concentrated s u lfu ric a c id and allowed t o stand over concentrated s u lfu ric acid fo r approximately 12 hours.

A fter separating the two phases* the ben-

zone so lu tio n was washed with water u n til free of a c id and i t was f in a lly d ried over sodium and d i s t il l e d from th e m etal. Chloroform- Both C.S.P, and recovered chloroform were dried over an­ hydrous reagent grad© potassium carbonate and d i s t i l l e d from the same reagent. D iethyl Bther- Commercial eth e r was mixed thoroughly w ith a c id ifie d f e r ­ rous s u lfa te so lu tio n .

This was followed by drying the e th e r over anhydrous

potassium carbonate and d i s t i l l i n g i t from th© same reagent. Ethyl Alcohol- 95 Per cent eth y l alcohol by volume fa s tw ice d i s t i l l e d . This alcohol was employed as the solvent in a l l spectrophotom etric work re ­ ported with the exception of th a t on lycopene, Skellv Solve B- Commercial (Skelly Corp*) petroleum e th e r (B.P, 60-70°C,) was ex tracted with two portions of fuming s u lfu ric acid .

This acid e x tractio n

was followed by a 10 per cent sodium carbonate wash and a 5 per cent a lk a lin e potassium permanganate wash, of 5 per cent of

(The potassium permanganate so lu tio n consisted

t h i s reagent in a 10 per cent sodium carbonate s o lu tio n ,)

These operations were followed by washing the hydrocarbon f if te e n tim es w ith water* drying over sodium and d i s t i l l i n g from the same reagent.

The f i r s t 5

per cent and th e l a s t 10 per cent of the d i s t i l l a t e were discarded* Activated Glycerol D ichlorohydrin- Glycerol dichlorohydrin* obtained from the Shell Chemical Corporation* was tre a te d with antimony tr ic h lo rid e and d i s t i l l e d according to the method of Sobel and Werbin (4&) The se n si-

10 t i v i t y of the reagent depended upon the quantity of the antimony which d is­ t i l l e d in the complex as well as possibly other fa c to rs .

B,

Dietary Substances

Vitamin A D eficient Diets Diet 301 (D 301) Composition

Source

P arts

Devit&s&nised Casein

9

S heffield Farms Co,, Inc, Hew York

Defatted Wheat Germ

9

Viobin Corp., M onticello, H I.

Cerelose

58

S a lts 12a

h

Brewer* s le a s t Irra d ia te d Y east, Std. 3&0 Crisco

Cdrn Products Refining Co., Pekin, H I .

Anheuser-Busch, I n c ., St* Louis 2

10

Fleisohraann*s, N.I.C. P rocter and Gamble, Cincin­ n a ti, Ohio

M e t 30 . (D 30) This d ie t had the same composition as D iet 301 with the exception th a t the 9 p a rts of wheat g em was replaced by 9 p a rts of vitam in A free Casein to make a t o t a l of 18 p a rts of t h i s material.. Diet 31 (D31) This d iet had the same composition as Diet 301 with the exception th a t the 9 p a rts of wheat germ was replaced by 9 p a rts of a crude casein low in vitam in A content which was not s u ffic ie n t to prom ote storage of the vitam in.

The crude casein was obtained from S heffield Bi-products Co.,

Hobart, Hew York.

11 Diet 32 (D 32 )

th is d ie t was th e ©am© as D iet 31 w ith th© exception th a t th e 58 p a rts o f cerelose was replaced by 58 p a rts of sucrose. All th© dry in g red ien ts were mixed and s o ft Crisco blended in to give a homogeneous m ixture. Stock Diet Composition

P arts

Source

Yellow corn, ground

50

Bloomington M illing Co.* Bloomington* Ind.

rthole wheat* ground

29

Bloomington M illing Co.* Bloomington* Ind.

A lfalfa l e a f meal

3

Bloomington M illing Co., Bloomington* Ind.

Whole iidlk powder

10

B essire & Co.* Indianapolis* Ind.

Crude casein

5

S h e ffie ld By-product e Co.* Hobart* New York

Irra d ia te d yeast* Std* 360

1 .7

Fleischm am ^s, New York City

Calcium carbonate

0.5

Calcium hydrogen phosphate

0.3

(CaHPO^)

Sodium chloride* iodised

0.5

S a lts 12a Composition Sodium chloride

147.0

Potassium dihydrogen phosphate

409.0

Magnesium sulfate (MgSC)^^)

87.0

Calcium carbonate F e rric c it r a t e Potassium iodide 5

P a rts

c^ ’or^’£is

400.0 35.0 0.9 4.7

12 S a lts 12a Composition {continued)

P arts

Zinc chloride*

1*2

Copper su lfate (C u S O ^ O )

0.5

The above ingredients were pulverized sep arately to a fin e homo­ geneous s ta te in a mortar a f te r which they were mixed thoroughly. *Both manganous s u lfa te and zinc su lfa te may be su b s titu te d fo r manganous chloride and zinc chloride.

C.

Preparation of Supplement M aterials

^-C aro ten e- The ^-caro ten e preparations employed were obtained from General Biochemicals* Inc.

One supply consisted of 90 per cent beta and 10

per cent alpha carotene$ and another from, the same source consisted of 05 per cent beta and 15 per cent alpha carotene.

The d esired amounts of the

commercial ^ -ca ro te n e preparations were dissolved in peroxide-free e th e r and these solutions ware mixed thoroughly with a determined amount of Wesson o il to give the d esired concentration.

Th© eth er was removed under reduced pres­

sure on a warm water bath and the re su ltin g carotene-Wesson o i l supplements were sto red in low -actinic fla sk s a t 4°C. under nitrogen. jfenthophyll and Lycopene- These supplement m aterials were suspended in Wesson o il in the same manner as the beta-carotene. Vitamin A- One p a rt by weight o f D .P .l, vitamin A concentrate* consisting of approximately 200*000 I.U . of vitamin A per gram* was dissolved in th ree p arts by weight of Wesson o il to give a solution containing approximately 50*000 I.U . per gram.

One drop of th is solution was equivalent to 1*250 I.U . or 277 micro­

grams of vitam in A.

13 Geraniol- Five grams of geraniol obtained from Elmer and Mend Company Has dissolved in enough wesson o il to make 20 ml. of solution and to give a solution containing 50 mg. of the substance per 0 .2 ml* Methyl lonone- This material was obtained from the Verona Company, H.Y.C. and i t was prepared in the same manner as the geraniol supplement* c j - and 3 -Ionone^- The req uisite amount of each substance was dissolved

in wesson o i l to give the proper concentration. Menthol** The desired amount of menthol obtained from Eastman Kodak Company was dissolved in warn Wesson o il to give th© desired concentration. Squalen© and Phytol- Squalene, obtained from D is tilla tio n Products, Inc. and consisting of over 90 per cent o f the unsaturated hydrocarbon, was dissolved in the requisite amount of Wesson o il to give the desired concentration as geraniol and methyl ionone. likew ise dissolved in

Phytol, from the American Chlorophyll Company, was

wesson o i l.

Vitamin B- A predetermined amount of alpha-tocopheryl acetate, obtained from Merck and Company, was dissolved in su ffic ie n t Wesson o il to make the de­ sired concentration of solution.

D.

Care of Animals

Method o f Preparing Vitamin I D eficient Animals Unless otherwise sp ecified a l l rats employed were albinos.

In experiments

dealing with vitamin A d eficien t animals, approximately three to four weeks old rats suitably marked for id e n tifica tio n were placed on a vitamin A d eficien t diet in groups of seven to eight rats per galvanized iron cage of approximately three * Both