Univers

ity of

Cap

e Tow

n

Univers

ity of

Cap

e Tow

n

The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non-commercial research purposes only.

Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author.

'l'o

~o:rcmia

" It is va1n to do with more what can be done with lesa.u

William of Occam 1290 - 1349

CONTENTS

PART I

INTRODUCTION ·

SECTION 1 The pzl!oblem ......... o ................... . 1

SECTION 2 The effect of dietary fats on the serum cholesterol level of' man •••.•• o • • • • • • • .. 5

SECTION 3 The effect of dietary fats on the serum cholesterol level of rato •••• 4........ 12

SECTION 4 A pilot e~erirnent on l'ats ••••.•••••• ,. 17 (EXperiment 1)

A. Introduction....................... 17 B. Experimental ..................... ~· • 18 c. Conclusions....................... 22

SECTION 5 Method of approach...................... 23

METHODS

INVEST!GATION OF TI:IE RAT AS AN EXPERIMENTAL ANIMAL

SECTION 6 The development of an accurate method for the total serum cholesterol deter­mination in 0.2 ml of blood............. 25

A. Methods based on the Liebermann-Burchard reaction,................ 25

B. The p-toluene sulphonic acid method ................ ID............... 26

S~!ON .. :Z

S.ECTION ~r.

o. The ferric ohlo:r.ide method •. ·•••• 21 D. ~e further refinements of the

ferric oh~oride method •• ~......... 28

A mioro .... me·thod fo:r the detern11nat1·on of total serttrri cholestl;lrol in 0•2 ml .ot· blood ............. • .. ~ ...... ·-· · · ·· 31

A. EXperimental . ~ •.• ~ •••••.•..•• • • 31 »• Rersul ts. . . . . . . . . . . . . . . . . . . . . . . . . 35 c.. J)1seo.ssion • . • • • • • • • • • • • • • • • . • • • 38

DeSign of a.n:tmal·e~pe:r!ments ••••••••

A., ~Ch~ioe. ot exper:tmental dtet .... . Be EXpei'imen~al deeisn ............. .

Effect tof. various "fats and o11s On the seX'tlm . eholeste%'ol. concentrat;ion of ttatf!J'.

I , . . .

41

41 43

46

. Pu .· Eft~ot of ~eplaeing ooaonut oil by sunflower :Seed oil . . . • • • . . . . • . 46

(Experiment 2) B. ·· ~ffeot · ot ·but te:t' ·:fat, .. bee.f tallow,

groundmlt oil. ·and fish oil...... 50 ·. . (Experiment 3) .

di Effect of small quantlt1~es ot dietart cholesterol . • . . • . • . • • . • • · $4

· (Ex.pe~iment ·4) . D;~

E. Discussion .•.••••••••• ~ ••.••••• Conclusions £rom .Part II •.•..••• . . - - .

57 6o

'l'HE HYPOGHOLESTEROLPEMIO EFFECT OF OF . StJ'N'FLOWER ,SEED o·IL

SECTION 10'

, SECTtON 11

Intt'od.uction ...•••••••.•••••. · ••..• · ••

Effect ot: whole suntlower seeds ....• (:&:~ertment 5.)

SECTION 1~ The chemical fractionation of sunflower seed oil ............... ., • 68

A. B.

c.

D.

Preparation. of f'.ractions •••.•• Effect ·Of saponj_fiable and unsapol11fiable matter ......... .

{Experiment 6) Effect of' interchanging unsap­onitieble matters .of coconut and sunflower seed oils ......... .

(Experiment 7) Discussion ••••••••••••••••••••

68

71

74

16

SECTION 13 The pbysieal fractionation of sun-floweJ."' oeed oil ••• -............... 80

A.

B. c.

Liquid propane segregation of sunflower seed o11 •••••••••••• Composition ~f p~opane fractions Effect of various fractions •••

(Experiments 8 and 9)

SEcn'ION 14 Investigation of the unsaponi:f'iable

80 84 89

matter by feeding pure compounds.. 95

A. Effect of' r.:, -si ·tosterol. • • • .. .• .• 95 (Ei:periment 10)

c.

D.

Etf'ect of· squalene ............ . (Experiment 11)

Combined effect of ~ •si tosterol, P.:, -carotene • c:J..- tobopherol and

s4ttslene ................... , •••.• ., (Experiment 12)

Discussion

98

100

SECTION 15 Conclusions f'rom Part III ........ .,.. 106

PART IV

THE HYPERCHOLESTEROLPEMIC PROPERTIES OF EGG LIPIDS AND THE ABSORPTION OF CHOLESTEROL

SECTION 16 Introduction...................... 109

SECTION 17 Effect of triglycerides from hens' eggs.............................. 113 (Experiment 13)

A. Choice of exper~ental diets.. 113 B. Preparation and composition of

lipid fractions............... 114 c. Results and Discussion........ 116

SECTION 18 The absorption of dietar.y cholesterol 120

A. Effect of large doses of cholesterol fed with and without sunflower seed oil............. 120

(Experiment 14a) B. Effect of feeding cholesterol

and oil separately............. 123 (Experiment 14b)

c. Discussion and Conclusions..... 127

PART V

DISCUSSION AND CONCLUSIONS

- THE SOLUBILITY HYPOTHESIS -

SECTION 19 Discussion and conclusions from Exper~ente 1- 14................. 131

SECTION 20 The solubility of cholesterol in triglyceride oils.................. 135

- 1n vitro experiment -

SECTION 21 The solubility hypothesis.......... 140

A. Statement of hypothesis........ 140

B. Consequences of the solubility hypothesis..................... 141

c. Conclusions.................... 145

SECTION 22 The solubility of cholesteror in sunflower seed oil ••••••••••••••••

- in vivo exEeriment - 146 (Experiilieni 1!5 )

Conclusions from Part v........... 149

PART VI

APPLICATION TO MAN

SECTION 23 The absorption of dietar.r cholesterol by man............................ 150

(Experiment 16)

SECTION 24 Application of the Solubility ftY.pothesis to man •••••••••••••••••••••••••••• 157 A. Effect of natural :rats and oils 157 B. In vivo solubility determination

{n man ••••••••••••••••••••••••• 159 c. The serum cholesterol ra!sing

p~operties of egg lipids •••••••• 161

1). Effect of ~-sitosterol •••••••• 162

E. Effect of hydrogenated oils ••••• 163 F. Effect of oils relative to a fat

free diet ••••••••••••••••••••••• 164 G. The rate of change in serum

concentration ••••••••••••••••••• 165 H. Discussion •••••••••••••••••••••• 167

SECTION 25 The rete of diffusion of cholesterol from serum to different :rats............. 169

A. The intravenous injection of fat 169 B. The diffUsion of cholesterol

from serum to different fats.... 170 (Experiment 17)

SECTION 26 Cholesterol metabolism as affected by different dietary fats.............. 175

A. Circulation of cholesterol...... 175 B. Intestinal absorption........... 177 c. Excretion....................... 179

D. E.

Liver synthesis •••••••••••••••••••• Conclusion •••••••••••••••••••••••••

SUMMARY . . . . . . . -· . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ACKNOWLEDGEMENTS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

PUBLICATIONS ....................................

REFERENCES ................ ' . ,. ...... -· .......... .

OoooOoooO

183 186

188

191

193

194

PART. I

l N T R 0 D U C T I 0 N

1.

SECT!ON 1

TH! PROBLEM

The or:tginal investigation Which led to the chain

of events of' Which the present thesis is a small part • tvas a

surveu of the incidence of ischaemic hea):'t disease among the

different raetal groups in cape Town. conducted. by Bronte­

stewart, Keys and Brock (1955}. Amo~ the results arising

out of' this work was the clisooveey that highly significant

di:tf'er-enoes in tne meen total cholesterol level exist between

the three racial groups, with the Bantu having the lowest,

the Europeans the highest serum cholesterol and the Cape

Coloured somewhere in between., ~hese differences correlated

with a number of factors among which was the habitual

consumption of fat, the intake of the European being more

than tWice that of' the Bantu, w.tth the Coloured again in

between. These authors conclude:

"The results of this suztV"ey support the bypothes~s that the dieta~ intake of fat influences the level of the serum cb.olesterolp ...... ,.and may be one of the major :factors influencing the pathogenesis of coronary heart disease. "

A fUrther finding was that Within each racial group

the serum. cholesterol level was highest in those with the

highest income and bore a parallel relationship to the intake

of foods x-ich in fat of animal origin, 'Whereas there 'ro.s no

correlation with the intake of vegetable :rat. This indicated

2.

that in addition to the quantity of fats consumed; the

origtn o~ qtlal:tty of these rats seemed to have an important

bear>ing on the resul t1ng serum. cholesterol .level produc¢d•

To investigate this aspect, Bronte-stewart, Antonia.

Eales and Brock (1956) fed a variety of different fa.ts and oils

to humans under strictly controlled conditions. studied the

effect on the se:rum cholesterol levels and concluded 1

••An:tmal fats and hydrogenated vegetable fat behaved. differently from vegetable and marine fish and mammal oils. 'l'he most likely common difference between these fats and oils is the proportion of unsaturated and saturated :ratty acids 1n the f'ate and oils concerned."'

Thus whereas relatively saturated fats such aa butter,

pal'tia:llY hydrogenated groundnut oil and beef muscle tat

raised the sei"'l.m cholesterol level, unsaturated oils such

as sunf'lowet' seed oil, groundnU.t oil and South AfPican

Pilchard oil had the opposite effect.

Due to their possible bearing on the pathogenesis

· of ischaemia heart disease, these interest:f..ng findings have

triggered off a vast amount of resear-ch in all pal'ts of' the

wovld• which has on the whole confirmed this work in

innumerable studies on animals and man. A considerable

portion of this woxak was, and still 1s; directed towards

attempts to explain why and how different dietar,y tats

produce. different sexrum cholesterol levels. Many interesting

experimental facts have been reported and a number of theories

have been proposed to account for these facts•

None of these theories; however~ bears detailed

and critical examination and marw- investigators would

probably agree that the factors 1n fats and otls responsible

for their serum cholesterol regulating effect al?e at present

(November, 1959) still ill def'ined, if not completely

unknown. :rt is the object of' the present thesis to contribute

towards a solution of this problem. This can perhaps be

stated more clearly as follows -

VJhat pt-ecist!ly are the chemical or pbysieal p~operties of different natural triglyceride fats and oils which, when fed to man, produce different serum cholesterol concentrations?

!n the following pages• attention will be confined strictly

to the solution of this limited objeo.tive. Many investigations

relating to this subject deal with the effect of different

dietacy substances on '•cholesterol metabolism", i.e. the

slnru.l taneoua effect is studied on a variety of blood lipids,

ta(;cal ste:rols, liver lipids, cholesterol synthetd.s 1 ·bil.e

acids, etc. 'l'he:ve is no doubt that suCh oomprehenaive studies

yield valuable results,. They often suffer f'rom the disadvantage,

however. of being dit:f'icul t to interpret ana are consequently

wastefUl of much ingenious and laborious experimentation. It

is :f'eltt therefore, that a precise ·definition ot the relation­

ship between two single variables (i.e• property of d:letaey

fat versus serum cholesterol level) mey be of value as a

foundation to more comprehensive studie.au.

The fi~st a1m-1n an investigation of this nature

is the creation ot the neeessaey e~pe.rirnental mach1neey, the

scope of Which is, of course, deter.mined by the resources

available. As the title suggests, the present study is mainly

4.

concerned with eXper1tnent.at1on on rats. To substitute rats

for humans requires extensive qualification and justification.

A major part of the work Will the.refore be ·eonoerned in

investigating the rat as an experimental animal and in

demonstrating that under certain conditions rats react in

a s.1milar fashion to humans as far as the biological mechanism

under study is concerned. It is believed that this has been

done satisfactorily. · It follows that acy- results and conclutdons

arrived at from experimentation on rats are likely to throw

stl'ong light on the parallel mechanisms under study in humans.

An attempt will therefore be made in the concluding parts of

this thesis to apply any new knowledge gained on rats to the

human sphere. This new knowledge:• after integration into a

theory (the solubility theoey) will then be offered :f'or

consideration as a new solution to the problem stated above.

Furthe~ remarks oonce:rning the general structure,

f'o:rm ot presentation and plan of this investigation will be

made 1n Sections 5 and 8; after the work of some ot,the more

prominent workers in the field has been briefly reviewed, and

the :resul.ts of an e:x:plo1 .. ator.v experiment on rats reported.

Against this background more detailed discussion \Vill become

possible.

.SECT;tON 2

THE E.FFEC'T OF . DIETARY . FATS

ON THE SERUM . CHOLESTEROL OF .. MAN

Although the serum cholesterol regulating e.ffect

of dietary fats has been known for a considerable time, it

is only oomp~atively recently that it became evident that

fats of different chen'iical compos:i.t1on and o:rigin behaved

differently in this respect. Up to about 1952 the View

was prevalent that all dietary :rate raised the serum

cholesterol and that the extent of this .effect was proportion­

al to the amount of fat fed, ana \vas unrelated to the origin

of' the :rats fed~ As recently as 1952, Keys discussed the

relative merits of different fats in raising the serum

cholesterol and came to the conclusion that •tthere is not

the slightest eVidence for a dif'f'erence between animal and

vegetable fat."

one of the first indications to the contrary

apparently- came from Groen et ar. (1952) who fed diets rich

1n different tats to humans and found that animal f'ats

produced higher sertun cholesterol levels than vegetoble fats,

Kinsell et al. (1952) actually produced a lowering of the

se:Ann cholesterol level in hosp1 tal patients consuming d.ieta

rich in vegetable fate Two y-ears later; Mayer and associates

(1954) followed with a report o'£ a greater serum. cholesterol

~a:t.sirig eff~ct of lard as compared with vegetable tats

eons1trting or· g~ounanut o:l1, corn oil and "t~ge1a.itbl·e

tt~argax-ine• h!"thet? ·Confi;Anat1on of the· ~iew that anttmal ' . '

fats behaved differently from V'egetable fats 'came from ' ' .

the work ot Harding and st~e (1954) who campaved the

eel"'1lt1 choie13terol lete.ls of' vegetariana and ·non,--vegetarot·ana ana. found the·lattel!' to ha.\l'e higher valuesco

A1 though·· no agreement had beeri. .f'ea~hed.. as te>

whethei'i' vegetable £ata actua~l:V 1qwered. the serum choleste.ttol

level or only produced a moderate lt'ais1ng et:re~t ~ it b~came

e'ttS.dent that in genei'al they behaved ditteX>ent).e front animal

that this aifferent behaviour is not due to the . - . . - - '

. srt~all Cholesterol ·content eommc:mly touna in t:luclt tat$ as butit;l!r, beer, tal.lovt; lax*!, etc, has been shotvn 'by: man3

wottke:rs, nota'bty Messinger et a:L• (19SG} and can therefore

b$ assumed to be of little' i.nlportanctb

tt is · a.gainat this 'back(srpunci · thAt Iron te*Ste\'iJaM

and his eQllaboPato~s (19.56) took one step :f\tvthets by

aem.onstrfiting that certain Qhem1ca1 p:rol>e:rt1es of fats such

as total unsa.tuttation, 11nole1e. a:eia content oti 'r$1ated.

prope~ies such. as the «>ntent of' 'essential fatty acidti ·

oorrelated well with tbe serum ~holestero1 'Vtalues as Aiscul3eed

in thf) previous .aeotionr~ ''!'his cor~lation1 tttrthermore, was ' '

inilepeJltient .of the or:tgtn of the tats a.nd ·oils :rea, tb.u.a

sup~~~e<iing the trantmal 'V'ersus "egeta.bten tat theoey. A

typical exp~;rfment is illustrated in Fig., 1.

Late:r in the same year§ Beve~id.ge, Oonnel. and..,

Meyer (1956, l9S7a) studiea·the prop~rt1es of butter arid

7.

corn 01~ and ~epovted that whereas ~tocophe~ol (Which is

present in corn oil) when added to butte~ did not abolish

·the elevating ef'fect of the latter, ~-sitoster-ol when

aam:tnistered in doses much in exeesa of. those present in

corn oil did produce a highly significant depression of

the serum eholestel:'ol, which eq1.1alled that of com oil

itse:t::r. These workers then went on to study the molecularly

distilled f'rsot1ons from both fats and found the lightest

:f'ttaction f'rombu.tter to have the most potent elevating

effect whereas the same fraction from corn oil had the

most depressing effect. They noted that these active

fractions were associated in both cases with high percent ....

ages of unsaponifiable matter consisting of mueh d.igi tonin•

preeipi table, tiebennan-B'IJ:.lf!ehard. positive sterols. As a

result of' this they suggested that the composition of the

unsaponifiable matter may be partly t>esponsible for the

potent depressing effect of eo~ oil. It must be pointed

out• however, that some o.f these differences could not be

shown to be statistically significant.

:It is at about this point in time (Marcht 1957)

that the present investigation was initiated, Mueh material

has been published since, Some of this will be discussed in

more detail in z-elation to the findings of tHe present study.

The more widely held views at present cant however, be

eummarised as follows:

Kinsell and Sinclair (1957) believe that the

hypooholesterolaemic action of unsaturated oils is due to

l!ll!i!!l. EFFECT OF DIFFERENT FATS ON THE SERUM CHOLESTEROL LEVELS IN A MALE BANTU AGED 37

3000------------------------------------------~------~ c~~uzooo ---~~~~~~~~~~~~~~~~,.~~~~ 1000

.0

200

F'(;Z~~u 100 0

140

IZO

Suttt!r

I.

$tRUM CHOLESUROI. 100

("'9./IOOml.)

:r/ - ... - t'----

I :

CD ,.. Cl ... e 0

·=

' 1: 80

60

I\ODY·WEIGHT 140

(I"' 13S l.-..o~.o~ow.w.l ......... .J:::r:::::t:::t::::~:::::::::::::::::::::=:±:::::::::::w_j Q 10 ~~ zo 2s ·30 45 50 55

D .. YS

&sed on Sronte-:St~w<trt, Antonis. Eoles & Brock (1956)

Example of experiment l)~ Bttonte-Stewart., Antonisj Eales and Brock, 1956.

15~----------------------~-----------------------r-----------,

AVERAGE PERCENTAGE CHANGES IN PLASMA CHOLESTEROL VALUES FROM

THOSE OBSERVED AFTER 8 DAYS ON DIET PROVIDING 35% CALS. FROM BUTTER

G-----------------. 0 5

---·-- -------· 6

----------o .... ~ ~ --~ -,

~~::-- -ell35% eels. I rom bul!erfat + - ............_.., ......, ---- ._ e 1

25% corn oi I ) ·---._ _ ----- -o 2 02135% eels. from butterfat+ sitosterol -._ · ----.0 3

in diet I ) --.

03{35% cals.from butterfat+ 25% hydrog. corn oill

e4 { 35% cola. from butterfat t 25% hydrov. corn oil t sitosterol In diet I l

05 (35% cols. from butterfol t25% cocoonut oil)

e6 (35% col s. from butterfat t 25% cocoonut oil -!-sitosterol to bring to level of diet I l

----. 4

-25~--------~----------~~--------------------~--------~ 6 12 16

Time- doys

EXample .of experiment by Beveridge, Connell, Mayer and Ha~~t. 19,58.

the presence of so-called t1e.esentj.al fatty acids",

:t.e. linoleic acid• linolenic acid and arachidonic

aoid .. , Kinsell,· Michaels, 'Friskey and Splitter '(1958}

concluded after the feeding of purified ethyl and

glycerol esters to healthy and unhealthy humans that

"the major active 1ngred1·ent in various vegetable fats

responsible for!' lowering the plasma cholesterol is

linolenic acid.•"

One of' the major criticisms against this

v.iew has been made by Ahrens et al. (1959a) who pointed.

out that fish oils which are the most potent cholesterol

lowering oils known are practically devo.id of essential

fatty acids. Even more recently, Keys, Anderson and

Grande (1959) actually l'eported an increase in serum

cholesterol after administering a p.reparation of arachidonic

acid to human subjects. Ahrens and eollaboratoPs (1957),

after conducting one of the most comprehensive investig­

ations into the properties of a large number of' fats and

oils, exPressed the opinion that the total unsatu:ration

as measured by the iodine value constitutes the best

correlation obtainable to date. They admit., hoWever;.

that there are serious discrepancies which Will be evident

from the inspection of Fig. 3 ..

"' z 0

160 ..,

140

120

100

80

10.

... _, -SAFFLOWER

,___CORN

,..._cOTTONSE.E.O o-4

-LARO ---PEANUT

SPk~"oE ~~HICKEN

Q ,..--LARO

6 0 PALM

-LARD

-SEEF _£

4 0 -BUTTER .3 ,--COCOA SUTTER

2 0

!--COCONUT

+3 +40 •SO ... 60 •70 ... a PER CENT -10 0 -tiO +20 0

0

·····----~~----__;....--------"""'

Relationship between iodine values of dietary tats and serum cholesterol .levels, expressed as percentage diffe~enees from base•lines established during ingestion of oorn oil. Open bat' at z.v. 126 = estimated reproducibilit;v of' corn oil base-line (from Ahrens et a.l.,1957)

Ke;vs 1 Anderson and Grande (1957, 1958), This

group claims to be able to pltied1et changes in .seJ:ltml cholesterol

level due to a change in dietarzr fat from the following formula:

L\ Chol. = ~. 74 l::l .s - 1. 31 D P

Where Ohol. = change in serum cholesterol concentration in

mg./J.OO ml s = difference between percent of total calories

pltovided. in the two diets from glycerides of sat1U'ated fatty

;· .aq1tls and. p being the co:rrespond~llS dtf:t·el'erice tn

giy,Certdes Of poiyunaatu.:rated fats"· 'ritta i¢nnu1a ~elegatea t • ' ' ' ·', . ' • ' • :' 'I ' I • .._ 1 '_' , f

the mono.;;.Ur1satura.ted o1elc acid to a completely neutre1

position, ' ana: tncidentail.y b~eelks dovm 1~ the. ease of 00~ '• '

: · ol'l. which l,rrodttees lowe~ choles·tero1. values than p~d.ictea

.. While '1\vdl?oganation. 'of oil~ 'p~~tiuees 'vaiu~s ~ot. ~$ hi~ 'as·, I

,ilr-ed!cteil,

· ' · · tseve~ttig~ ·et ~i.J · (t958a j' discu~sing some · further

·" wof.k with i)utteJ!'1 cooonut ¢11 .. natural ana.. llyd.X"ogenateti ;

' ,. CGtn Q{l.·· .eonel:ude ~'ftteJ'6 oan 'be no doubt .theit' .in th:e·.e~S~ 'j . - '· . j, ·"{, _,., ' ' • ' •• ~ • •. -~ • ' • ) -~ .._ ' • • ' ~ ' • : ·: ~ "";

.. 'of eom ·()tl at le~~t. ;plant a1le:r~1$ such. ,a$ ~1t~~i;erol, . ' • s •• i • I '• I' .' ; <.~-~.~. ~<J•••. '· , .i" , , ••

jl

,crt some. other .substance associated. with S.t in t.he 'll1U~aponif1able :, > f·; ,'. ( ',. ,'. • ' t • , •• ' I! ' •' ' '',· ' ' . ', •

matt~r; a¢CO'f.Ult .fo:r mt1ch of this (cho;lestezoo).. ;towef>iM) '. ,

1 ', 1 J' 1 •· , • 1

1 •· ' ' • 1 • ,·,

activity"• After f.!nding only- a minhtlal eftect with th.e . , ' > I ·' .·t I"' -,, '. 1 J •. , t , ' ' :

f •' I .

arttf!Qial. £$atu.rat ion or hy.dro~enat ion o.f' com oil they

. ' ftttiothe~ ~tate that n •••• thel'e 1.~ no eLnple relationship

between degree of saturation ana the ab11ity of a .f'at to

depress plasma oholesterol leYe1a. tt

!'rom the. above discues1on 1t is evident that ~

present state ot knowledge ot the subject is high1:v unaa~ia•

:faotoey and. btasmentat"y. It ean aat'elsr be said that th~

ttactora .in dietary rats and oils which inf'luenQe t:ha serum.

cholesterol level a:re at p~esent st:itl unknown. To venture '•

an 1nt~lllgent guess as to a poasible mechanism bjr which

dietary fats behave as they do!l is theref"ore premature a.t

this stase•

12.

EFFECT OF DIETARY . FAT .ON TH:E SERUM CHOLESTEROL

LEVEL .. OF THE . RAT

"lhe amount ot work that has been devoted to the

study of the effect of dietar~ tats and cholesterol on the

s~rwn cholesterol level of animals 1s pt-otU.gioua-. No doubt

this is due to its growing interest in relation to the

pathogenesis of athevoeclerosis. Another reason may be

the comparative ease with "V~rhieh cholesterol is determined

in the laboratory• To give even a brief outline of all anirnai

work 1$ beyond the scope of' this study.. l::f attention is mostly '

confined to experimentation with rats as experin'lental animals

the output of work is still gl'ea.t and the results obtained al'e '

bewildering. Fortunately, good reasons can be found for making

an· even narrower selection of mate~ial tor closer examination.

tn the great majority ot animal eXperiments in which

dietary factors are studied in relation to their eff'eeta on

the se:~JUm cholesterol level, the diet itself' was .supplemented

with cholesterol* In the case of the rat this was often

tuz-ther supplemented with othe~ substances such as cholic ac1ch

Thio'llracil and other d~a were oocaeionally ad.d$d to lower

the basal metabolic rate. The purpose of' these manipulations

was to produce or aocentuate effects not normally o'bse:rved.

with .motte natural diets.

'.r4ere can be no doubt, that in the ease of man at

least, dietarz.fats .... mostlJr tree from cholesterol- have a

ehal'acte:ttistic effect on the serum choleste~ol level. It is

p!:>eoisel;y this effect, however, which is under study and large

doses ot cholesterol must therefor-e be e~cluded from the diet

1f conclusions arr11red at from animal experimentation are to

have any- application to man. Attention \rtill ther-efore be

focussed mainly on report~ of feeding experiments with more

natural diets.

It is curious that the claas1eal work of Sohettle~

(1948) ts rarely quoted in more modern texts. He ted ·various

animal and plant fats togetber with a basic .diet consisting

largely of skim milk to mice. . Dog depot fat, lard and tallow

cleE~r.ly raised the blood oholestero1 Whereas the more unsattll'ated

. sesame and oliVe oils had little influence. Although these

results antie:ipated the e:f":f'eets now accepted ·tor hu.man$, no .

attempts we:re mede by othel's to conf1JOm or extend these studies.

The work of' swell et al. (1955) on rats illustrates

the very d~f'.ferent effeets obtainable by feeding a fat (olive

oil) under different conditions~ Mnoh greater elevating

effects on the serum cholesterol level s.re obtained when

cholesterol and sodium taurocholate are included: in the diet~

In the same year, Shapiro (1955) obset'Ved. highe:r serum values

with saff'lov1er oil than hydx-ogenated vegetable 'tat in the . unsupplementea diet of rats·. This is not in accordance with

later human exper1en~e.

One year latel'. Olson, .Jablonski and Tatlo:r (1956)

.fed cholesterol-free diets to 1-ats but added various amounts

of methionine and choline to the rations• All ~Sel"Wn lipids

were depressed bUt .,. ••• "this effect was not influenced by level

or kind (larda corn o.il• butter) o.f diet.aey fat." S.imile.r

resul'ts w.i th natural diets v1ere obtained by Okey and Le:ym.an

(1957)., who were unable t9 find cona~ste~t tU.ff'erenees between

tote.l cholesterol levels in coconut oil and eottonseed -oil-fed.

animals.

e;x:tend.ed. thei~ :Ln'\restigat1ons.to oth~r f'at~S which were fed wlth ' . ' i ;

and Without cholesterol .supplement f'or se'ren weeks _.after weaning.

Again the serum cholesterols _produ~ed by 1.3 d~ferent saturated

and un-saturated. fats showed no co.rrelation. With results obtained

on hUmans. 'i'he eame results wei'e, _tor example~ obtained with

corn and coconut oil.

Grunba1.1m; Geary, Grande:• A~d~rson and Gl:ick (1957)

aotual.ly :obse~ed higher sel'Um values with sunf1owerseec1 o11

as eompat>ed with butte.r when fed to !rats on a e)lolesterol-free

Pu~ina. CJhow diet. The ~imals of A:f'tergood, Deuel and Alf.:in­

Slater (1957) ro.e<FJ.ired 24 weeks • ,.reeding under similar conditions

to demonstrate a signiticant ditf'erenoe in ~etf'ec:t between .dietary

lard. and cottonseed o.il; ;the latter oil. producing the iower

values., Diller, Woods and Harvey- (1957) :tailed to demonstrate

any 1nfluenee of' hydrogenat·ion on the e:t'f'ects of corn and.

Safflower Oil in the cli-ets ot ratS;o

More recently Best, Lucas; Pat:terson and Ridout (1958)

.studied the ef'f'ects of ten different ed.ible o.ils a.nd fats on

total h,e_pat1c lipidS and S~r'U11l cholesterol in the presence o.f

;rarious amounts of dietary Choline and concluded that Ji & •uno f'a.t

or oil produced a level of se:t'Wn tiholestet>o.l that -diff'·ered

sighifieantly .trom that produced by ·any other11•

Nath:; Wiener, Harper and Elvehjem (1959) developed

a diet containing 25% or hydrogenated coconut oil., 1% of

cholesterol and o.s% of oholio acid. Tbey claimed that this •I ~ - ' •

diet is 'e · •• "suitable f()l' the study of nutritional faotcrrs

af'f.eeting sei"Um eholesterol eon()entration in the ratt•. 'i'hese

workers do not state, however., exactly .what nut.r1 tional :f'acto.rs

are to be ;studied under these a.rtifictal condit1Gl~• Ne.:tther

dietary cholestet'Ol nor f~ts are apparently envisaged~ The

former .is included in the basic ·diet. whereas of the latter 1 t '. is said that t~•••"the variations in t.he fat level of the diet

had 11 ttle effect on the .serum cholesterol .eoncentration° •

Even a b.riet review of the subject is incomplete

without referring to :t,he extensive work on rs.t.s of.' negsted. and

his associates. Although these investigators fed cholesterol.

choline and chol:i.e acid along with fats in the diet theit>

results ·are nevertheless wo.rth noting. They have recently

(Heg.sted;,. Gotsis., .Stare and Wo~cester, 1959') .summarizea thei.r

findings in the f'om of an equation., . log., serum cho1. = -o.-oo648s + o~o2105M ·~· o.o2415P

+ 2.4836 ;.

This relates the serum cholesterol level in mg.~/100 mi with

the pet'cen.tage of .saturated fatty acids (S), monO<o-Unaaturatea

tatty acids (M.) an(i poly-unsaturated acids (P) in the dieto

This equation suggests that the monQ .. unsatttrated acid raises

the serum cholesterol level while saturated and po1y--satuxoated

acids reduce it. Unfo.rtunately these autho·rs proc$ed to quali.ty

this by disclosing that other equations ean be derived. which

give as good a "fit" as this equation.- 'It ia thus .not easy

to see what conclusions oan be drawn :from this work. Nor is

16.

the relationship of these findings to the human sphere clear.

Thus comparison of this equation with that derived by Keys et al.

from wot-k on humans (see Section 2) reveals that opposite effects

are attributed to saturated fats when fed to ~at and man.

It would appear from the above brief review of the

literature that rats are not suitable experimental animals fo'r

the study of fats and oils in relation to their different

effects on th.e serum cholesterol level of ~·

SECTION 4

A PILOT EXPERIMENT ON RATS

A. Introduction

From the brief literature sui-Vey given in Section 3,

any attempt to reproduce with rats the effects of dietar,y fats

on the serum cholesterol of humans would seem to be doomed to

failure. An attempt was made, nevertheless • for the following

reasonst

1. Rats are omnivorous animals which thrive on a variety of diets and are easy to keep.

2. Unlike rabbits or birds. they do not easily respond to dietary cholesterol treatment and a!'e thus more like humans in this respect.

3• The visualised fractionation of oils in the laborator.y with a view to isolating a bypefcholesterolaemic concentrate or substance is likely to yield only small qnantities of oil. If statistically valid results are to be obtained• a mininn:l.m of six test animals per dietary group would be desix-able. Six 200 gm. rats on a 20 per aent fat dietwould consume about 130 gm. of fat in a test period of, say, 7 days. This would seem to be a reasonable quantity of £at to handle in the laboratory.

4. The amount of blood obtainable from the tail of rats without etra.in is approximately 0.2 to 0.5 ml or 0•1 to 0.2 m1 of serum. The cholesterol in this quantity

· can be estimated· with suitably modified conventional methods• obviating the use. ·of ul txoa.•m1¢:romethods. reqntring expensive ·eq\11pment,

With these· o•erwhelming advantages in m1tui ;tt

would app~a:r that a re~exarnination of the x-at aa ·an e~~r1menta.l

·animal is wol'tb w'hilefj

' ' t:!J.etaey fats and a ~at-free diet on the semrn cholestel:'Ol

concentration of' flata· has been exPlot'ed• Coconut fat and

sunflower seed. ot.l were· chosen as test ·f'ata. 'the serum ' ' ~ til

Cho1esterol rafs1n.g properties .of the former and lovtering

propet-ties Of: the latterappear'to ,be well d~cQ.ni~n~ed from

huma.n eXperiments.

Method! An approx.imately seven months' ·Old colony ·of 24

male \Vhite albino rats, previ<:rusly aubais.ting on stt:u1dard

labo~ato~ chow was d1vt4ed-at the beginning-of the e~er~ent

into tour diete.t7 g~ou.ps of :S'ix r~ts each• Group I was .

offered an esaent.:tally fat-tree diet while the remaining groupe . , .

received lei per cent of comb:tnations of coconut tat ana sunflower seed oil .in. the diet. Details ot the diets :are

· given in Table Ia• In addition, a mirie:ral aJ?.d 'Vitamin

supplement was: given to a+l g~ou.ps. The dai.l;r ratio:p. pe:r rat

was 20 e;m. for Groups li to IV and an iaocalor1o 23G2 gm ..

to:r Group l. The animals were ke])t on their experoimental

tU.ets tor 3.3 days after whieh the adt.lib• J.abo~atory ohow

waa ~estotoed. to them tot! a fUrther 18 daye1 obse:rvatlon-. .~

Skim milk powder . p.otato starch Laboratol";v chow ·Coconut !'at Sunflower seed o!l

Dtetar.v gttC)UpB

PSl'tS by Wl)i.ght ! I! Ill IV

Go 60 Go Go 4G i4 14 14 '10 10 l.O 10

0 16 12 8 0 0 4 8

. 'begtnning of· th~ expal:\iment1 and. after 241 :53 and 51 days.

Serttnt choleste];$;0ls w~re estimated. according to the method

of ~latJd.$" Zak and Bo;vle (1953), which was adapted to~

this single expe~iment to :Cope With the "ery small amo'Unta

of' serum available.

The t"esul ts are tabulated in ·fable 1.b . end illu:s tr•

ated 1n t;t:ig~ 4~ Qnl.y part of' the :food ott"£a?ed to the animals

was .accepted. for the first few days 1 resulting in an initial

loss in weight ot up to 17 per cent 1n some casesit Subsequently

·the tull rations were eonsu,nted with consequent steaay gain in

weight• ·Group I on a fat-:tow diet produced a considerably

lower W$1ght eurve ·than the Othe:tt groups. l.l'he g.ro\vth" of' th~se

groups we.e remarkably uniform• $uggesttng equal absorption of

coconut and sutrtlower s~ed of1s.

!.ABLE Ib - (Expe~1ment 1)

pAN SERUM CHOLESTEROL CONCE1'1TRATIONS IN MG./1 00 ML OF GROUPS OF SIX

RATS ON COMBINATIONS OF COCONUT FAT AND SUNFLOVVER SEED OIL DIETS

DAYS ON DIET

GROUP DIET 0 24 33- 51

1 ttFat free" 84.2 ± 8.8* 113.8 ± 3.5-H + 92.2 - 5.2 115.0 ± 3.6

2 16% ooconut f'at 85.2 ± 9.8 141.6 ! 6.9 + 139.0 -11.7 126.1! 6.7

3 12% coconut fat B5.o :!: 5.3 116.5 ± 4.8 + 110.7 ± 7.8 4% sunflower seed oil 110.3 - 3.9

4 8% coconut :fat + 79.3 - 4.0 112.2 ± s.4 + 101 .. 1 - 7.1 113.8 ± B.o 8~ sunflower seed oil

* standard error of mean iBf eXperimental diets were replaced by laboratory chow after this day.

Mean Percentage Changes in Weight

GROUP DAYS ON DIE'r

0 12 18 28 33!£ 57

1 0 -15.4 -17.6 -17.2 -15.2 -2.5 2 0 -10.2 - 9.6 - 3.2 - 1.4 +4.0

3 0 - 9.8 - 7.5 - 1.5 o.o +6.3 4 0 -11.6 - 9.2 - 3.2 - 2.0 +4.0

l

t\)

0 •

·21.

The mean serum cholesterol levels .of' all groups

rose considerably above their starting values du~ing the

first three weeks• Pure coconut fat produced the greatest

elevation of' 67 per cent whereas the quarter- and hs.lf'­

eubstitut1on of coconut tat by sunflower seed oil resulted

in rises of only 37 to 42 per cent. The differential gain

of approxtmately 30 per cent; due to the pure coconut f'at

over all other diets was sta.tist1oally significant at the

2 per cent level, or less. After another two weeks a

tendency for these differences to increase was noted. The

reverse happenedt when the experimental diets were withdrawn

for a fUrther 18 da1S and replaced by the original laborator,v

ohow.

r-----1

150

..... ~ 120

"' .... , 1G 110

..J 0 0

10 MAY

THE EFFECT OF DIETARY FATS

f I I

16'7. COCONUT FAT

ON THE SERUM CHOLESTEROL OF THE RAT

.16 28. 3 JUNE

9 IS 21

FAEE

27 3 JULY

'-----------------~~---~---~--....)

Fig. 4. The effect on the serum cholesterol level of the partial replacement of coconut fat by sunflower seed oil. (Experiment I)

22.

Conclusions: Although by no means a model of experimentation,

this pilot study hos proved encouraging. Lack of previous

experience With animal work fortunately proved to be no undue

handicap in effecting the various manipulations. serum

cholesterols were estimated with an improvised method, with

improvised apparatus. The animal colony wns far from uniform

with ~egard to age and weight. Yet the substitution of only

four per cent of coconut fat by sunflower seed oil in the diet

ot the animals was reflected by an eas.ily detectable difference

in the serum cholesterol level.

SECTION 5

lmTHOD OF APPROACH

Before embarking on a definite course of action

it may be permissible to record a few leisurely reflections I

concerning the plan of investiRotion and mode of approach to

the problem stated in Section 1. This m~ conceivably be

attacked in a rrumber of ways, depending on the inclinations

of the investigator. The physiologist might be inclined to.

favour, as a first step, the elucidation of the mechanism

Whereby dietary fats - any fats - and cholesterol are absorbed

from the intestine and thereafter influence the cholesterol

metabolism as a whole. The biochemist might prefer to start

by studying the effects of a large number of different fats

on the serum cholesterol concentration under strictly controlled

experimental conditions in the hope of relating these effects

to a substance known to be present or absent in these oils

from existing analytical data.

The chemist, on the other hand, is likely to neglect

biological mechanisms altogether and direct his whole attention

to the chemistry of the oils themselves. To him an experimental

animal is a tool only, a kind of indicator or reagent vmich

enables htm to recognise, isolate and identifY o compound

having certain specified biological properties. He uill thus

probablY prefer to tackle the systematic fractionation of one

single oil to track down a specific compound suspected of being

present :ln this su;\;stance.

lt ia the last of t-hese alteronatfve appWJt:tohes that

'has been adopted in the p:reeent study., l:t is also the method

that has been least eJtl)lotoed ·by others !n seeking an Eil"'lswer to

the sante questions .•

!t is difficult at the outset to pred].ct whidh Of these

policies· is most likely to lead. to a solution of t'he problem. The

truth, of course, is that no pal'tieul~ branch ·of science o, . . . ·~

approaeh has a ·priority in this respect. The cou:r$e taken by the

present study is, in fact, an excellent example of ~he ster1~-;i.ty

of a •• sectionalist'' outlook& As will be seen, th~ third applt'oach ' ..

led to ·a promising start.~ 'tt will also 'become GVid~nt_, however,

especially ·from the later pa~ts ot this thesi.s that this line of

investigation evel'ltually led to an impasse from whicl'! on1;v- the

pl'lysiologi.cal approaeh could .rescue 1 t.

!he eneour~ing r\9su1 t.a o'btained f~om t~~ pilot experiment

with rats (Expe~iment 1)., if. confirmed and. con:solldat~d:~ ,afford an

e~celient opportunity to_d.evelop th.is animal as a.aensitive chemical . . . . . .. ..

utteagent" in the above sense., . :tf su<:eesstu.l., th_is Wi11 create the

Pl"econditions for embarking on the chemist·•s approach" namely the

ehemiesi o.r p}lystceJ. f"raettonn·tton of an oil· to isolate o~

~oneentre:te therefrom ser:u.m cholesterol regula.ting substances,

assurningt of course, the.t aueh entities do~ in fact, exist-s

~S'riGNl'IQN . O,l ... ~~ . RAT

AS. AN

EXPERIMENT~.:. ANIMAL

SECTION 6

THE_ D~OPDN'l' OF :811 ~COTJ,RATE J4~HOD FO! .:a'rtE TOTAL SERm! CHOLESTEROL D~'r:ERMINATION IN 0.,2 .. ML ... OF BLOOD

A seal'ch thJ?ou.gh the med.ioel and eliem.ical abstracts

~eveals the existence at a bewildef'ing and ever faster growing

profuaton of methods tor the esttmat:ton of cholesterol. Man;v

of these methOds are advertised as being simp1e ~ rap:td and

reliable., On closer tnspectlon snit espeeially on .trial in

the l.aborato17, however, the trostrated newcomer find.s that

the aocuraey of most methods ts·:tnvel'se1y proportional to

their speed and the amount ot trouble taken to $nsure good

results.

A compromise has to be found. Since the determination

of total set1.ttl'i Choleste~tol of rats forms the basi~ analyt~ca.l

procedure in this thesis, Qazoet\tl attention has been given ·

to the selection and development of a. suitable methoC.i.o: In

addition to the obviously desirable attributes, thia method

must be capable of being scaled down to compensate :f'o:r the

small quantity of serum available and the comparatively low

serum cholesterol ooncent:rat1on of rata~ The latter ran.ges

nonnall;v between 45.and 70 ms-/100 ml.

At. Methods based on . the t1ebermann-llu.rcha.r4 reaction

· !he Lie'berm..<mn-Bur<:hard. reaction (188$ '""' 1889),

:t.e. the.production·of a g:reen colour when cholesterol :ts

26.

reacted With H2s~ in acetic anhydride is the basis of most

classical methods. It is also the basis of a method due to

Abell, Lev,, Brodie and Kendall (1952), which has been practised

with success in our laborator,v for some years. It was discarded

as a basis for the development of a micromethod on account of

the relatively low intensity and stability of the colour

produced by the Liebermann-Burchard reaction. It must be

admitted that some of the tempting short-cut methods offered

more recently, influenced this decision.

B. f.Pe p=toluene sulphonio acid method

This method, first developed by Pearson et al. (1953),

together with its variations, e.g. Turner and Eales(l957), has

the advantage of producing equal colour intensities with both

free cholesterol and its esters and does not, therefore, require

a saponification step. It has been given a thorough trial but

was eventually abandoned for the following reasons:

1. The handling of minute volumes of liquid, made necessar.y by the low colour intensity proaueed by the p-toluene sulphonic acid reaction and the small volume of serum

. available (25 r l ) necessitated the use of micro absorption cells in a Beekman Ultraviolet Spectrophotometer. This was uneconomical as a routine method.

2. No less than five different pipettes are used in the method described by Pearson. This required the construction of a set of five micro constriction pipettes with capacities ranging from 0.025 ml upwards, an extremely laborious task even after considerable skill hnd been acquired in the glass-blowing technique used. A single breakage of these delicate pipettes caused a del~ of some days.

. .3* Reprodu.e1bil1ty was poo:- in. spite of the elaborate equipment usea •

. !he r$act1on of: cholesterol with te.M-ie Ohl.oride

has fir~t been used by .Zlatlus, Zak and Boyle (1953) to ·

detamine serum cholesterol concentrations• !his methoa.·

depends on a measul"ement at SGo mp.. ot the eo~ou.r inteils:ity

produced when serum dissolved in glacial aeet1c acid t:s

· treat~d directly with a colour reagent .consistihg ot a

. !;l,olution of Fe013 in ·Qonaent"ra.ted sulphu:ri e acid• · the

.following advantages vender this method att~adtive:

1,_ EXtreme sirnp1io1ty. X,ike the p ... toluene sulphonic acid metbod. 11 n() pl'evious extraction of the cholesterol tz.orp. the serum 1 s requi:rea.

2l!b Equlmolecular concentl'ations of choleste\L'Ilol and ;

cholesterol esterj3 ;vield eqU.al colou.~ intentdties, thu.s obviating the saponification of the ester~J.

3~ fhe purple colou~ is relatively atable.

4~ The co1ou.~ intensity produced is appreciably highezi than that ttue to either the Liebennann•!u.Nhartt o·~ p•tolu~ne su1phon1e acid. reactions ..

The last point in particular> decided the adoption of. this

methOd as a starting.potnt for> scallng-down operat.tona•

Fol1oWinS the direction given ·by z·;tatld.s et· ai. (.1953) 1 but

Setiling down solvent. and z-eagent volumes to a half P..nd serum

volume to a quarter (0.025. ml)froom a constrict.lc.>n micropipette),

values fotl' human sera v1ere obtained which were .trom 60 to 7CY/o

higher than when detefinined ~Y the Abell method·; Obviousl;y

substances other than oholesterol contribute to the colour

production. !his is in agreement with the high values found

by Best et al. (1954) with this method~ Poor repr~du.cibility

was also noted. ·'!'he obvious advantages of this method,

howevezo, warranted further efforts at deVelopment.

n. i'he further refinement. of the FeCl,; method

An obstacle in the above procedure 1s the high

viscos:t t1 ot the colour reagent due to the concentrated

sulphuric acid. 'lhis renders the quanti ta.t:ive <U.spensing . from small pipettes uncertain. A related factor is the

sluggish and inadequate mixing of' the viscous reagent with

the acetic acid•serom solution. ·Local overheating 1-n the

resulting exothermic reaet1on.is diffiClllt to avoid.. For

these reasons; FUrst and Lange (1954) criticised this method.

It was .felt, howevert tha.t a potentially p:rom:tsing reaction

cannot be rejected for what are hardly insurmountable

difficulties. one of these dif':f'.1eulties was met by the use

of all•glass svringe pipettes. A detailed description of

these pipettes appears .in the next Section. The mixing

probletn was overcome by bubbling a stream of air into the

reaction mixtut"e from a f'1ne capilla:ey glass tube and was

operated by means of a hand rubber bellovts. ~he intenni ttent

a1r stream was cushioned by inte~ediar,v passage through an

empty winchester bottle. These modifications 1mpl"'Ved the

reprodUCibility of' the method withou.t eliminating the

comparatively high cholesterol values obtained ~th it. The

1a~ter tlef~~twas realised by Zak et al.• .(1954) who f'i~st

filtered oft the se:ru.trt proteins obtal.nea. by pt-lleipttatton

With a 50/50 $Ol.ut1on o.t a.leoho1 ~. acetone, !'hey then appl~ed

the oolou.~ :reagent to the dJ:"ied.alcohol ... acetone ext~act

di$solved. in glacial acetic acid. These authors repo~t ., .

extensive ~ecove17 experiments to illustrate the aceuraey ;

ot th~it> method. It 1s $eldom realised; hovrever, that ~ood:

. recoveries often g:l:ve .more fnform.atio:n on the skill of the

opere.tor.than on the original amount o:t cholesterol present

.in a given sample. ' In the hands ot the au. thor the sealea ... aown method.

of Zsk et al• still produced re&m.l ts consistently higher

than those obtained by the A'betl method., Attempts wex-e made ' • 't

-to f~nd. mota~ selective tat solvents. Usit.~g the method of

~ak et, a.1 •. ~t emp1oy1nS ditfe~ent ~olvent systems, the·

:f"ollowfns e}lo1esterol concentrations were founa for a ~serut:n " '"' .

w1 th a~ ''Abell •• choleste~ol value of 152 ms~

TABLE .2

'rh,e efticien¢Y' of diffe~ent solvents on the $Xtt"aotion of! eholesterol ~om serum

' ':t _,

liot acetone/ethanol 1:1 Cola acetone/ ethanol 1 ~ .1 dhl.orofotm""'tltethanoi ltl Ethano1•ether 4:1 Meteyla:l

mg./100 l'lil

.181 184 186. 1S4 !1.98. 18S 185 1S2 1$.5 182

t;fhe ,~qual etficteney of hot or cO:id. a~eton~ethenol

as extraction media confirms the views ot Mertens and Albers

(19.53). ·These :raslllts lena no support to the often held View

ot the alleged superiorit;v of one ;particular comz:non tat

solvent over others; fo·r the extraction of oholeste:rol :from

On searching :f'.or a. reason tor. the r.esiclunl discrepancy

'between t.he Zt:tk and Abell methods, it was ,iiealt,sed. that in tbe·

1atte:r ail saponifiable materlal is removect by sapon.ificat;i.oll•

That the saponifiable lipid contributes to the eololU' production

was proved by detellninin.g "-cholesterol" in a sample of fatty

aoids frQm which it had previously been removed bu aaponifieation . ,,..· . '

and -ext:raction of the ·unsaponifiable matter. .small positive

t'etildings were obtatned$. $ubsti tuting the solyent extraction

ot lipids directly trom serum by the ext.raation of ·unsaponffi.able

matter f'rom the saponified ,eerum failed to eiiminate the

d.:i!SCre:pancy,.. ifhe saponification p;rocedure had other veey

desi'!Nable advantages however. tt solved the ·p·ro'blem ot

zoemoving solvertt precipitated proteins by filtration or

centrifugation. thus increasing the repl'oduc1~~11ty- t;>f the

method.· As will be shown below• it also correiated well with

t'he Abell method and was therefore finally ado~ted tor .su'bse®,ent

work on rs.ts+ The final tom of the method 113 desert'bed in

the next section.

31.

SECTION 7

A MtCRo-!tlETHOD FOR THE DETERIIINftTION OF TOTAL

SERUlf qHOLm'l'EROL IN O. 2 ml OP BLOOD

A. Experimental

Preparation ot S!!Rle

The rats ere oaged 1n tight fitting Q¥11ndr1cal

wire mesh containers stoppered at both ends b.y means ot

rubber bunge, on~ of whioh is proT1de4 with a notch to allow

the tails to protrade. The latter ere suspended in water at

.38 - .39°0 tor 20 minutes. A:f'ter careful drying, about 2 Dill.

is cut trom the tip of the tails and the cage is clamped

vertically in a retort atand. The blood is gently "milked",

drop by drop, into micre-oentrifuge tubes. The wounded tails

are treated with Priar's Balsam, which baa on no occasion

tailed to etfect satisfactor,t hesllng within a tew d~e.

. . .. . The mioro-oentr1tuge tubes are specially made

tor this purpoae and hold approxtmstely o.4 ml

ot blood in the narrow shank ot 4 mm. I.D. In

Pig. 5 a tube, mounted in a oentrituge

container, is illustMted. After spinning at

3,000 r.p.m. for 10 minutes, the supernatant

serum is transferred to stoppered micro-test

tubes constructed trom 5 mm I.D. glass tubing.

In this condition the serum is stored below

o0c, ate~ for ana1Jreia.

32, ..

B..eagents

h SUlphuric ac:tdA.Ro (Merck)

2. Glacial aeetic acid A.R. (Me~ok)

3~ 33% aqueous KOH aolut ion

4~ ·Iron stock·solution. 6f'o grn. ot Feel~ an.h• and 4 ml of disti~led water made up

0to 100 rn:r with glaoial ,

aeet:io acid. Store below 0 C and thaw when reaui.rea. .. ,. . """' ;·

0 Petroleum ether, 60/80 c.

Cholesterol standard.. 200 mg. re•ceystalliaed drieCi cholesterol in 100 m1 of glacial aeeti.c acid solution ..

Procedul"e

Thoroughly stir the serum contained. in .miero .... tu'bes

by means .of glass t-od dravm out to a thin point., 'Using a

oonstriction mic~o.-pipette (see below) with a capacity of

25 to :35~l; transfer serum accu.rately to the bottom of 5 ml

glass!""stoppered. {13 10) test tube. Add exadtly o.; ml ot

alcoholic KOH stock solution, ma.de up freshly by diluting 2 ml

of XOH stock solution to .30 rnl 11vith absolute al-cohol ..

Saponify fo~ one hour in water'bath at 4o0c~ After

cooling, add exactly o.s ml of distilled water ~ollow.ed by

3tO m1 of petPoleum ether, Replace glass stoppet> and shake

thoroughly .for one minute.. ·Thirty tubes are con'\renientiy

shaken simultaneously in a test tube ra·clt.. The tu1;>es are .hold.

in place 'by pressing a fos.m rubbe:r lined board against the

stoppers.,

Allow to settle~ then transfer 2.0 lill of: $Upernatant

petrQleum ether layer to micro colorimeter tubes. Blow to

dr,yness in waterbath at 6o0 c with stream of war.m air, To the

dried residue add 1.5 m1 of glacial acetic acid from all-glass

~ringe pipette (see below). Continue heating in weterbath at

6o0c until uneaponifiable matter has dissolved. This takes about

3 to 5 minutes. Allow to cool.

From a second all-glass syringe pipette add 1.0 ml colonr

reagent. ~hie is made up freshly by diluting 1 m1 of iron stock

solution to 100 ml with concentrated sulphuric acid. DDmediate1Y

after adding the colour reagent, bubble a gentle even stream (see

below) of air through the solution by means of a capillar,r tube to

mix. Measure absorbance 1n colorimeter at 560 ml" within one hour.

A distilled water blank and cholesterol standard, dispensed

from the same micro pipette, receive identical treatment.

Notes on Agparatu~

I

An enlarged close-up photograph of a micrc­

eonetriction P1Eette constructed by the

author is shown in Fig. 6. Some experience

is necesear.y to get the volume within the

required range (36.2 rt in specimen shown).

Some practice was also required to prevent

the constriction from collapsing altogether

when the required minute opening was

obtained. These constriction pipettes are

operated by mouth by means of a suction tube

and del1Yer a very accurate and reproducible

volume.

Fig. 6 Micro-constriction pipette.

. 34. . '

fte bot-atr J!l!ldtol4-•Ser be!h p'btptlgp waa

me.4e ,ae 11luatftlte4 in Jl:I.B• 7. tram whtoh 1 ta, oonetru.ottan

Ia ..-s.tentt ftta appeNtue wee alao round oonve!llent ~or

the .,apo1'8t1on ot · Ohl'elllfltoBNph!o tract tone.

A aet ot a.J.l•ll!Je &r!M! ~';est t,a !a me4e 't\1

eeortn:otng a p.yru Wfl.nse, Nom the bat'Nl or which a lerasth

o't oaptllan tubtDC le 4rawn. '1'hNe tnOb portlou ot the

latter a11e sealed to the outlet ot elllilal' tntaet artfna-a

o't the eame lllllke.

lll• z. Hot atr meD!to14-waterbath combination.

These are then mounted in fittings Which

allow the plungers to travel a fixed but

adjustable predete~ined distance

(see Fig. 8). The syringe and fitting

in turn are held in a retort clamp in a

vertical position, making their operation

with one hand possible. An accurate,

fast and highly reproducible deliver.r

of small volumes of viscous liquid is

thus possible which is 1ncictentall.y also

independent of the room temperature.

The air stream for miXing the colour

reagent was produced by a hand rubber

bellows. 'l'he pulsating air :flow proc!uoed

by the latter was cushioned b7 intermed-

1&17 passage through an empty winchester

bottle, thus produotng a continuous flow

from the capillary.

Pif' 8 Adjustable al •ilaaa s;rringe pipette

B. Results

The present method has been in use for over two years,

in the course of which over 2,500 serum samples have been success­

fully analYsed in duplicate. TWelve sera 1n duplicate are

con'Yenientl.7 included in one batch and can be estimated by one

eXperienced operator in six hours. Details of expertmental

results are given in Table 3.

TABLE 3 (See p 37)

A B c standard error between Standard Deviation Coefficient of Correlation duplicate readings

~ample No. S!IDJ?le a samnle b Samnle No .. L.B. FeC13 -(X) (y) 1 59 6o 195 253 1 158 186

... 2 54 54 197 255 2 150 173 3 47 48 201 259 3 95 112 4 51 54 194 260 4 91 109 5 59 61 197 260 5 111 134 6 63 65 195 262 6 119 134 7 53 54 198 257 7 128 151 8 69 71 200 257 8 130 160 9 31 33 202 2g6 9 120 155

10 37 38 199 2 l 10 116 145 11 48 49 200 257 11 133 156 12 47 46 202 256 12 249 287 13 58 61 199 261 13 172 205 14 44 43 . 14 182 223 15 44 43 S.D.= 2.7 S.D.= 2.9 15 264 320 16 44 42 16 133 163 17 56 56 17 209 255 18 65 64 18. 225 270 19 46 45 19 221 264 20 46 45 20 249 296 21 54 55 21 120 140 22 44 43 22 150 180 23 48 46 23 223 262 24 54 53 24 178 220

S E = 1.1 I 2 (2~02 r = 0.997

r2 '2 )C. -

SE=~ S D = n n .:z.. ::X."-J - 2:: ~ 'E Y r-=-2R n-1 V n ~X.'" 4 :lcl \I n'E~,_ -(z ~)~

The standard ert~or between duplicate readings

on 24. different rat sera determined on different d~s was

1.1 mg./100 m1 (Table 3A).

The mean and. standard deviat:to·n o.:f' 10 succeasiire

detenninations on.dif'ferent d!\'{s over a period of one month

of the same human sei'UlTl was 197.8 ± 2. 7 mg./100 ml. Individual

values ranged from 194 to 202 mg./100 ml. For a different

sample of' serum analysed 10 times on different days these

figures were respectively 258.0 ± 2.9 (253 - 262).

(see Table 3B).

To deter.mine the relationship between the values

obtained 'by the present procedure end those obtained by

the now classical marco-method. of Abell et al. (1952), the

cholesterol content of 24 different human sera were deter.mined

by both methods. The results (Table 30) are summarised in

a scattergraph reproduced in Fig .• 9.. From this it is eeen

that the correlation ( r = 0.997) between the present method

and that due to Abell et al. is excellent.

The eq~1ation for inte~conversion is:

y = l.l82x + 2e139

where ~ = FeCl3 value and x = L.B. value.

Fig. 9/

38.

1.1.1 250 0 ti 9 0 ~200 a:: w Lt.

ISO

100

y: 1.182X + 2.139

(r=0.997}

150 '200 LIEBERMANN - BURCHARD

250

Relationship between the sel'lltn cholesterol concentration aa de~e~ined by the micro ferric chloride method and the Lieber.mann-Durchard reaction as described by Abell et al. (1952).

-c. D1scu.ss1on,

·A difficulty encountered in most methods employing

the ferric dhloride sulphuric acid reagent is the sluggish

and inaccurate dispensation of the·viscous colour reagent.

this difficulty, accentuated b:V .the small volumes employed ' in the present case, was overeomeby·the use of' special all-

glass syringes. The accurate meaaurem.ent of volumes of' serum.

of the order of 20 to 40 f" 1 is even more importnnt. Unless

this can be done successfully, care in subsequent manipulations

is obviously not tully effective. The micro-constriction

39 ...

pipette was found to be capable of deli'tl'ering highly l'leproducible

"VOlumes of 11®1d.

'lb.e sapon.itication step is not theoretically necessary,

. G:lnee equ.imo:terular amounts of t~ee and esterified cholesterol . .

have 'been sholm b;v Zlatkia et al. (1953} and Zak et al•· (1954)

to.produce equal eo1outt intensities with the ferrio chloride

reagent.· The saponificati~n step; adapted f'rom the Abe11 method

ha$• however• been included to remove the saponifiable matter.

This makes a small but sigtd.fica.nt contribution to the total

absol:'bance.. A I\irthel'l great advantage is the neat separat( ion

ot prote!ns ~.t affords. Neither filtration nor centrifugation

are required .•

A similar comparison between the .terrlc chloride

reaction and. the Liebennann-Buroha~d reaction as described.·

:t"espectively by Zak et al. on the one band and Mertens and .,

Alberos (1953) anc:i dolman and McPhee (1956) on the othe~. hae

been made 'b;v Sml t and Weymeye:P ( 19.57) • ~eir ·~eporteti

regression equation, af'ter Jt:ntet'changtng"'ar1ab1es.\ to make it

eomparable 1st

w:t th a correlation coeffictl.ent of 0~9f7fi Except for the . .

constant term whlcn is lat'ge:r in thei:P ca.~e the two eqttations

are in good agreement.

It is believed that the .excell$nt rep~oducibfll.ty

obtained with the present micro method is att~1butable largely '

to tha .special attention that has been paid to the experimental

details attending the aceurate measurement and hand11ng of sm<~.l1

·Yo1umes of liqUid. While no o:t'1g1na11ty is dla:tmed. tor any

·of the appa~!tus used here, no reports eould be toi.md whf;lre

. similar appa.r~.tus have been described. fof! the mie:ro est.imation

;:,t cbo1estero1 in $eNrn • .. -

..

SECTION 8

lphe choice of the non-fatt;v part of the

eXperimental Q.iets would be a comparatively simp1e

matte:r if it wet'e knovm that proteins, capboh,vdrates;

m1neroa1a and "fitamins plasred ne role in the· regulation

of·the serum cholesterol concentration. Unfortunately;

this is not the case• After spending a day or two in

the library one might, in fact, be. tempted. to conclude

that· it is di.fficul t to find a chemical subst.an-ce knovm

to dietit!.ans which has not 'been cla1mec1 by someone, at

some t1n1e, to influence the serum cholesterol of some

species in one wa::s. or another. .

l'or the p~esent purpose thase various influences

are of little concern except :tn so far .as tlley might inter­

tette With the mechanism by which dietary t:t'ligl;veeride fats

· as au.ch undoubtedly exert their etfeets.. In the absence of

this knowledge, 'the a ;e:rlo:ri Choice of a sui table basic . .

diet necessarily invol-ves some :guesswork.

. 'i'he bas.ia diet listed in Table 4 has been chosen

as a compromise between the following desil"a'ble chl;ltoa.ateristi¢s -

1/

1.. Minimum number of ingredients 2- Nutritional adequac,r 3,. Easily proourable eomponents at reasonable cost 4. Keeping ~ality at room temperature .5. Acceptability 6. Unit'orm1 ty .of components from batch to batch 7. Negligible fat content 8. Probable absence of components likely to attsci

the serum cholesterol level seriously. The importance of excluding Cholesterol, eholic acid, thiouracil, etc~, has already been etzsessed 1n Section 3.

T,ABLE !.f;

BASIC FAT-FREE COMPONENT. OF EXPERD1EN'TAL Dl]:l:TS

Skim milk powder Dehydrated lucerne

Parts by weight

G6

Brewer's 3feaet De-fatted sunflower seed husks Salt mi~ture (NaOl containing o.l% Feso4 , 7H2o and o.ol% ou.so4 .. 5H2o

fo complete exper~ental diet add tat unael' test

Total

8

so.o 20.0

100.0

Although deficiency symptoms &.z>e not likely to S.l'"ise

in short ter.m exper~ente, thie diet was found to meet the

req~irements of teats in all the more important vitamins a.n.d

minerals with the possible exception of iron and copper which

were added a.s a supplement in the salt mixture. This basic

43.

diet, togethe:r W1 th fat, did i.n :ract promote sa:t is factory

growth as will be shown in a long term experiment (No.3)

with an eXperimental butter diet. The growth cu:rve in

this instGnce (Fig. 12} closely t>esembles a s:tmilar cut'Ve

described bf Weeks (1957) to illustrate normai gtiowth·:tn

rats,

l:t is more important to meet a possible criticism

that serum cholesterol levels may- be influenced by essential

fatt:v acid deficiency. !he linoleic acid. content of the

most saturated fat fed, i,e .. coconut fat, howeVeF 9 assured

the animals of a minimum daily intake of' 50 mg• ,an enti.rely

adequate guentity. Aaes .... J:fDrgensen et al. (19.58} moreover,

have since shown that this deficU.enC"tJ is vel?! difficult to

· induce :tn adult rats.

Manasement . ot .x:at.s

With few exceptions, the animal ex:periments to be

described in these pages follow the same pattern. ~e pilot

experiment described in Section 4 served as the basis for

the d.evelopm~nt of a standard e:mer:tmental desisn which was

as followst-

fhe animals used for each expe.rlrnent cons~.sted of 24 white male albino t-ats reared on labotoator;v. chow. They

were selected from a larger group fo:r uniformity of weight and appearance. ·At between two and three months of a.gej the eolony· was split up randomly into four groups of six rats each~ The animals were individually marked and housed in

1a cages, two rate to each cage• The distribution of. the four dietary groups among the 12 cages followed a definite systematic pattem. The first group was assigned to Cages 1, 5 and 9; the second group to. Cages 2, 6 and 10, etc. This served to randomise possible va~iables arising out of unequal distribution of light and temperature and the considerable time 1nterv~l between bleeding the ~ats in the first cage 1n the early morning and those in the last cage in the afternoon •. ·

The animals were bled as desei'ibed in Section 7 at the beginning of' the experiment while still on a laboratory chow diet and thereafte;r at weekly intervals. Great care

·was taken not to exceed the mintmttm necessary quantity (0.2 to 0.3 ml) of blood drawn for analysis. Recentlyt Coleman and Beveridge (1959) have likewise drawn attention to the importance of this point. The no~sl duration o£ a routine experiment was two or thi'ee weeks, a:f'ter which the colony was discarded.

Unless othePWise stated, the experimental diets were fed ad. lib. and. consisted of 80 per cent of the fat .... f~ee basic diet described under A above and 20 pe~ eent of the lipide to be tested. All rui:tmals we:re weighed at weekly intervals during the test period.

Presentation of results

' We~ights and sei'tlm cholesterol concentrations on the

eXperimental diets containing the oils under test were recorded

as percentage changes from the initial values, i.e~ those . obtaining immediately betore splitting up the colony into the

experltnental dietary groups, while still on a labomtor.v chow

diet. Ueans were calculated from the individual percentage

4$.

ehange of each animal. For stat.istical convenie-nce the init.ial

val·ues we:re taken to be 1 00 per cent •

The method ot ·working with changes in serum conoentx-ntion

rather than w.ith absolute values produces better statistical "p·••

values by smoothing out disturbing ef'f'ects due to abno:rn'lally high

or low serum coneentrations which some individual animals have

in their no.rmal state.

The statistical methods and f'ol'mU1a · used were tho.se

de.scribed 'Qy :Bancroft (1957).-

ttt'~ value's were ealculated from the :fo.rmula

... ... x .... y

t = ----s

n1 n.2

n1+n2

where s J) the standard de,:iat:ton f'or smell samplet! is der.ived. f':rom

... -,g-

"P'' values, i.e. the p:robab11ity that the dif.ference in the change tn serum cholesterol concentra'tion of any two groups of' :rats due to two different diets ar~. identical were computed and are reeortled with each table ot resultse Variations from mean values are recot>ded. as standard er.ro~ o:f the mean:

s •. E .. = s

Regression lines and coe:f'fioients of correlation were likewise computed as direoted by Bancroft,

46.

SECTION 9

THE EFFECT OF VARIOUS. FATS MID O!LS ON THE

SERUM . CHOLESTEROL CONCENTRATION OF RATS

Although the results of the pilot experiment

reported in Section 4 are Gncouraging, the demonstration

of the rat as a suitable e.xper-im.ental animal requires

confirmation. It will be remembered that the object of

this study is to contribute to an understanding of the

effects of dietary fats and oils on the cholesterol level

of humans. The interest in rats extends only aa far as

it can be shown that these animals can be induced to

imitate these effects. How well can they do this? In the

following three experiments, rats are given a more rigorous

test under what are hoped to be improved conditions, and

using the improved experimental techniq~es described above ..

The ef·f··ect of rellacing coconut oil by sunflower seed oil . . EXperiment 2} .

E;Perimental

From human studies it is known that of all the

common oils, dietary coconut oil produces most probably the

highest serum cholesterol levels. Assuming that rats behave

similarly, all animals were placed on the experimental diet

conta.ining coconut oil (see Section 8) for 17 days in order

to produce uniformly high serum cholesterol levels. (It is

.seen that in the present experiment the n standard experimental

design" had not yet become fully established but the deviations

41·

therefrom are fUll~ deacribed)•

Arter this · pre11mirialJ:'Y .treatment the colony waa

split up into four d1etar,v groups~ one of wh1dh was lett . . .

unchanged ae control.. .The remaining three gi'Qups rece1:•ea

a. qu.erte:r$ halt, and total replacement of the coconut oil

by sunflower· seed oil (s.s.o.) •. 1he coconut oil was alkalt

r.et!nea., bleached and stesm-deodorised: and had an 1odbie value

of ·9~2, Whereas the· S~r si!i o. was a crude pr-oa.uet ot ·iodine value ,

132 ana 11tt(l)1ete· aeiacontent of 65 per cent (alkali isomeris,..

ation metbod'of'.Herb and Riemenschneider; 1953)1 wh.ich'hs:d

bt:len eltJ)l'esseti :fr.om the seetis· WJ.thout tuztther treatmentii

A.f'texo 36 d~s the s~-s.o. replacements we~e·a:tscontinued

and the animals kept·tora furthe~ 27 d.S:Vs on the experintenta1

eocon,ut oil diet. Senam cholesterola were det~m1ned a:rter 9,

36anil·63 days.

Results I ,

-. ' t t '

· !he !'Osults ar-e tabulated in Table S and illustrated

in ,Figli 10- tt is seen tliat only 9 days after the ihtl'oduction ~ . !.. . .

o.f the·s.s.o. in the diets there was a progressive drop in the

se~ cholesterol level with increas~.ng proportions ·Of Sos.o.­replacemen.ta•· .· Although in thts instance the effect of' one

qual'tex- subst1 tution of cocormt tat b:v s. s. o~ could not be.

shovm. to. be $1gn1ficant, the hal!' and total replacements .. . -- ..

produced depressions of approximately 20 per cent and 3.3 pe~ cent

. sisni:f'icant(~ respectivelY. at l per cent and less than 1 par cent.

After a turtb.e.r 27 ·a.ays ·on the experimental diets these $tat.1$t-' leal f1gures det.erioratea. It is of interest. however. that the

48.

serum values had not yet converged af'ter the subsequent

27 day per'iod. 'in which the s.s.o. replacements had been

d1 a continued,.

Fig. 10.

Comment:

9

•

I

-------------------1

-36

S,F.O. REPLACEMENTS DISCONTINUED

DAYS ON DIET

-63

I

I

The hypooholesterolaemic eff'ect of various concentrations of s.s.o. in coconut oil (Exper~ent 2). · --o- 20% coconut fat .. 15% coconut fat, 5% s. s. o •

• 10% coconut f'at, 10% s.s. o.

• 20% s .. s.o •

When this ex.periment had been completed, it was

realised that not much is gained by pre-feeding the colony

with coconut oil. No ,doubt the serum cholesterol nlowering"

aetion of s.s,,.o. could have been as well detected without

special pre-feeding if' e.onsidered relative to a coconut

TABLE 5 - (Experiment 2)

MEAN PERCENTAGE CHANGES IN SERUU CHOLESTEROL COJ'TCET'TTRATION OF GROUPS OF SIX RATS AFTER 20% OF COCONUT F.AT IN THE EXPERIMENTAL DIET IS SUCCESSIVELY REPLACED BY SUNFLOWER SEED OIL {s.s.o) THE

Im:PLACEMENTS BEING VIITHDRAWN /IFTER 36 DAYS . (STARTING VALUE = 100%)

GROUP 9 36 63

bays Days Days

I 2Q% Coconut f'at 102.0 :t 6.4H 110.2 :t 5.3 108.2 :t 3.4 (diet unchanged) {102.5)m£ {110.0) (117.8)

II 15% Coconut fat 91.8 + s.s 113.1 + 1.2 116.0 - + 3·3 - -5% s.s.o. (100.9) (106.7) (114.0) III 10% Coconut fat 81.0 + 4.4 102.3 + 5.5 107.9 + 7.4 - - -

10% s.s.o. (101.6) {105.9) (114.0) IV 20% s.s.o. 68.6 + 4 .. 0 86.0 + 4.2 95.4 + 4.2 -- - -

(100.7) (105·9) (111.5)

Significance of differences (p - values)

I- II 0.30 0.90 o.so I- III 0.03 0.40 0.90 I- IV o.o1 o.ol o.os

if. - Standard ewor of mean u = Mean percentage changes in weight of rats

so ..

control diet. The previous dietary history is o:r little

concern. · !t was also learned fr.om, this eXperiment that

nothing is probably gained by prolonging these tests beyond.

two or three we eke. With these points in mind the final

standard experimental design was adopted.

B.. The effect of butter rat, beef tallow, groundnut oil and f!sh oil {Eeperiment 2)

Exl?el'1mental

Of' the many oils tested on humans there is fail'

agreemen·t that butter, and to a lesser extent beer tallow,

raise the serum cholesterol• whel'eas highly unsaturated fish

oils have the opposite effect. Thex-e is some controversy

concerning ••semi-unsaturated'' oils sueh as groundml.t oil.

!n the present experlment, thefollowing four representatives

of these thl'ee classes of oils were tested on rats in a

standal'd. experiment of three weeks' dux-ation (see Section 8).

Grou;e

I

II

I.Il

IV

!odine value . -· . -

Butter fat

S.A. Pilchard oil (sardtna '7 182.1 oacellata ;c~pensis)

k\1-tl Beef tallo 1 1 39.6

Arachis oil

After the experiment prope:r, the butter and fish groups were

maintained for a fUrther 28 weeks on their experimental diets

to study long tenn ef'f'eota

51. '

Resu,lts . (aee T~ble 6 and 'F1ga6 ll and 12),i. ·, ' '

. ·rJte results allow that after ·only l!h)Ven .days Qn

the experlmental diets eaoh. group attained a Oharaoterist~~

serum,Cholesterol .level Which was maintained fQ~.th~~~ week~~t

1,'he.btttter and fish oil groups maintained the~ lGvels ' ' . I . • ' . •

~s~e~tiallY. un<lhanged tor. ·the duration o~ the exp~flimen.t.~ ~·· . .

1~e. 31 weeks~ This fact will be tound useful when cU..scu$sing

the. relation between. d:ie:ta:ry fats and cholesterol metabo11.sm

1n Seetton 2G.-

From the statistical f1gut-es ·supplied with fable 6

it is :Seen that the d.itfe~nees betwee11 these group averages

stand on tinn foundations 1n spite of the small num'bei" of' rats ..

t.rhe small differences in tha growth rates s.how no

co;r>relat1on With. the sertllll cholesterol levels up 1;0 9 weeks•

This did not apply after this time,, however. 7he :fish g~oup

ceasea to grow atte.r ~orne months· as 1s illust~ated in Pig• 1l2

and did in fact begin to l·ose weight atte:r a fu~thett period:

ot time~ !he question of whether this ia· ·due .to an ess~ntial

:ratty acid de:f'icieney 'or some othe.:r caus~. wlll. not be pursued

since this would. deviate from the stX>aight and narrow path

adopted .:in this work.,

TABLE 6

MEAN PERCENTAGE CHANGES IN SERUM CHOLESTEROL COifCE'ITTRftTION OF GROUPS OF SIX RJ'lTS AFTER CHANGING TO EXPERIMENTAL DIETS CONTPINING 20% OF DIFFERENT FATS (STARTING Vfl.LUES = 100%)

GROUP Dift WEEKS ON DIET Iodine 1 2 3 9 31 value

I Butter 31.9 127.5 ! 8.1* + 8.3 116.3 ! s.6 117.6 ± 7.0 + 119.7- 113.9 - 9.5 (100.9) !HI (109.9) (112.8) (137.6) (157.9)

Fish Oi1 182.1 73.4 ± 4.5 66.0 ± 4.5 69.2 ± 5.1 74.8 ± s.o 82.1 ± 4.4 II ( S.A:f'r. Pilchard} (98.6) (107.4) (108.8) (133.0) (135.3)

39.6 115.4 :!: 3.3 + + III Beef 109.5 - 2.8 113.2 - 4.9 - -

(96.2} (103.2) (108.3)

IV Groundnut 95.6 101.1 ± 5.8 101.1 ! 4.4 + 98.1 - 5.2 - -oil (97.7) (19lf.9) (109.9)

-Significance of differences (p - values)

I -II 0.001 0.001 0.001 0 •. 001 0.01 I-III 0.20 0.20 o.6o - -I-IV 0.03 0.03 0.03 - -

II -III 0.001 o.ool o.ool - -II -IV o.ol 0.001 0.01 - -

III -IV o.o6 0.20 o.os - -

ft = Standard error of mean a = Mean percentage changes in weight

\J1 1\)

•

7 ~ DAY$ ON DIET

The effect on the serum cholesterol level of var.lous fats (Experiment 3)

--o-- Butter f.at x Beef tallow & Groundnut oil • Fish oil (s.A. Pileha~}

I I

J I I

I I

l ' I .. I I I

l

54·

80

70

60

Ill

~50 <( :r: u

40 ,_ :r: C)

~ 30 FISH OIL

~ z ~ 20

a: Ill'" 0. 10

0 ---------------------~-------------------

50 250 300 350

Growth our-ve of rats on die.ts containing butter fat and fish oil~ e~ressea as per cent .increase from weight at two months of' age (part of EXperiment 3).

I

o~ The effect of small qUantities of di~taey cholesterol • .. .. • HI O

0 (Etme:riment I !:i:l ... .. . .. .. . . . .~ ...

There is a good. deal ot evidenoe that 1n man the

effect o.f diffel"'ent oils on the serum cholesterol level ts

la:rgelN" independent of the small amounts of .gholesterol

associated with animal fats sueh as butter and beef tallow.

'this aspect wa.s therefore studied in the !iat.

The standard experimental design was followed..

'the diets were:-

Grcmp I

%!

III

Experimental coconut di~t (20% ct>conut fat)

Expertrnental s.s~~o. diet (20% s •. s.o. h Ae·rot' :tl plu$ 0.2% eholestet'ol {1% or oil) added 1!1 or.yst~ll!ne tonn to aiet.

As tor tt pius o.2% cholesterol (1% of oil) in dissolved form in s.s.-o .•

'!'he animals were ble& at the beginning of! the eXperiment e.nd.

· a.tteto one.; two end three weeks on the expettimental diets,

Neithet- form or cholesterol administration with

S;.S·~ o. could. be shown .to pl:ooauoe .effects wb.ieh cU.ffered

sign.if'ieantly from that of pux;e s. s. o. in the diet. The

experimental coconut diet; on the other" hand., pl>Oduced. sel'Um

choleaterol. leveie W'h~Ch were 1teey ed.gni:f'icantly higher. It

.is noteworth;r that higher serum cholesterol 1e;re1s were . ' ; '

found 1n this experiment than in thoae of Experiment 2!i T.his

is a·ttributed to the different basic leirels from whiCh these

changes were calculated~ 1$e. eXperim.ental coconut diet in . ' ' .

Experiment 2 and laboratory ehow in the present one.

TABLE Z MEAN PERCENTAGE CHANGES IN SERIDi! CHOLESTEROL CONCENTRATION OF GROUPS OF SIX RATS AFTER CHJ..NGING

TO EXPERIMENTAL DIETS CONTAINING 20% OF DIFFERENT FATS (STARTING VALUES = 100%)

GROUP

I

II

III

IV

I-II I-III I-IV

II-III II-IV

III-IV

DIET

20% Coconut fat

20% Sunflower seed oil (s.s.o.) .

20% s. s. o. ' o. 2% dry cholesterol

(1% o:f oil) 20% s.s.o. containing 1% dissolved cholesterol

1

131.8 ! 3-4* (100.4)9

110.8 ± 5.2 (101.8)

120.1 ±. 4.5 (99.5)

114.1 ! . 6.4 (102.2)

WEEKS ON DIET

2

6 + 121. - 3.5 (105.3)

+ 97.0 - 3·5 (106.3)

+ . 101.5 - 3.6

(104.3) + 93.1 ... 4.5

(107.6)

S1gn1f'icance of differences (p - values}

2 = Standard erztor,of mean

o.o1 o.o6 0.03 o.ao 0.70 o.so

o.ool o.o1 0.001 0.40 o.6o 0.20

ftft - Percentage change in weight

3

126.0 ± 5.6 (110.7)

102.3 :t 4.5 ' (111.3)

+ 105.3 - 4.8 ' (111.2)

+ 97.3 - 5-3 (113.0)

o.ol 0.02 o.o1 0.70 o.so o.,;o

'

\n 0\ •

51 •.

OIET ·

The effect of small quantities of cholesterol (Experiment 4) ---o--- 20% coconut fat~

• 20% s.s .. o. >< 20% s. s. o.. plus o. 2% eho1. ( cry.s tal line) * 20% s.s.o. plus 0.2% ehol., (dissol:ved in

· S. SoO)•

n. Discussion

:tn the present ·pilot study, using a simple rind

tneXpensi'V'e tH.et 1 devoid ot artificial additi?es; no difficulty

· was experienced in demonstrating significant. ·differences between

the effects of some saturated ana unsaturated :f'ats on the serum

cholesterol levelii tn one case (Experiment J) these differences

sa.

were maintained essentially unchanged for 31 weeks. The

serum cholesterol concentrations produced in rots with isix

dietary oils showed similarities with those produced in

humans with the same oils.. Thus, in Experiment 3, 'butter

produced the highest letrela1 followed by beef' tallow and

groundnut oil and ending with fish o.i1 at· the bottom of the

scale. 'l'hese oils were placed ,in the sa.'ne order by Ahrens et

al. (1957) who fed the s:;une oils to humans. The high and

low levels produced respectively by coconut oil and sunflower

seed oil are likewise well established f'~om human experiment­

ation .(e.g. Bronte-stewart et al., 1956).

These findings are not in agl'eement with similaX'

studies on rats, discussed 1n Section 3 above. A report by

Fu.nch, Nielson and Dam (196o), appe~1ng at the time of

writing is likewise at variance with the present results.

A r1se 1n se:rum cholesterol is descl'ibed after changing fxaom

margarines and butter to maize oil in the diet of rats.

The choice of suitable compounds for the non-fatty

par.t of the expera1mental diet may be responsible for obtaining

the p.resent effects. That this.has a bearing on the subject

becomes evident from the work of Portman, Law~ ana Sruno

(1956), who :find that dietary corn staroh results in lower

cholesterol levels than glUcose in mice. Similarly; diff'erent

proteins (Olson, .Jablonski and Taylor, 1957} an.d vitamins

(Hsu and Chow, 1957) are claimed to have an influence.

It may be significant 1n this respect that the

early success of Schettler's work on mice (see section 3) was

like,vise achieved with skim milk as the basic non~tatty dietary

component.

It was st~essed in Section 3 that the omission from

the diet of cholesterol, cholic acid, thiouracil, etc. is of.

prime impo~tance 1f the effect of t:r1glycer1de fats, and fats

alone, are to be studied. The present series of expe:t>iments

has shown that these addi ti\res aroe not ~equired to imitate

human expel'1ments with I'ats.

One of the problems encountered in some of the earlier

experiments (Expezoiments 2, 5 and 6) was the tendency ot the

serum. cholesterol to escape from the 1n1tial influence of the

different dietary fats·. A similar ph$llomenon has been described

reeently by Seskind and his oo-worke:rs (1959) who observed that

the "•••elevation (of serum cholesterol) appears transient tor

the less saturated. oils but is more sustained for highly saturated

f·ats. u The l'eason for this is not clear. A possible explanation

is that temporary lipaemia may result from excessi\l'e bleeding.

The elimination of this trouble in later experiments by improved

cholesterol detennination methods and C]Ot:sequentl;v milder

bleeding techniques tends to support this explanation. In a

given experiment, hov.rever, this diffieul ty does not vitiate

the discrimination between oils and their fractions; which is

the objective. To counter this and other difficulties it was

d.eH3ided to carry at least one, but preferably two control groups

(experimental coconut and s.s.o. diets) through each eXperiment.

Since, without exception, coconut oil produced significantly

higher serum cholesterol levels than s.s.o., the effect o:t

60.

the unknown oil fractions could then be assessed in relation to

these controls.

E. Conclusions from part II

It was felt at thia stage that the experimental routine

maohiner,v had been brought to a state o~ sufficient refinement

fo~ its practical application to the main problem• without fear

of breakdown. Although the lipid metabolism of t"ats may differ

in some important respects from that of humane, concU.tions have

been created under which ths serum cholesterol r~gulating

properties o~ different oils may be similar for both species.

If such 1s the case. any clear understanding of the lipid

metabolism ot the rat !s not immediately essential tor the rat

to be used as a test animal for the study of these oils b7

fractionation or other- techniques~ The sensitivity of the present

rat assay method can best be judged from its application. In: '

Experiment 12 (Section 14}, tor exarnpl.e, significantly' different

e:ff'ects (P = o.oo1) relative to a standard f'at have been

demonstrated with as little as 35 gme. of oil after seven days'

feeding. .Furthexmore, it can be predicted with confidence from

the results of Experiment 4 that under the conditions of these

exper~ents an1 manipulation of the serum cholesterol of the

rat by dieta:ry fats is unlikely to be materially affected by

such small quantities of cholesterol as are pres~nt in most

common animal fats.

P A H T. III

;!'HE. HYPOCHOLESTEROLAEMIC EFFECT

OF Btn."FLOWB:R SEED OIL

61.

SECTION 10

INTRODUCTION

Careful thought has been given to the selection

of a particular fat or oil for closer examination. Preference

was given to a typical cholesterol-lowering oil since this

appeared to hold out the prospect of isolating from such an

oil a substance Which might by itself be capable of depressing

the serum cholesterol level. If sufficiently potent in this

respect, the practical value or this hypothetical substance X

might be considerable.

Of the existing number of oils of known hypocholester­

olaemic p~perties, sunflower seed oil (s.s.o.) was selected for

detailed study for the following reasons:

1. The effect of sunflower seed oil on humans is Vlell established (e.g. Bronte-stewart et al., 1956, Williams and Thomas, 1957, Gordon and Brock, 1958).

2. The fatty acid composition of sunf'lower seed oil is ver,v stmple, consisting of four fatty acids only. Should a fractionation of the fatty acids become necessary this fact is important.

3. The various inter.mediary products and by-products from the extraction and expression of the seeds to the refined oil were obtainable fram a local Pefiner,v.

4• The author had some experience in the technology of this oil.

62;.

.'l'he chemist tv of su.nf'lqwe~. seed oil -~.

The oil eXpr-essed from the seeds of the euntlower

plant (Ke)J.anthus annuua) contains tour f'att;y acids onl;y-, ' namely palmitic* stearic, oleic and. linoleic acids. The

. '

relat.ive proportions of these acids in the oll val'ies widely

aeco~ding to the cl!nlate in which the parent pl.ants are grown.

Tropical eltmates favour the p..roduction of mot'e saturaated

acids whel?ees the r>e?e:rse holds tor more temperate regions,,

Varoiattona ot ltnol;eto acid content from the eJ<ttt'emes of'

30 to 75 per cent are ,encountered.. From the author·• s

ex;pel':i.ence, S0uth African grown plants yield o·ils 'tiv:t th a

linoleic ~cid content .ranging from 57 to 66 par cent a.s

detennined by alkalf.•i.somerisation techniques (Am, Oil·. ·

. Chemists' Soc.; l95::5h With the modern methods of gas:...liquid.

pa~t1tion ch:rom.atogra.phy' used :t.n Secti·on 13; considerably

higher values were obtained. This is in agreement vvith the

work ot Or>a.tg and Murty (1959l. These. :investigators likewise

fpund that the older analysis · o.f Se s. o. .had. to :bf3 :re·v:tE::ed r­

and report the following figu.x-es for. some oils ranging·. in

iodine value 'between l38o$ and 141.6:

Fatt;r a.cid,

Palmitic Stearic Oleio Linoleic

% Composition

6.6·~ 7e2

3.7- 4·1 15•!5 -l6o2 73·9 ·"78·3

L.ike most vegetable seed oils• eru.de siis .• o. oontains small

quantities of phosphatides. These are, however; normally

removed in the refining process. Less is known of the

composition of the unsaponif'iable matter, but sitosterols;

carotenes, squalene and tocopherols l'lave been reported.

These will be considered in'more detail in Section 14.

The chemical constants and full analysis of a

sunflower seed oil used in these tests will be found 1n

Table IIb of section 13.

SECTION 11

!tHE EFFECT O:F . WHQ~E St!mPWER. SEED@. ( EXperit.ment S)

suntlowez. seed oil· (s.s.o,·) is obtained industrially

by the expzaession. o~· solvent extract.ion o.f kernels which in

most ·cases are onl37 very in·completeiy separated from husks •.

Crude s .• s. o* in fact, develops a ·Oloudinesa on stan<Ltng which

can 'be traced. to a wax, cet7l. eerotate, originating f.rom the

husks (l3a:r-enther, .1923).. Any claim to a systematic search for"

h9pocholesterolaemi,c sub.stances should, therrefore • include not

only the p.usk, but also thenon•:f'atty protein: portion of' the

$eea.s. ln the present ,expe1"1rllent ,. the relati'V'e serum eholestet>ol

depress~ .. ilg properties of' husks, eXpressed o11-·and the pl?otein

f~actton of the whole seeds were tested•

Diets

Due to the·nature ot the substances to be tested, the

diets fo~ this eJq>eriment dev.iated somewhat ':f'l'"orn the standaro

pattei'"nt and we!\e as f,ollowet

Group t '!J.rcmp l.I :

Group III:

.10 per cent of coconut fat of egperimental coconut diet repl~ced' by crude s~S-eO• 10 per cent of coconut f'at.ot experimental coconut diet replaced by crude s~s.o.·given in the term o:f' kernels. As for Gt'oup l plus 5 per eent of' finely ground sunflower seed. husks •

1'he Group Itt l."attons necessitated a.n adjustment of .th~ skim

milk component . to keep the. diets :tsonitrogenous .• The ·:resul ti.ng

65.

deficiency of carbohydrate was made up with potato starch.

Other departures from the standard experimental

design were as follows:

1. All an1rnals were pre-fed with eXperimental coconut diet for 15 days before the beginning of the experiment.

2. To ensure equal ·consumption of these heterogeneous diets, rations were l"estr.1eted to 20 gm •. per day,

per rat.

I. !

Husks were excluded f.:rom the diets .of Groups I, II, and III.

14 26 35 DAYS ON DIET

Fig. 1~ The effect of various fractions from whole sunflower seeds (Experiment 5).

---<>-- Exper.imental coconut diet (20% coco.·) • - 10% cooo., 10% s .. s.o. (pure oil) • · 10% coco., 10% s. s. o. (as kemels) A 20% coco., 5% hUsks.

TABLE 8 - {Experiment 5)

MEAN PERCENTAGE CHANGES IN SERUM CHOLESTEROL CONCENTRATION OF GROUPS OF SIX RATS .AFTER THEIR EXPERIMENTft..L DIEri+ CONTAINING 20% COCONUT F.AT IS MODIFIED AS SHOWN

(STARTING VALUE= ~00%)

GROUP DIET DAYS ON DIET

14 26 35

I 20% Coconut fat 105.8 + 3-7• 105.8 + 3.7 103.3 + - - -(diet unchanged) {101.5)& (102.3) (100 .• 8)

10% Coconut :rat .+ 4.2 101.7 + 4.9 104.4 + II 81.4 - - -

10% s.s.o. (pure oil) (102.2) . (102~9) (104.4)

III 10% Coconut fat as. a + 2.7 96.6 + 5·1 96.6 + - - -10% s.s.o. (given as kernels)

' (100.0) (lOQ.O) (98.1)

20% Coconut fat 100.9 + 3.2 + 6 mar 94·4 + IV - 111.1 - 3. I -

5% sunflower seed husks {98.4) (99.0) (99.3}

Significance of differences (p - values)

I-II o.ol o.so o.so I-III o.o1 0.20 0.20 I-IV o.so 0.30 o.lo

II-III o.so . o.so 0.10

+ = HUBka omitted from Basic Diets of Groups I - III

3.8

3.1

2.7

3.6

2 = standard error of mean H = Percentage changes in weight of rats iBDl = Changed to Diet II from 26th day.

Results {See Table 8 and Fig. 14)

After 14 days on the teet diets there was a mean

drop of 16 per cent in the serum cholesterols due to the

unsaturated oil diets of Groups II and III whiCh were not

significantly different from each other (P = o.so) but

significantly different (P e 0.01) from the control Group I

and the husk Group rv, the latter two again being similar.

As after 26 days differences tended to disappear,

it was decided to switch the husk group to 10 per cent s.s.o. {as for Group II) for reassurance as to the sensitivity of

the animals at that stage. The consequent drop of 15 per cent

confirms the initial observations. The dotted line in the

graph represents the mean value of this secondar.y drop.

It is concluded from this experiment that no

appreciable Cholesterol depressing substances are present

in the whole sunflower seed other than those due to the oil

itself. The transient nature of the response of the rat

exhibited undei» the conditions of this experiment does not,

it is believed, detract from the validity of this conclusion.

68.

SECTION 12

THE CHEMICAL FRACT£0NAT!ON OF SUNFLO~VER SEED OIL

The next step in the study of the serum cholesterol

lowel'ing property of s.s.o. is to separate the latter into

its two basic parts~ i.e. fatty acids or tr1glycer1des and

the unsaponifiable matter (U.M.) and examine each fraction

separately. Although the unsaponifiable matter constitutes

only approximately 1 per cent of the whole oil, an "active"

substance, 1f such exists, might conceivably reside in this

fraction.

A. Preparation of fractions

The starting material was an alkali refined, bleached,

steam-deodorised and cold filtered product with an iodine value

of 133 and linoleic acid content of 62 per cent (alkali

isomerisation). To ensure maximum protection of labile

unsaturated substances from isomerisation and oxidation the

following mild techni~e was developed to separate the unsaponif­

iable matter:

930 gm. of oil to which 2 gm. of hydroquinone had been added as antioxidant, was shaken vigorously with one volume of 5 N. KOH and two volumes of freshly distilled (over zinc poWder and XOH) ethanol.

Under these conditions, saponification was complete in a few

minutes at room temperature. The conventional and possibly

har.mful refluxing of the saponification mixture for hours at

Pis. 15 Apparatus for the extraction of unsapon1f1able metter.

70.

elevated temperatures was thus el~1nated.

The resulting soap solution was diluted.1mmediately

to 15 litres with distilled water and extracted with freshly

distilled dietbyl ether in an apparatus adapted :from an

extractor described by H1ld1 tch (1956). 'l'he diethyl ether

was kept free :rrom·peroxides by continuous distillation over

alkallne pyrogallol (see .Fig. 15).

10

:: 5 ;;. z ... 0

220 240 260 WAV€. l.€.NGTH

FiS• 16 Ultra-violet absorption spectra of eth.yl esters of s.s.o. fatty acids (see text).

After 36 hours of continuous extraction time;

96.5 per cent of the 1.05 per cent of U.M. originally present

in the oil was thus removed. The etheral extract was !'reed

from pyrogallol and hydro~inone by repeated partition with

o.s N KOH and distilled water. The ether was finally removed

71.

by distillation and vacuum•

The potassium soaps were converted via tho free

acids to the ethyl esters in 97 per cent yield following

the procedure described by Hilditch.

A second batch of ethyl esters were pl'epared. as

above but without removal ot the U .. M. The iodine values of

the ethyl esters (.122.5 and 12,3.3) agreed within experimental

errox> with the theoretical .value calculated from the i.odine

and saponification values of the original oil, d.emonstrating

no appreciable deterioration of the acids. That the extraction

step eau.sed no structural changes in the oil was fUrther

confirmed by the negligible difference found in the absorption

spectra in the ultra-violet region ot the two samples, :t.e.

the extracted (sample A) and unextt-aeted {sample B) esters.

The two .spectra are illu~trated .in FiS• 16.

B. The eff'ect of' saponifiable and unsaponifiable matter . . · .. · .. . .... (Experiment 6) • .. . .· ... · . .

The two fractions pl'epa:red as described above were

used to pl'epazoe the following experimental diets.

Group I· 20% coconut fat (experimental coconut diet).

II. 10% coconut fat, 10% s,, s.o •.. , ethyl eaters, U11Me removed.

III. 20% coconut fat containing 2% (0.4% o.f whole diet) ot·U.M. separated from. s ... s.Oti; in dis.sol ved f'ol1Jl .•

IV. 10% coconut fat, 10% s .. s.of)~ ethyl esters, u. M. not removed .• ~ '

It will be noticed that in Group III the quantit;v of u.s. fed with coconut fat is app~oximately twice the concentx-ation

in which it is present in s.s.o. This was done to accentuate

possible e:t'f'ects ..

Results (see Table 9 and Fig. 17)

As on previous occasions the e:xperimental coconut

control diet produced the highest serum cholesterols, whereas

the erode s. s. o. esters (U.s. not removed) pr>oduoed the ·lowest.

Extreme~y interesting, however, is the low cholesterol eu:rve

produced by Group III. receivi~ s.s.o. unsaponi£1ablca9

This group closely resembled the behaviour of Group IV and

was, in fact 1 statistically indistinguishable fl:'om it, but

signifioant]Jt lower than Group I after nine and sixteen days

(P = 0.01 and o.04). In view of this resttlt it is not

surprising tbat Group II receiving purified s.s.o. esters,

i.e. substantially free from u.M., oocu.pted a position inter­

mediate between the Groups 1 and tv. As in Experiment s, under the conditions which obtained here, the responses of

the rats to these diets appeared to.be of a transient nature.

Fig.l7/

TABLE 9 - ( E;periment 6)

MEAN PERCENT .AGE CHANGES IN SERUt.f CHOLESTEROL CONcg..rTRATION OF GROUPS OF S !X RATS .AFTER CHANGING FROM A LABORATORY CHOW DIET TO EXPERntENT.AL DIET CONTAINING THE FAT PREPARATIONS LISTED

. (STARTING VALUE = 100%}

GROUP DIET DAYS ON DIET

8 16 23

I 20% Co-conut fat. 125.0 + 6.o* 122.5 + 4.6 111.7 + 3.3 - - -(97.o)iE* (101.6) (99-5)

II 10% Coconut fat 105.8 + 5.4 104.0 + 4-3 103.6 + 2.9 10% s.A.o.esters unsap. - - -removed (99.5) (106.5) (108.7)

III 20% Coconut fat 96.8 + 5.6 107.4 + 4 .• 2 114.0 + 5.2 - - -0.4% unsap. from s.s.o. {96.8) (100.8} {103.5) IV 10% Coconut fat 90.9 + s.o 101.5 + 3·6 lall ..• l + 6.7 - - -

10% s.s .. o. esters unsap. {96.5) (98.5) (101.0) not ztemovea - -

Significance of dif':terenee (:p - values)

I- II o.os o.o2 I- III o.ol 0.04 All I- IV o.o1 o.o1 N.S. II- IV 0.07 0.70

.III- IV o.so 0.40

H = Standard error of mean tif = Mean percentage ehanges in weight

~------- -------------

8 16 23

DAYS ON •o1E.T

'----~--~--------------~--

The effect of the unsaponifiable matter from s.s.o. - (Experiment 6}.

~- 20% coconut fat

I ,

I I

l ~ r

I

· • 10% coconut fat + 10% s. s. o. esters u.M. matter removed

• - 20% coconut fat plus 0.4% U.M. from s.s.o.

• 10% coconut fat + 10% s.s.o. esters, U.M. not removed. -

C. The effect of interchanging the unsaponifiable matters of coconut and sunflower seed oils - 'Ex~eriment Zl.. " ,

If' the serum cholesterol regulating substances/in

fats and oils reside in the u.u., it should be possible to

reverse the effect of coconut and snnflower seed oils by a

total interchange of unsaponifiable matters between the two

Oils. tn the proportion of their naturol oeeu~nce. This

wns ette~npted here •

. mel'imentol

Using the t~chn1ques described above, the following

four f~actions we~o p~epnred and tested on rata;

1. · ~M ethyl e. etora .. hom oo. conut :tat .... tv aotao, t.e~ iilthOit t the removr.:ll o.f 1 te own u .. t.h

a. Purified ethYl esters from eoconut tatty eotda contnin!ns ltOS% tl .• r.t~ trom s-s.o.

3.. Qmdp ethyl esters trom s.s.o. flltt:v acids. 4. i!9.£1f1ed etey].estero ·tram s.s.o. tattu notds

con~alnlne o.S4% ot u.u. tram coeonut tnt •

.J

811

!·~~~~~ !~L-·--------~----~,~~--~------------~.~~

OA.YS ON DIEt

PlS•. 1,8 The. effect of tnterohongtng the unsv.ponlt1ob1o .mnt·ter between coconut nnd aunf'lower seed. o·il etl\V'l entetas ... (EX,Perbnent 7). ---o--- Coconut eetern • coeo, u. n.

• • Coconut estern • s.s.o. u.s. • s.s.o. eaters + oooo. u,s. • s,s.o. asters + s.s.o. u.s.

Since insufficient quantities of material were available•

these fractions· were fed as 15 pe~ cent on.ty in the basic

tat•f'ree diet.

'the replacement of. the u. M. of s.s. o. by that of

coconut fat had no demonstrable ef'f'ect •. i'he reverse procedure,.

however. t.e. the exchange ot u.:M. of coconut tat by that of ' ' ' . .

s. s~ o. resulted in a complete r.epresaion of the serum cholesterol

raising prope.rty of the c:rude coconut fraction f.!-ftel' 9 days and

about 60 per cent repression after 18 days • feeding. These

effects were statistically secured respectively at two and

thl'ee per cent. The results of the present experiment (like

those o£ Experiment 6) suggest that the feeding ot fatty neids

in the f'ornl of' ethyl este:t's instead. of trigl;vcerides does not

materially change their oharacteristie effects •

. D. Discu.ssion

tt can be concluded from the resui~s ;Crt Expe~J.ments

6 ana 7 that there is little 'doubt that the Q.nsaponifiable

matter of' sunflower seed oil. 'ts somehow implie~ted in the serum

regulatU'ig pl:'Operty of this oil. Exa<:tly 'to what extent this

is the case it is C.U.ff'icult to say at this stage. l:t appears

that the ll•M• iS pvobabl;v not the only responsible factor Sinee

1t was possible to demonstrate a partial reversal only, of' the

effects of two oils by the interchange of their unsaponit:lable

matters. In the later pa.x>ts of this work (Part V) art explanation

will be givo:en for these observations.

TABLE 10 - (p;xper1ment 7)

MEAN PERCENTAGE CHA11GES IN SERUn CHOLESTEROL CONCENTRATION OF GROUPS OF SIX RATS AFTER CHANGING FROM A LABORATORY CHOW DIET TO EXPERIUENTP.L DIET CONT.AniiNG ETHYL ESTERS OF COCONUT OIL MID

SUNFLO'l"IER SEED OIL FATTY ACIDS WITH INTERCHANGED ID1Sl\PON!FI.~'\BLE MATTERS (STPRT!NG VALUES = 100%)

GROUP. DIET DAYS ON DIET

9 18

I 15% Coco. esters + coco. u.s. 114.3 + 2.9* 127.0 + - -(94.7)!1Hl (97·7)

II 15% Coco. esters + s.s.o .. u.s. 100.7 + 3.8 109.3 + - -(96.3) ·(96.0)

III 15% s.s.o. esters + s.s.o. u.s. 94.6 + 3.0 93.0 + - -(95.6) (96.9}

IV 15% s.s.o. esters + coco. u.s. 91.1 + 1.4 97.2 + - -(95·3) (95·3)

Significance ot differences (p - values)

I-II II-III II-IV

III-IV

~ = Standard error of mean ~ = ¥ean percentage changes of weight

o.o2 0.03 0.20 o.o1 0.05 0.04 0.30 0•40

Coco. = coconut fat s.s.o.= sunflower seed oil u.s. = unsapon1fiable matter

5.1

3.8

3.1

3.2

78.

Jones§ Reiss and tfuffinan (1956) were among the

:tirst to d.raw attentton to :f'aoto.~s other then ths fatty acid

They :fed cor.n oi1 !lnd an .I

equ.i valent · amount o£ com oll in the fom of . c:orn germ to

cholesterol :f'ed eh.iCkS and found the· earn germ· to be

considerably more .active as. :a se:ruffl .. :choleste'~o'l d~pr.easa?J.t, . . . . ..

. . I - .

suggesting the pr-esence of' ad.d.i tion·a~. faot:ors in the gem . .

other than the o1.1 .i tse·lf! •.. t:rhi.:s pr1nes;ple did not ap:Pl:V '· <., . ' .

t'o 'the Vthol~ .aunflowel?· see:d .and the 'oil· e;xpr.ease~ .. thel~e.from ~ . . . . ' \ . . .

~ 1: . '' ' ' .. '• ' ' . '

a.nd tested w1 th rats .as .lin EXperiment 5• Sones ·and his

as.soc.iates also rea~ranged the propo.rtions of. fatty t?¢1ils

p.resent .in cottonseed oil to simulate the oomposi tion of'

corn oil without being able to reproduce the depreas.ing effect

ot the latt~r. They ·conclude "It seems unlikely that this

difference. •• ~·could be e:xplai.ned by di.:rf.erence.s in fatty acid

composition."

Experimenting on the human scale With a variety of

different oils,1~~l'1Ils5!?~=~-~t ·--a-0 -~(1957) came to similar'

conclusions: ,.~"~the cholesterol depr-essant action of' corn

oi.l towe::.Nls humans was not fully aee.ounted for by 1 ts degree

of unaatu:ration or eon tent of • !!Ssential ·• tatty a.c:tda. u The

unsaponifiable f.raetion .of this oi.l was latet> i~~estigsted.

by Grandet Anderson and Keys (19$8}, Who fed o.es sm •. of "~om

oil tJ. M. daily to .man ana recozoded a mean decrease of only

:;.2 mg~ of cholesterol per .100 n1l ot serum, wh10h was not

sta.tlstically signif'ican.t.e

Ahrens and associates (i957), af'te!P a s.imilar-' study {see Seetion 2) find no di:f'f'erenee in the activity of

corn oil after :reducing the unsaponifiable mattel? from

1.2 to o.~ pel" cent, admitting, howe\tel:', that their

..... ~readiness to accept the unsaturation hypothesis as

finnl:V proven has been tempered by the realisation that

none of the exper~ents performed here or elsewhere has

ruled out the possibility that the :factors sought may lie

. in the non-gly-cer:td.e po:rtion of the ted tats."

The work of Beve~idge and his group with humans

has been discussed in Section 2 and elsewhere. Although

the p~esent studies were conducted independently from, and

approximately parallel in time to those of these investigators,

sim.ila~ities of.expe:rimental design, results and interpret­

ation are evident. This applies more particularly to the

importa.nce attached in both studies to the :role of the

non~glycerid.e fraction.

It may be'objected that the chemical separation

of' saponifiable and uneaponifie.ble matter constitutes a

drastic and perhaps illegitimate interfevenee with the

properties o'f natural oils. This difficulty does not arise

if' the composition of the unaaponif'iable traction of

sunflower seed oil is changed by physical means.

80.

SECTION 1,2

THE PHYSICAL FRACTIONATION . OF SUNFLOWER . SEED OIL

A. :the l!gp.id ;propane se.sresatton .of sunflower ~eed o,i,ls

The physical fractionation ot tPiglycerides is

cU.ff!cult on 1:u:count of the small d.iffet:'Emces in pbysioal

propertie.s between triglyceFide speciee found in most common

oils, A comparatively crude separation into mo.re saturated

and unsaturated fractions can be effected b;r low-temperature

Oey'stallisation in its various modifications. Of the more

efficient methods, howe~er; only molecular distillation and

segregation in liquid propane have been used on a large

scale. The latter process is of comparatively recent origin

and is still little known in the oil industry~ Only a \n.""1ef

description will be given since it has been fully described

among othe~s, by Mettikow (1948), Pasa.tlno (1949) and Stubbs (1951) ..

The separation of different triglyoerides b;y liquid

propane depends on their selective solubility in this 11qu:ltied

gas, at temperatures neal" its critical po:tnt. Propane, a gas

at nonmal temperatures and pressure, is easily liquified by

compression to about 10 atmospheres. At 1ts critical temperature

ot 95.6°0 it has a critical pressure of 43 atmospheres

(632 lbs./sq .• inchh The solubility of glycerides at a given

concentration, i.e. a given propane to oil ratio, is a fUnction

of the temperature. Its solubility decreases with 1ncree.$1ng

te..1ltperatu.:re and beoomes more and more selective with respect

to moleoula~ weight as the temperatures approaches the

critical temperature of 95.6°C., In ge~eral, molecules of

lower molecular weight and unsaturation ha~e a ~1gher solubility•

and vice versa.

In practi~e the efficiency of this simple separation

process increases many times in a fractionation tower in which

~oth e. temperatuM and concentration gradient exists. The oil,

premixed with propane at a suitable temperaturee is pUmped und.$r

high pressure into the tower somewhere near its middle.

Equilibration of the const.ituents between two liquid. phases

takes place at each point along the length of the tower, the

heavier ph_a.ae dropping to the bottom. ~he pFoec:u~a resembles

in some respects ordinar:v fractional distillation :u.nder reflux

except that in this case two liquid phasea are present and the

highest temperature is meintB;ined at the toE· The different

fractions a:r-e bled ott at appropriate points along the tower,

the high molecula:t' weight components nesr the bottom, an.d vice

versa. The propane is flashed of:f and l"'e1iquit:ied for fu!'ther

The liquid pr-opane segregation process (Solexol

process) was first applied for the concentration of' vitamin A

fx>om fish liver oil by Mar1ne 011 Refiners of' Africa Ltd.. at

Simon's flown. Vitamin A and its esters, with a comparatively

low molecular weight (286) concentrates together with the

. greater part of the unsaponif:iable matter in the light (overhead}

fraction. The rest of' the unaaponi:fiable matter tends to

concentrate in the heaviest (bottom/ f'~act:ton.

While the concentration of' unse.poni:fiable matter 1s

82.

relatively easy, the fractionation of the triglyeerides them­

selves 1s more difficult on account of the smaller differences

of molecular weight obtaining between different t:riglycerides.

Fish oilt however, consisting of esters of' fatty acids with

. widely different chain lengths can be segregated into high and

low molecular weight fractions which \vill incidentally also be

respectively of high and low unsaturation.

Attempts to segregate sunflower seed oil into high and

low iodine value fractions meet with difficulty. This will be

evident from the following table. This summarizes the results

obtained in the laboratory by the low-temperature fractional

crystallisation in diethyl ether (do\m to ... 70°0) of sunflower

seed oil according to the method of Hilditch (1950).

TP.BLE lla

CHEMICAL CONSTANTS OF GLYCERIDE FRPCT!ONS OBT}.!l'lED BY LOW- H TEUPERATURE FRACTIONAL CRYSTALI.IS.ATION OF SUNFLOWER SEED OIL

Glyceride Weight Linoleic Iodine Sapon. Glycerides fraction (gm.) acid % value equiv. % mol.

A 13.6 36.2 '78.8 291.0 6.9 B 38.1 53.3 111.2 291.0 19.3 c 33.2 61.5 133.4 293·5 16.6 D 20.9 70·5 142.8 291.0 10.6 E 60.0 72.2 11.14.1 29.3.0 30.0 F 18.2 68.2 147.7 297.5 g.o G 16.0 65.8 155.0 310.5 7.6

Total 200.0 64.0 1.33.8 293.0 100.0

While the iodine values ot the fractions range from

78.8 to 155 the mean molecular weights of fatty acids as reflected

in the saponification equivalents show comparatively little change.

a These resul te were obtained b;y the author in the research labor­atories of Marine Oil Refiners of Africa Ltd., and are reproduced from Laborator,y Report No. 356, dated October 27, 1953.

The glyceride composition of 'Sunflower seed oil can be calculated

.from thene data. This showed. that the distribution of fatty acidS

in the glyceride molecule follows ·closely Hildi teh • s law of e'Ven

distribution. This is another unfavourable factot> against ,the

successful propane segregation of sunflower seed oil• Th.eoe

theoretical considerations were fully borne out in practice.

Even unde:r the most selective plant conditions, vecy little

effective :fz>actionation of glycerides was achieved.

With regard to the unsaponif'i~ble matter, however, the

situation 1s different. ln the case of·V.itamin A bearing fish

i:tyer oils~ .f'or exrlrnple, fractd.ons consisting of over '70 pe~ cent

of'. un~aponi:f'iable mat'ter aan be obtained u.nder optin1ttm eondi tions. I ,

. I . . . ·soth from theoretical conaide.tta.tions ;ana past exper.i~nee, the

' • propane segregation o.f' sunflower seed oil would be expected. to

37ield f'ra.ct1ons of un.ifom glyceride .compo.sition, but differing .

in both quantity and composition of unsaponifiable matter4

Through the generosity ot Mar~ne Oil Refiners of

Africa Ltd~; a liquid propane segregation pilot plant, together

with operating staff., was made available f'o.r experimentation ..

Ninety gallons o.f sunflower seed oil were also donated for this

The sunflower seed oil was fractionated in liquid

p~opane und.er comparatively uneelective condi.tions; i"'e.

comparatively low temperature (165 .... 1'75°F) and low propane to

oil ratio (40 : l). This was done to minimise effective

fractionation of triglyceridee wh1.le still allo\Ving the easier

fractionation-of unsaponifiable matter to take pla.ce. Th:t-ee

84.

sunflower seed oil fractions were obtained; a light (A),

inter.mediate (B), and heav,v (C) traction comprising

respectivelY 35%, 57% and 8% of the charge.

An impromptu attempt was also made to use the

propane plant for the large scale extraction of unoaponifioble

matter from aqueous alkaline saponified sunflower seed oil.

While the saponification of 45 gallons of sunflower seed oil

according to the method described in Section 12 proved

comparatively easy, the "unsap." extracted by liquid propane

could hardly be recognised as such. It consisted mostly of

mineral grease, pipe sealing compound and other unidentified

constituents. These were leached out from various parts of

the plant by the alcoholic and strongly alkaline soap solution.

The expertment, performed with unsuitable equipment, \?Os

lT.ritten off as a fiasco.

B. Composition of propane fractions

The results of the chemical analysis of the three

fractions and the original sunflower seed oil are set out in

Tablellb. The deter-mination of the usual chemical constants

followed conventional lines. Sterols were deter.mined by

precipitation of the digitonide from 95% ethanol. The fatty

acid composition was deter.mined by gas-liquid chromatocrraphy of

the methyl esters. The latter were prepared as described in

Section 12. The stationary phase wao 15% Glutarate-ethylene

glycol polyester supported on Bo-100 mesh oelite. The 200 am.

by 0.5 cm.column was operated at 217°C under an argon cas press­

ure of' 52 lbs./sq.inch. The instrument was a Barber-Colman gas

as.

TABLE 11 b

(E;pertments 8 & 9)

Chemical COffiPOSition of Liqgid Propane

Segpegated Sunflower Seed Oil Fractions

Fractions

A B Original (OVerh.) (Interm.)

% Free fatty acids (as oleic) 2.36 3.33 o.67

Iodine value (Wijs} 132.6 131.0 133.8

Saponification value 169.0 191.3 188.2

% Composition of H fatty acids Palmitic 5.7 5.9 6.6 atea~ic 2.1 :;.9 4.:; Oleic 17.5 18.3 16.9 Linoleic 74.8 71.9 72.2

% Unsaponifiable matter 1.08 1.52 o.64

%Digitonin preoip1t-able sterols 0.403 0.·705 0.210

%Carotenes (as p,.-oarotene) o.o11 trace trace

dete~ned by gas chromotography

. c ·,

(Bottom)

2.39

131.3 185.5

4.5 3•G

15.7 76.2

1.19

0.096

0.23

~ I

sj 1

~ - ..

86.

----,....---;;-:--.---=-~.......:_+---7-~i-:--t-+---l-~-l---+--''-'--+-r--·--,---;--- - - -;; ·---~L-~~~~-+~~-~~+----~~~----~~~

Oaa-11qu1d chromatog~ of ~ethyl esters from 11qu1d propane eegregated sunflower seed oil fractions.

I

:( ~~ 100 oo 8' 7o "'

., "' "' 10 • 10 "'~

-"" .. 30 I .. CD

I > 'I

.. .8 • .• 3 . " "' "' I ~~ l ··t·•

:, .. • ;::: ,. .

I V • ~ 'Xl "" ... 0

t-g '

' '" "' ~ ~ ll CD . • ··I· ; ! aa-

tJ • ~ (!) t(Jtl,...

"" '!" O;!it<D

~~t! f t

!t-4

I c.-. : .::s.,..

t ..... 1 •••

l i•o •

c ; .. ,._ I s..-vo:s

CIO 100 00 70 .. i ........ c g~g . ... J. - .• ..tiiO · o ,... ... o ., •.'S

-· c!eaS , I . 70 "" I "' " ., u

" .. "' N T

~ u Q 1

CD 0 l

0 -~~,

10 "" 30 "' L 0 10 "' "' ·-""'

88.

chromatograph. The setting up of the instrument and the

supervision of' the analysis by Drs.s. Lipsky and J. Lovelock

.is reflected in the neat chromatograms obtained• These are

reproduced in Figs. 19a and l9b.

l:n F1g.o 19c the absorpt·ion ·spectra in the visible

region ot the unsaponif'iable matter of the sunflower seed oil

tractions in eyolohe:xane are reproduced~ The presence of a

0.07

0.06

> 0.04 ,_ "' z UJ 0

..J <( u

~ 0 0.02

340

Fig.. 19e

0.018

0.016

0.014

0.012

.• 0.010 ... ·· .. t

...... f3·CAROTENE 0.008

0.006

0.004

360 380 400 420 440 460

WAVELENGTH (mJt)

Absorption spectra of' the unsaponif'iable matter from liquid propane segregated sunflower seed ·oil fractions in cyclohexane. Fraction A, B & charge: refer to right scale Fraction c:. ref'er to left scale a-carotene: arbitrary scale Extinction coef'f1ci.ents are calculated from concentration in whole oil. ·

~aroteno1d pigment and its app.roximately twenty-fold concentr­

ation in the heavy fraction (fraction C) is unmistakable. The

dete.rmination of the abso:rption curve of a pure sample ot ·~ •carotene in the same solvent (dotted. line on a.rb:i.traey scale)

did not oo~respond to that of the oil fractions. The latte~

probably contained a mixture of Ol 1 ~ and 1 carotenes a:nd othel'

chromogenic carotenoids.

An inspection of the results reveals that ea:r-lie~

hopes were fulfilletl. Three uniqUe sunflower seed. oil tract ions l

have been created. The fatty aci:a composi t1on of all f:ractions I

shows no significant deviation :from that of the original oil.

This is also reflected in the similarity of the iodine.values.

The unsaponifiable matter; however, is seen to differ radically, .

both qualitatively and quant1tat11Tely- in all fractions. !hua

sterols have concentrated in the light fraction whereas cal~otenes

have collected .in the "bot toms••. It can be reasonablY .. assumed.

that a s~ilar faie bas overtaken other undetermined consti tu~nts

ot the unsaponif'Utble mtlltter. Effective fractionation of the

~saponifiable mat~er has been achieved without materially

· affecting the composition of' the saponifiable matte!'.

c. ~e effect. of' ,various f'!'a~tions (~eriments 8 and 9) . . .

The three liquid propane segregated sunflower seed oil

fractions were tested in the usual way. i.e .. 20% of the oils in

the experimental diet were fed together with a similar coconut

oil control (Experiment ·8),.

Encouraged by the results thus obtained the experiment

was repeated under more difficult conditions. In the tr>epeat

eXperiment (Experiment 9) only half of the oil in the diet

consisted of the test oils, the remainder being coconut oil.

Results and Discussion

The resul te of Experiment 8 (Fig. 20, Table 12)

show that fraction C produces lower serum cholesterol levels

than either.:f'raction A or fraction B. There appears to be no

significant difference between the two light fractions (A and B).

Their properties are intermediate between that of coconut oil

and f'ract.ion 0.

7 21 DAYS ON DIET

The effect of liquid propane segregated sunflower seed oil fractions on the se~ cholesterol concentration. (Experiment 8) --o- coconut oil control

• fraction A ~1·· igh. t ) .. • fraction B medium) • fract.ion 0 heaVy)

TABLE 12 - (Experiment 8)

MFJI,N PERCENTAGE CHA:t-TGES I:t-T SERUM CHOLESTEROL CONCE"~TRJ\TIOl:r OF GROUPS OF SIX RATS .AFTER TRANSFERRING FROM A LABORATORY CHOW DIET TO EXPERIMENT.hL DIET CONTJIINDIG THE SUNFLOWER SEED on, FRAO'l'IONS LISTED

(STARTING VALUES = 100%)

GROUP DIET WEEKS ON DIET

1 2 3

I 20% Coconut fnt 137.3 ... 7.5* 134.6 + 9·7 144.5 + 7.1 - - -{103.9)3Bt (102.8) {107.6)

II 20% SUnflower seed oil 113.8 + 5.9 112.3 ... 3.9 115.1 + 4.6 overhead fraction - - -

(Ft-action A) (106.8) (104.9) {109.9) 20% SUnflower Seed oil

111.2 + 3.8 105.2 + 3.3 112.1 + 3.4 III intennediate fraction - - -(Fraction B) (109.1) (105.6) (108.4)

20% SUnflower Seed oil 95.2 + 2.7 89.4 + 3.1 109.0 + 3.5 IV bottoms .fraction - - -

(Fraction C) (99.2) (100.3) (103.0}

Significance of differences {p - values)

I-II 0.03 o.o6 o.o1 I-III o.ol 0.02 o.o1 I-IV o.ool o.oo1 o.oo1

II-III 0.7 0.2 o.6 II-IV 0.02 o.o1 0.3

III-IV o.o1 0 .. 02 o.6

• = Standard error of mean !0£ = Mean percentage changes in weight of rats

\0 .... •

The same trend is apparent · in Ex.peiJim.en.t 9, &xcept

that the Cholesterol curves .1n this instance ere rttnning closer

to the cot:onut aont:r-ol.. This would indeed be expected from

the smaller proportion of test oj.ls which were .offered to the

animals •

. . '

7 14 21 DAYS ON DIET

Fi,s,.. 2.~ The effect of liquid propane segregated sunflower seed oil :r:r:oactions on the sertm1 cholesterol conc.entration (Ex-periment 9) o!

--o- coconut oil control} . . •. . f·.ra. cti. on A: ~light) half substitution · • fraction B medium) see text.: • · fraet 1on c · hea'Vjr)

The d.ifferenee of the serum cholesterol levels

obtained with fraction C as compared \vi th that of the othe:r

two, is statistically sound.. It .is believed that this is the

first time that statistically significant differences have

been found between the ef.fects of' ph;vsically ft>act1onated

TABLE 13 - (E;.periment 9)

I

MEAN PERCENTAGE CHANGES IN SERUM CHOLESTEROL CONCENTR./\TION OF GROUPS OF SIX RATS PFTER TRPNSFERRINl FROM LABORATORY CHOW DI:ET TO EXPERnmNTAL DIETS CONTAINING THE SUf\lFLO\VER SEED OIL FR.liCTIONS LISTED

. (STARTING VALUES = 100%)

GROUP 'DIET

I 20% Coconut :tat

II 10% Coconut fat 10% :fraot ion A (overhead)

III 10% Coconut fat 10% fraction B (intenn.)

IV 10% Coconut fat 10% fraction 0 (bottom)

1

+ H 127.1 - 5 .. 2

(98.7)Hit

118.0 :!: 3.8 (100.8)

125.0 ± .3.3 (97.6)

112.8 ± 2 .. 7 (99.1)

WEEKS .ON DIET

2

133.7 ! 5·7 (103.3)

115.0 ! 2.8 (104.6)

118.8 ! 4·1 (101-.9)

Significance of' difference (p - values)

I-IV II-III II-IV

~II-IV

a = Standard errol"' of' mean

0 .. 05 o.ol 0.20 0.90 . 0.30 0.20

o.o2 0.10

HK = Percentage change in weight

3

124,6 + 4.4 (1o6.7)

+ 117.2 - 3.7 (107.6)

116.4 ± 4.1 (103.5)

o.o1 0.90 0.05

o.1o

parts .of the same oil. It will be remembered that in the

human experiments of Beveridge (see Section 2) the .effects

~eported with moleoularly distilled butte11 and com oil

fraetionswere not supplied with s'tstistical criteria.

Conclusion·

Two important eonelu$ions emei>ge from Experiments

8 and 9• The firat ia that the unsaponifiable pert of' sun£1owe:r

seed oil when modified by liquid propane segregation also

modifies the effect of this oil on the serum cholesterol level,.

. 'The :t>esul ts of Experiments 6 and 7 (Section 12) w1 th chemically

separated unsapon1fiable matter have thus been conf'ix-med,

The second conclusion is that there appears to be a

limit beyond which it is impossible to modify the properties of

su.n:f'lower seed oil by manipulating the unsaponifiable matter•

It was not possible fo:r example to imitate the extraordinary

h.ypercholesterolaernic effect of coconut oil. This a__~ain confirms

the partia.lly negative (or positive) result of Experiment 7

(flection 12).

SECTION 14

INVESTIGAT10N gF THE UNSAPONIF!ABLE llATTER BY FEEDING PURE COtfPOt:J'NDS

A. Effect of' ~-Sitosterol (EJmerimeD;t ):0)

The results of the experiments described in

Sections 12 and 13 leave little doubt that the unsapon1f­

iable matter (u.u.) of SUnflower seed oil is somehow

implicated in the cholesterol lowering properties of this

oil. To pursue this aspect further, one possible method

ot approach would be the fractionation of the u.u. itself

and the testing of the resulting tractions in the usual w~.

An easier ~ ~ be the testing of such substances as are

known to be present in the u. M. of SUnflower seed oil in

the for.m of pure chemical compounds.

The largest single constituent in the U.M. oB

sunflower seed oil, 1n common with many other vegetable oils,

is ~-sitosterol, Which has indeed been shown to produce a

depression in serum cholesterol values 1n humans if administered

in massive doses. Inspection of Table 11 (Section 13) reveals

that about 0.41% of this sterol is present in SUnflower seed

oil. In the present experiment 1% of ~ -sitosterol has been

dissolved in coconut tat and tested together vdth the pure

control coconut fat and SUnflower seed oil groups in the usual

way. The ~-sitosterol was extracted from a commercial prepar­

ation and re-cr,vstallized three ttmes from 95% ethanol. It

probably contains traces of the di~dro derivative. The

latter substance is, howevel'", unlikely to interfere" since

it has been shown by Siperste:tn et al. (1953) and Beher et

al. (1957) to have cholesterol lowering properties resembling.

those of the main. constituent.

Results

The results revealed. by Fig. 22 and Table 14 show

cleaxoly that 1% ot ~-sitosterol in coconut .fat had no material

effect on the behaviOllr of this oilt or conversely that the

serum cholesterol depressing effect of Sunflower seed oil is

unlikely to be due to its ~-sitosterol content. It is of • interest to note that due to wide fluctuations of cholesterol

values tn this exper~ent it required 18 rats per group to

produce sound statistical figures.

-;;;­~ "'105 0 s,oot~-...-i.;i;iia:*~~~~;;~;;;;;;;~-g-~-i ----------------------~ 95· Ul ~

~100 w

"" .., w .J

~ u 90

eo~--------~,o~--------~20~--------~ao~--

DAYS ON DIET

F1g•22, The Effect of 1% B•sitosterol on the Serum Cholesterol Concerltration (Experiment 10). --o- Coconut oil control

X 1%~-sitosterol dissolved in coconut fat • Surtflower seed oil control

TABLE 14 - (:Experiment 10)

MEAN PERCENT.AGE CHANGES IN SERUM CHOLESTEROL CONCEi\TTRftTION OF GROUPS OF EIGHTEEN RATS !FTER CHANGING FROM LABORATORY CHOW DIET TO EXPERIMENTAL DIET CONT.AIN!lTG 20% OF THE FATS LISTED

(STARTING VJ.LUE • 100%)

GROUP

I

II

III

I- II

I- III

DIET

20% Coconut fat

20% s.s.o.

10

. 83.4 + 4.8

(97.0)

20% Coconut fat containing 101.7 ± 4.8 1% dissolved (!--sitosterol (97.8)

I

DAYS ON DIET

20

103.9. .:!: 3.6 (94.8)

88.5 ! 4.2 (96.2)

107.2 ± 4.4 (98.8)

Significance of differences (p - values)

o:o1 0~70

o.o1 o.6o

30

+ 104.2 - 3.7 (99.4)

6 + 8 .1 - 4.1 (98.3)

. + 107.0 - 3.2 (101.6)

o.o1 o.6o

:11 = Standard error of mean a. = Mean percentage weight change

~ •

98.

8. Effect .of Squalene

Another constituent of the 1:lD.Saponif1able- matter

common to many unsaturated oils, including fish oils in small

a.rnounts • is the highly unsatu~ated hydrocar-bon sq11alene

o30 u50• This compound was first isolated and identified

in Shark Liver Oil by 'l'sujimoto (1920) and has since been

found in Olive Oil b:V 'l'horbjamarson and Dl"Wmlond (1935)

to the extent of 30 to 64% of the unsaponifiable matter

and by Fitelson (1943) in most natural triglyceride o.ila.,

i I.

DAYS ON DIET

Fig. 23. Effect of 1% Squalene in coconut oil on the Serum Cholesterol level. (Experiment ll) ---or-- eooonut oil control

& l% squalene in coconut oil • sunflower seed control

TABLE 1!) (Experiment 11)

MEAN P:ERCENTflGE CHANGES IN SERIDJ CHOLESTEROL COrTCE!rTRATIOT-T OF GROUPS OF EIGHT RATS AFTER CHJINGING FROU LABORP.TORY CHOW DIET TO EXPERIUENTAL DIET CONTJ\ INI1 TG 20% OF THE FATS LISTED

(STARTING VALUE = 100%)

GROUP DIETS WEEKS ON DIET

1 2 3

I 20% Coconut :fat 133.9 + 5.211 115.1 + 4.5 106.7 + 5·3 - - -(97.2)m£ (100.0) (104.3)

II 20% s.s.o. 104.6 + 5.6 98.2 + 5·5 91.3 + 5-3 - - -(95.8) (97.7) (103.0)

III 20% Coconut fat containing 122.7 + 2.5 111.2 + 5.6 106.6 + 2.4 - - -1% Sqnalene dissolved {96.6) (98.4) {107.1)

Significance of differences (p - values)

I- II o.o1 0.03 0.05 I- III o.o7 o.6 0.9

~ = Standard error of mean ~ = Mean percentage weight change

100.

In SUnflower seed oil Fitelson .~eported a concentration

of 12 mg./100 ml. This substance also derives interest

from the fact that Langdon and Bloch (1952) believe it to

be an.1ntermed1ar,y in the synthesis of cholesterol from

acetate in the liver of :rata. Ohanno:n {1926) has demonstr­

ated that squalene is absorbed by this animal• a fact whieh

McGuire and LipskY (1955) confirmed with humans. In the

present experiment squalene was tested in the comparatively

massive dose of 1~ of the coconut oil 1n dissolved form.

It is seen :from Fig• 23 and Table 15 that the

serum oholesterol depressing activity of squalene• if real

at all; (p = 0;,07 atte:tt one week) was extremely weak. In

the trace quantities present in Sunflower seed oil its

effect can no doubt be neglected altogether. It is note­

worthy, however, that the squalene curve rona below the

coconut eont~ol at all times. La:ttger doses may well have

a more p~onouneed influence•

c. 'l'he Combined Effect of B •Sitosterol. (3 -carotene • ~-Tocopherol and Squaiene (EXperiment 11}.

ln this eXperiment an attempt was made to create

an artificial ttun.sap• ", comprising substances known to be

present in Sunflower seed oil• In addition to ~-sitosterol

and sqUalene, Lange (1950) has reported 70 rng./100 gm.

a-tocopherol in Sunflower seed oil whereas carotenes were

identified in the unsaponifiable matter by the author as

101.

reported in ~able lo (Section 13B). ~·tocopherol is moderately

well absot-bed by the rat (Shantz, 1949) whexteas Ahmad (19.37)

Showed that r-carotene iS almost completely utilized b:9' the

rat provided 10% of fat was incorporated in the diet. In

the absence ot fat this provitamin A is not utilized.

~he folloWing amounts were dissolved in coconut fat

and tested for serum cholesterol depressing properties: •

~ -si tostero1 o. 7% J ~-carotene o. 3% ; .. [)(-tocopherol o. 2%;

squalene o.2%t creating 1•4% of artificial "unsap.u additional

to that already present in coconut oil. Except .in the case

.of ~ -.s1 tostel'ol these amounts are necessarily very arbi tral"'Y

and far in excess of those naturally presen~ 1n Sunflower

seed oil.

\

•

7 14

OAYS ON OIET

----~--~-------------'

.fig. 24. The Effect of' artificial unsaponifiable matter and -of the propane segTegated ,.Fraction C" (see Section l3B} on the serum cholesterol level (Expertment 12). ~-- coconut oil control

A artificial unsapon1f1able matter (See text) • Fraction "O" from SUnflower seed oil (25% in

coconut oil • SUnflower seed oil control

;ABLE 16 - (Experiment 12)

MEAN PERCEffr.AGE CHANGES IN SERUM CHOLESTEROL CONCENTRJITIOJIT OF GROUPS OF SIX RJI.TS .AFTER CHANGING FROM LABORATORY CHOW DIET TO EXPERIMENTAL DIET CONTJI INING 20% OF THE FATS LISTED

· (STARTING VALUE = 100%)

GROUP DIET DAYS ON DIET

7 14

I 20% Coconut fat '128. 7 ± ~· 7* 128.8 + 3.7 -(98.3)" (102.5)

II 20% SUnflower Seed Oil 100.7 + 4.0 98.7 + 3·1 - -(96.7) (100.8)

III 20% Coconut fat + artificial 116.2 + 3.4 114.9 + 5·3 unsap. (see text) -{97~8) (101.2)

15% Coconut fat + 103.6 + 4.6 IV 5% Propane segregated 105.2 - 3.2 -fraction "C" (97 .. 5) (100.8)

Significance of difference (p - values)

I- II 0.001 o.oo1 I- III 0.02 o.os I- IV o.oo1 0.001 II - III o.o1 0.02 II- IV 0.40 0.30

K = standard errox- of' mean *- = Mean percentage weight change

I I I

I I I

I I

I ! I ' I I l

I

I

-0 f\)

•

tn a~ditton to. tht! above ·thli:l ·t~stre~tn1' or the

most a¢tlve f!Jt the liq11ld. p;ropa.ne aegrega/ted :SU,nf1ow~r stli!ecl

otl txta~t!ona (Se13tion lJ ,.B). was. cOnipax'~d tn a .. ~lf;lnd, ot 25% • ' < ' ' ' • •

tn' 4()0~t .o.iJ. (t:.v:. ·Of b~¢n4· :=· 4J•4 ) with. pure :~unt,'lcwe~

eeea Gil•

!he ·diet aontalnins tbe <n)Qonut tat' 'wttll art!tio1a.1

"unBap. tt l>~odti~ti a eel"Urn chollest:et>Ol Ctep~19sston t>e)..ative to

pUtte ctt¢onut otl. of a.pptoq~ime:t~li ~o%. ·Although s igntft cant"

e9nd.ftiona with Suntlower beeeJ: oil• · fhe SUntlowe~ seed oil fx-aetto:nQ ..... eoeonut oil·

blend ~, produced values n~t st,gniticru)'t1Y: different· ~m

those due to plll"'e SUnflower aeeli u11.

' In the prea!3nt .series ot th~$e expel'imen.ts it was

not possible to track down ·the c'ho1esterol lowering aetivi:tY'

of Sunf'lOW$~ eeed oil to an;r par>ticttlar ·~ornpounti coniainetl

,in the unsapontfla.bl,~ matte~. the negative ·e~:reot:B ·obtained,

bY teecU.ng the maf.n qonstitltent., i•<;ll• r•,ttt·os.terol, in :nea~

natttra.l <lose.$ ~e Qone1stent with the Jiieaults cf Experiment$

8 an.d !J in Se4tion 1) wheve the most at)ttv~ pro:pan~ segt>egated

~action from Suntlowe:r seed ·oil eontain~d the loWest amount ' - -· . . . - '' - - --- . ' . - ' -, - -- ---- '

o.t algttcm~n precip1tabt~ ste1;'01s.- When aonsi~ered. aga.S.n£3t the

baek,ground ot con tra-cy· 1 .. e;pot>ts front othG~ workel's, ho'We'\ter, ,

the pr~aent ttnd.ings are surprising"'

,ete~$011 (1951) was the ti~st. to show that when

chlQks we~ fed ~"""~itosterql alo~ with cbQle~te~olt bne~

"hoieatevo1a$11.ta dld not d.evel()p• Poll~ (1953) :to].lo'll1etl ""' ,. ..

with s!milal' studies on rabbttait! lPbf> th~ ¢omp1ets attpp~easton . \:

of ob:olest~~l~lnuuoed. hype~holesterola~mta he. had to te'*d these a.rdma.;ts the OQ10ssa1 dose of ? .~ of ~·si to~te~ol.

pet «•~ ,. .

A ·cons16.e~ab1e amount of wo~k haa been Q.on~ w.ith

this steli'o1 on »at~, \"l'lus aernand.es ana c()*wo~ket-s (19:5.3)

adtr.lf.n:Lstet'ed labelled. eholestePol to t>ats. and t~una that th<i

abt:?orptlon ot this compound was ~Ciuced by the simul tane()Us

a.Cimtrd.st~a.tton of ~ •sitosterol• 'losemru:m., })eyet>s and

lrr$.<:u1rnan (1946}, using dt'ff~tient exper:tmenta.l ·®nd.itions

tatlea to r:ontim thta. 'he atidi t!on of ei:the~ 2% or 10%

ot $oybean sterols to the cU.et ot ~ats <U.a not :vevel."$e th~

bne~cholestel"o'laemi'a prqd:t1cc;ni in the$e animals 'bY feeding

wtth a% oho1eatero1 ana 1% Qhol.!o &.(li.d• !t ls ~r-tcus,~

howe'ft:!l'.~ that the same 1nvestigatots obtained pQsitiv~ . . . •; '

' . results with tlihyttrocholeste~o1 und~r similar conC1itt9ns.

Al~:tn•Siater, We1ls~ Afte~gi)oa. and tJeu¢'1. ·(1954)' lt~ew~J]e . . ' . - . . . . .

~n 40ntrMt:f3tin<:'!t1on to the at)ov~., otb~r wo~k:ers

have J.teported. posltl 1.1"~ results with p•aitosteroi Jn mo,r~

~ecent pu~lfoation$• , .t1.rttong these are .:a~~~ $nd. Du,nd!an (1957),

He~nnan ·(1959) and ltath ana Harp~t- (19~9) \YorJd~~ w!th rate, and :.f'ina1!.~9\• ®~on:t Stoltenberg attd Q'eld.enialt Who used the

IIon.goltan gerbil as an e.tpet'imenta1 antma.1~ (1~59)

tl\ .assess.ing the. S18flil':1eanoe . o'f these.· *tllsul. t$

in relation to tlt~ p~sent findings It l11.lla·t be pointed out ' ., -

that .wtthout ~~¢eptt.on. the~e tn:veSttiet~ons \VdP'~ ~a~~:t~a .

()Ut W1 th animals ·which had 'beeni rend.e:Pe~ ~erchoieate~olaemie '·. '1. ' ' •.· .• '.· ' ' . • . . . ' . .

by the .f~eai~ of massive. dc>s~a of ~ho1estero1 ·ana.' 'fn.mB:n:r '

cases cho.llic acid as. Well.. As tfi:if as cou1d be ae'eertatne<!t . --~o .a~~efii~ ct.$~~ c1lo1~$terols hasabeen fbepo.~t~~ with .

f • ' - •• ' '_- •

ant.rn.als ()n a nat'f.nttn tat-rich· Citet.,

lt1 sptt$· ot the negative t:-eSti:u~$· obtain~ Wi'th

~-._sltostel?0.'1o !n EXPel'im$nt lO the~e $s p,q tiou.'bt th~'t under ' . ' . .

se!'Um cho~est~~o1 of ~trh . 'the poin~ !$ ma,rl.e h11~e~ h<>ws1te~t

th~t ~-s:tbor;;te~l· :ts riC)~. bel1$vea to ''be the mtb$tance in • 1 • • •

Sttnrtower< (U~~cl Oil Which is sa1e1y :reJ?pQnai'ble to'i! thr;l ' '

S~:VWn dep~<e~Us1Dg effect of this otlt., i'h.e (:SS$ Oft p ~sitoster>.ol ' '

• . ' j . ';

in at1~ma1 ~ats.- -.rhis ste~ol· only p~odtte!e$ ·ae·teata'ble Ge~ . . . . ~

choleat.e~ol t"a:uu.ng f!Yf£e¢t:a tt f$4 in. massive 4ose• when

dtssolVt:~d in oil. 'his v;;tll be :shown. 'in ·f;lub~H~qll.ent E}~e~irnents. ' .,> ,, • •

Utt1e ean be satd about the spcitioub ·activity· . . ' . . .

tou.n:a wt th squalene, at. no sirnil~ stu<ti.es are· (;)n -recoJ?a--. '

Mo~e 11\t(i:rea:ttng~ hoWE\lver1 ~ ·the results obt.ainea, with the . . .

ua~tifitiial Ul'isap••.- in ~er!ntent l2~ .'!he sign,it:tcarroe ct - . th~ small ael"t,Uft cho1estet>t)1 depi'esston obta:tn~ V!!S.th th1tl.

<~:ombinatzt.on of' subt:Jtari.~es Wil-1 beQotA$' appaz>e:pt ~ti Pat"t v.~

106.

SEOTI,ON 15

CONCLUSIONS

One of the primary objectives of this thesis was

to find out by means of experimentation on rats w~ dietary

sunflower seed oil produces law serum cholesterol levels.

It must be stated at the outset that this objective has so

far not been achieved, At the beginning of this work it

was believed that the special properties of Sunflower seed

oil must be due to oheem1oal substances contained in this oil

and that the,v must inevitably be exposed by some systematic

fractionation process or other, much in the same way as an

inorganic "spot" is analysed in the ehemistcy class. The

initial success obtained with the unsaponifiable matter in

Experiment 6 seemed to confirm these ideas. The search for

tteubstance xn had apparently been narrowed down to the

unsaponifiable matter.

The first discordant note in the "unsap .. n theory • t .

was struck by the next experiment (Experiment 7,), however,

where the total interchange of unaaponifiable matter between

coconut oil and Sunflower seed oil did not completely reverse

the effects of these oils on the se~ cholesterol level.

The following two experiments (8 and 9) with liquid propane

segregated Sunflower seed oil fractions undoubtedly favoured

the "unsap ... theory. Experiment 12 w1 th the art1fic1al ttunsap."

again pointed in the aame direction and so did the similar

107.

effects on the serum cholesterol produced by two oils with

radically different fatty acid compositions and unsaturation

in the same test. It can be concluded, therefore, that if

the results obtained in Part III can be relied on, there can

be no doubt that the unsaponifiable ma.ttel' plays a major role

in determining the properties of' dietary Sunflower seed oil

as a serum cholesterol regulating agent.

It also follows from the above that no tatty acid;

combination or fatty acids or their compounds in the form

of triglycerides in the saponifiable part of the oil, can

by themselves account for the vthole of the cholesterol depress­

ing action of this oil. This conclusion is in contradiction

to the views held at present by the majority of researchers

in the field.

There is a disturbing feature about the partial

success obtained with squalene and. art1f1·oia1 "unsa.p. n of

Experiments 11 and 12, the emphasis being on the npartialu.

Perhaps the indecisive argument of" •sap~ versus 'u.nsap .. '"

can be extended to the unsapon1f1able matter itse1f and one

can say that the activity of the "unsap." is not due to a.ny

particular chemical compound contained in it but rather to

some property of this fraction as a whole. The indications

are that thia would be a p}l;vaical property. ~ IJ.ihus Weitzel

and his co-workers (l95G) attributed their success in

preventing atherosclerosis in chicks by treatment with

Vitamins A and E to the emulsifying powers of these substances.

the startling variety Of chemical compounds not normally found

tn f'at.S; not the least imp~c;!SSiVe Ot' Which is' :N'.-.( 1-methyl•2;

.s~d1~P-chl.J:>~ophent:t) - maleamic acid (Sa.ch~ ·et ·al,. b t959hwhi.ch ·

hav~ been cl.aimecf to have JWpochole~ttitrolaem!,o prope~tles

:wh~ point fn the $8me di~ctioni

!1-he.fa.et t:n~t neither the present 'ts:tudi nor- many

sirnilal- investigations app~a.r 'to ~~e 'ehovm beyond :reasonable

doubt that the sei'tlnl cholesterol Pegulating ·p~opel'ties o:f

oils can be adequately ascribed. to .aey single ehendeal traction'

· o:r entity in the wbo1e oil, l~aves room to~ dot1bt , whether

there ar~ in f'act snch entities, or whether in addition some

pbysi~al characteristics of' these o!ls as a wbol~ may not

be tmplicated.t~ .

'It is doubts euoh a.s these wltieh deeided against

tlle continuation of' work o:n the fut.'the:r t't>actionation of the

\Ul$apo.nif':table matte!~ of Sunflower seed. oil in spite of the

fact that some p:rogrees had. all'eady be·em made . ·in .a rrwuber of'

pilot studies with elund.na ana reversed phase part ion ....

chrom.atog:raph3'• Sinttla!Q.;v-1 a t"J:u?ee-stage 'molecular still. ·

for the fractionation of' f'a.tty a.eids threatened :to become

redtandant be:tore its cor.u~truction had been complElted..

An altemati~e route 1$ the search for meOhat.tisms

rathe~· than su"bstanees, ~he study of a lipid 'ir.uiu¢1ng

J;v:per@o!testerolaemia such af:l egg· tat 1 proved: mot-e attt"active

.in this connection• This w·ill be repo~t('id in Pa~ tv of

this ~bests~

T,H~ •. BYP~CHOLESTEROLAEMIO PROPERTIES . Q.F.. · EOO, ~'I~n,{§1

JUW ... THE . Al3S.ORPTlON OF .CHOLE~TERO'J1

109.

SECTION 16

INTRODUC'l'tON

It .is known that the consumption of hens' eggs

by some animals and man results tn an extraordinary increase

in the serum cholesterol concentration. This has been shown 1

in the case of man by Okey and stewart (1953), Messinger, I

Poroaowska and Steele (1950), Cook; Edviards and Riddel (1956),

Bronte-stewart et al. (.1956) 6 Gordon, Wilkens 'and l3I"ock (1958)

and othe:rs. Rabbits also show a moat remarkable response to

diet$.ry egg treatment. Thus Pollak (1957) fed one egg daily

to these animals :and observed an increase in serum cholesterol

level of three to fourteen times the normal value, depending on

the method of preparation of the eggs. No repor.ts of simil~

experiments conducted with rats ha.ve been found.

There are a number of reasons for the choice of hens •

eggs as objects for study for the examination of oholesterogenic

mechanisms. Neither the "d.egree of unsaturationu hypothesis

nor· the "some substance in the unsaponi:fliable ma.tte.r 11 theo~

appears to be able to account for the behaviour of' eggs., !£

the asstimption·is made that the serum cholesterol raising ef't'eot

of eggs resitles in the triglycerides contained in the yolks; it

would have to be accepted that a fat with en iodine value of

between 70 and 80 has exceptional hypercholesterolaemio

properties.

110 •

. The fatty acid composition of hens' egg· triglycerides

is to some extent dependent on the diet (Cruickshank, 1934).

The following are quoted as representative analysis:

fatty acid Cruickshank

(1934) Fewster and Young

(1959)

% (Gas.chromatography)

% myristic palmitic stearic palmi toleic oleic linoleic linolenic

l 32

47 20

2

0.3 23.1 3.6 4.4

53.4-15.1

The cholesterogenic effect in man of such a fat,

according to the "degree of saturation" hypothesis would be

expected to be less than is the case with a quantity of whole

egg yolks of comparable fat content.

I iO

~--~....; 100 t-;1:::

~~ 90 ~"­~l 80 ~!;_;, :::s~::

~-- 70 V)

.... 60 ~

10 egg

whites ................ - -:

10 egg yolks iodine value

72

10 special 10 special egg yolks eggs iodine fried in value sunflower 100 oil 100 g.

. ,•;(' ;.:· ...

~-- 140 ~~ ~~----~--~~,---~----~-----­~~ 130 ~ ~~---L~~~~~--~~~~~~--~~ CQ 0 s 10 15 20 25 30 35 40 45 50 55

DAYS

Fig. 25 Serum cholesterol level of male Bantu age 34, during ingestion of egg yolks

(From GOrdon, Wilkens and Brock, 1958)

Gordon et al. (1958), further.more. have shown that

the artificial modification and unsaturation of this lipid

fraction from iodine value of 72 to an iodine value of 100

does not materially alter the cholesterol raising properties

of these "unsaturated eggs" (see Fig. 25). This modification

of the fatty acid composition of egg lipids was achieved by

manipulation of the birds' diet with high proportions of

dehulled sunflower seeds.

In addition to 59% neutral fat, hens' egg lipids

contain about 35% of phospholipids and 6% of unsaponif'iable

matter which is largely composed of cholesterol. Winterstein

and Schon (1936) quote 1.342% of this substance in total egg

yolk. The latter is thus seen to be the most concentrated

source of cholesterol in common foods.

It seems close at hand to implicate the abnormally

high cholesterol content of egg yplks for their.hypercholesterol­

aemic properties. This is, however, b~ no means generally

accepted in the interpretation of human exper~ents. This

common sense view has been rejected on account of the belief

that dietary pure cholesterol has little or no influence on

the serum concentration. Thus Cook (1958) discusses the work

of a :rmmber of workers in the field and concludes "In man

the plasma cholesterol is largely independent of dietar,y

cholesterol.u This aspect will be examined in more detail in

Section 23 below.

From the above brief discussion it would appear that

neither the triglycerides nor the cholesterol content of egg

yolks are responsible for their oholesterogenio action 1n the

case of man. Of' the lipids there remains only the phosphatide

f.raction and the tirmaponitiable matter other than eholesterol

to be examined$ . 1'he $ystematic fractionation. of egg lipids

with the object of isolating the eholesterol ·raising factor

or discovering its principle is obYiously desi~able• This

will be described in the next aect:t.on.

113.

SECTION lZ

THE. EFFECT OF TRIGLYCERIDE$ FROM H$NS; EGGS

A. Choice of eamer~mental diets,

First d.iet The triglycerides of egg yolke are the obvious

choice .f'or a :first examination of egg lipids. lt is these

compounds in other foods that almost invariably exert some

so:rt of effect on the serum cholesterol level. '!'he unsapon­

ifiable matter (in this. case largely cholesterol) .is here

included in this fraction since it accompanies it in nature

as well as in most chemical extraction processes. ~his

fraction will be called crude triglyeerides.

Second diet For the second diet of the standard four-group

experiment the triglycerides we;re chosen from whi·ch the

unsaponifiable pa:rt had been t'emoved, i.e. the test lipid

consisted of' pure triglycerides.

Third diet trhe lipid. portion of this diet again consisted

of pure triglycerides as in the second diet, but was in th.is

case supplemented 'by the amount of cholesteflol present in the

unsaponit'iable portion of the crude triglycerides of' the first

diet. As in all p;roevious experiments the supplement VIas

dissolved in the pure triglycerides.

Fourth diet The :rations tor this group of animals contained

sunf'lower seea oil as control. The remed.ndet' of the diets and

111+.

the conduct of the exper~ent followed the standard pattern

described 1n Section s . . It nm.st be admitted that the design of these tou~

diets :for an experimental test is not strictly systematic:

It betrays an a priori conviction that the sermn oholesterol

raising property of eggs can be attributed to nothing more

mysterious than their high cholesterol content., 'lhioview

v;as held in spite of' the prevailing evidence to the contrary

discussed in the previous section~ One reason :ts that

Experiment 16 (see section 23) preceded the present one in

time. Fro.m the results of this experiment it will be seen

that in man dietary cholesterol does in. fact raise the serum

cholesterol provided it ie dissolve{i in. fat. Another strong

ind.1cation oomes from the expe~iments of Pollak (1957) already

mentioned. The remarkable rise in serum cholesterol of rabbits

on egg diets is paralleled by the equally remarkable ease with

which these animals absorb cholesterol (.e .. g. Cook and Thomson,

1951). !f the same response cannot be obaerve(l to the same

deg~ee in other species tt seems ~easonable to postulate a~

least a vestigial parallel ef'f'ect in rats and man.

The isolation of triglycerides f'rom egg yolk has been

described. by Rhodes and Lea (1957) who showed that the cold

acetone {0°0) extraction of dried egg yolk yields a materi:fil

consisting of 97% of total neutral lipids Which will be called

crude triglyce.r1des. No attempts \ve:re made to :remove the

remaining three per cent ot phospholipids repoPted in this

~

11.5~

fxoact1on. The au. thor is indebted to Mr. V. M. Wells f'or ·

preparing a supply of this material• The phospholipids

were isolated .from the residue by repeated. extractd:on

with a mixture of chlorofo~ and methanol.

A thi~d of the acetone soluble fraction was

left. unchanged. The remainder was saponified. and the

unsaponifiable matter extracted and pnrified,. using the

apparatus and procedure described in Section l2A. Th~

regenerated acids were re"'!testeri~ied with glycerol according

to the method of Hilditch (1956h The reaction rn'ixture

was ·alkali refined in dieth,vl ether solution to remove

excess acid.Es. The re-synthesised troiglyce:rides diffe:r

from the natural product in that the acids.are now randomly

dietributed on the glycerol molecule,. In View of' considerable

evidence, that triglyceride.s su:f':fe~ a certain amount of random

intaresterif'icetion in the intestine before and during

absorption. this is no serious di.eadvant,ag,e. The fatty acid

composition v;as determined by Fewste~ and Young (aee Section 16) ..

t;tthe remaining results a~ tabulated in Table 17 (a). On the

3£

'TABLE 17§

COMPOSITION OF EGG YOLK LIPIDS

Triglycerides (!.V. 69)

tl'nsaponi:fiable matter (u.,M.)

Digitonin precipitable ~ee ~ steii>ols (in tJ.M.)

Phospholipids

2i of ;zolk

19.10 {

1.82 ~

1.57

10~t20

only traces of' sterol esters \vere detected

orude triglycet'ides

116.

. . - . ,, basis of the figure of l,o342 quoted by Winterstein and Schon

for the cholesterol content of whole egg yolk the digitonin

precipi teble sterols were la.:rgely composed of cholesterol.. In

the absence of more precise .information a cholesterol content

of six per cent iri the crude triglycerides was accepted for

evaluation in the animal test.

c. Results and Discussion

The resu.lts are recorded in Fig. 26 ana Table 17b

in the ur:m.a.l way and can be summarised as follows. The pure

ISO

• I -,

-------------------------~----------------

6

1 I I I

12 -----DAYS ON DIET----....

19

The .effect on the serum cholesterol concentration of' hens., egg triclycer-ides (Experiment 13).

• pure triglyeerides • c~de triglYcerides • pure triglycerides p1us 6%

cholesterol --o-·- sunflower seed .oil control

TABLE 17b - { §Xperiment 13)

Jr!EAN PERCENT .AGE CHANGES. IN' SERm.l CHO!.~ESTEROL CONCEi1'TRl\TION OP GROUPS OF SIX RATS

AFTER CHJINGING FROU I.ABORATORY CHOW TO EXPERU!EN'Tftt DIErS CON'TJ\INI!fG 2Q% OF THE

LIPID FRACTIONS LISTED (STPRTING VALUE = 10Q%}

GROUP DIETS DAYS ON DIET ' (LAB. CHOW) I

'

6 12Dli 19

I Crude egg trigl.ycerides 142.4 ~ 6.9_ 151.9 ± 10.6 106.2 ! s.s II Pure egg trig1ycer1des

(9~· 7) . <1so.4) . '. ~1l8.8) 115.1 - 4.6 114.3 - 5·3 105. - 3.7

III Pure triglycerides containing (10~.4) . (1~2.5) (1~1.0)

154.7- 8.6 134.3 - 9.2 108.9 - 6.0 6% added cholesterol (100.8) (100.4) (121.1)

IV SUnflower seed oil 94.8 ± 6.7 (100.8)

81.8 :t s.o (98.0)

+ 97.2 - 7.2 (118.6)

Significance of differences (p - values)

I- II o.o1 o.o1 o.9 I-III 0.3 0.2 0.7 I- IV 0.001 o.ool 0.3

II-III 0.01 0.1 0.7 II- IV 0.03 0.001 0.3

III- IV o.oo1 o.oo1 0.2

!l Standard error of mean 181 Mean pei"centage changes in weight 3!!1!1 Experimental diets discontinued after 12th dey.

•

triglycerides produced a mo(lei>ate rise in serum cholesterol•

reloti\l":e to the sunflowett seed oil control. ':Phere is no f'eaaon

to believe that the extent of this elevation is in any 'W8..,V

abnormal if the composition and iodine value o.f this :rat is )

taken into consid.e~tion. The Cl"Ude cholesterol .... rich.

triglyceridee 1 however, produced the most dramatic elevation

so far encountered in this $eries of Pat assays. This elevation

was not significantly different from that due to a diet of

pure 'triglyceridea to whi¢h an equivalent amount of cholesterol

had been added,. A. notable feature of this test is the pl'ompt

~eturn to baaeline of the cholesterol curves after disdontiriuation

of the experimental diets.

The conclusions to be drawn fro;m this eltperiment are

obv:I.Qile. A. gl9eate:r part of the veey marked eyper'Chole.sterolaetn.ic

prope~~ of egg yolk is probably due to its cholesterol content.

The design and outcome of the Whole experiment seems in :tact

tri'\t:J.al. were :1t not f'or the persistent light 1 t throws on the

question: if d1etaey cholesterol When dissolved in :rat ra1ses

the serum ooncentr:at:ion, how oan fat devoid of this substance

have a similar if leas marked influence?

It may be cr1t1eised that it has not been shown that

the feeding of whole egg yolks Pl"'oduees the same effect as that

ot an equivalent anount o'£ crude triglycerides or pu.re

triglycerides plus cholesterol. 1'he zrta~ked differences in

ef'f,eet on z-abbi ts, of eggs prepared in different waye, would

niake a strictly quantitative comparison dif'fieul t.. 'rhe ·

· physical state of the cholesterol in yolk is presumably ot

importance.

The investigatiC>n of the phospholipids and the

unsaponi£1a.ble matter other than cholesterol has likevrtse

been neglected in favour of a more detailed study of the

conditions under Which cholesterol is absorbed.

120.

SECTION 18

T~ ABSORPTION OF DIETARY CHOLESTEROL

The title of this section introduces a subject

on which much has been written. In spite of this, however,

no clarity exists about the precise conditions und·er which

cholesterol is absorbed by the .rat.. !n Section 17 it was

shown that cholesterol, if dissolved in egg triglycerides,

is easily absorbed.. The question arises whether the presence

of fat is necessary for this to occur. It ie the object of

the next two experiments to contribute to an answer to this

question.

A, The effect of larse.doses of cholesterol fed with and without sunflower seed oil (E!J?eriment 14a).

Eawerimental

It will be remembered that 0.2 per cent of

cholesterol in the diet either in crystalline form with

fat or dissolved in .fat could not be shov!n to influence

the serum cholesterol significantly (Experiment 4, :Section 9C).

The dose was accordingly increased to 2 per cent and tested

both in the absence and presence of fat 1n the diet, the

latter consisting of' 20 per cent of sunflower seed oil. In

order to make this experiment comparable with Experiment 4,

a diet with a total cholesterol content of 0.2 per cent

(1 per cent 1n sunflowe~ seed oil) was again included as

121.

was also the sunflower seed oil control diet.. In all cases

except in the fat-free diet the cholesterol was dissolved

at 150°C in the sunflower seed oil before incorporation in

the remainder of the rations. A certain portion, no doubt;

subsequently cr,ystallized out again before the food ~s

consumed ..

Results

see Fig. 27tt. and Table 18a.

The results appear to give a decisive answer to the problem

ot the role of fat in the absorption of cholesterol as

I (

i

6 f2 DAYS ON DIET

Fig. 27a The ·effect of large doses of cholesterol f'ed in absence and presence of oil (Experiment 14a).

• 10% cholesterol in sunflower seed oil (2% of total diet)

• 1% cholesterol in sunflower seed. oil ( o. 2% of total diet) * 20% sunflower seed oil control

--o- fat free

TABLE 18a - (E!Pertment 1~al

MEJI.N PERCErJTJtGE CHANGES II1 SERU!'t CHOLESTEROl, CONCErfTRrTION OF GROUPS OF SIX RPTS .f\FTER CHANGING FROM L/I,BOR."'TORY CHOW TO EXPER:mENTJL DIETS CONTA!NDTG 20 PER CENT OF THE LIPIDS LISTED

(ST/IRTI'f'TG VftLUES A 100%)

GROUP DIET DAYS ON DIET

6 12

I Basic fat-free plus 2% cholesterol 84.0 ± 3~· 88.8 + 5.41 -{95.9) (92.5)

II Sttnf'lower seed o11 109.6 + 4.70 110.8 + 5.37 - -(101.8) (101.8) III s.s.o. containing 1% cholesterol 107.1 + 3.16 112.9 + - - 3·15 (0.2% of diet) (101.4) (100.0)

IV s.s.o. containing 10% cholesterol 159.5 + 149.2 + 7.79 - 11.9 -(2% of diet) (101.0) (100.0)

Significance of differences (p - values)

I-II o.o1 0.02 I-III o .. oo1 o.o1 I-IV o.ol 0.001

II-III 0.7 0.7 II-IV o.o1 o.o1

III-IV o .. o1 o.o1

K = standard error of mean o = mean percentage change in weight

mea.suFed by a change in sel"U.l"ll concentration. The difference

in serum concentration of' nearly 100 per cent on :feeding the

same amount of cholesterol (10% in fat, 2% of diet) with and.

w.ithont .fat, ie impressive. · Ae in Expel:i:ment 4 a comparative­

ly small amount of sterol (1% in the tat~ o.2% of' the diet} in

the diet is without deteet$ble influence* thus confirming the

earlier experiment.

Of considerab1e interest is the :raot that the fat .....

free but cholesterol-rich concentration cu!"Ve is si.ftn_itieantly

lower than that dtte to a diet containj.ng aunflower seed oil

without cholesterool which is considered to be a. cholesterol

lowering oil in the ca.ae of man. The small loss in weight

of the animals on the fat tree ration, however, indicates

some ca"Q.tion in the interpretation o:f these data.

:B. '!'he e:tfeot of feeding cholesterol and oil, separ$.telz (Ex;pe:r>iment l4b)

The last experiment revealed what appears to be

a profound !nterdependenoe of dietar,y cholesterol and dietacy

fat. Neither substance alone will produce ma.xittnlm 'effects.

1he aspect which requires olal'ification is whether the rise

in serum cholesterol is simply due 'to ino~eased absorption

of cholesterol fed in a fatty medium or due to ·some other

mechanism 1-equiring the supply to the body of both cholesterol

a.nd f'at.- !'he latter al temative 1s likely to be the mol'>e .

complicated concept and also difficult to Visualize. It

would be desii>able to rule it out if possible ..

An elegant way to supply both :f~act:tons to the

bod:v,. out of contact With each other. would be to feed

cholesterol by mouth simultaneously w:t.th an oil em:u.lsion

by intravenous injection. To do this with ~ats would probably

~equire a high degree of eXperimental .skill. A compromise

on a less ambitious level is the feeding of both cholesterol . and oil by mouth but separated in time. ·This has beeh done

in the present experiment, which differs from the preceding

one by the change of two diets only.

Experimental

P.irst diet. Stmflower seed oil containin.g 10 per

cent (2% of diet) of cholesterol .as far as possible in

dissolved fom.-

seoond t:U.et. As first diet foX' seven days. There­

after the cholester-o1 was added in crystalline form to the

:rat-free diet and ths oil fed separately twice daily (at

8 hours inte%*\l'al).. This was done directly into the mouth

by means of a pipette.

Third diet. SUnflower seed oil ¢ont~ol diet

(without cholesterol).

Fourth diet. Fat free diet (without cholesterol).

See Fig. 27b and ~able 18b.

The ,results of Experiment l4b are essentially the sam~ as

those of Exper1ntent ll+e. except for one important point~

The separation of cholesterol and oil in the diet of' one

group of animals after the seventh day was reflected in an

immediate and significant drop in serum concentration

relative to a control, where no separation was effected.

This lowered concentration was only slightly higher than

that due ·to a ehole$terol-~ee diet with equivalent oil

content.

Fig. 27b

,..-------~~-------- ------- ·---~-----,

I.

.J 0 a:

175

160

130

~ liS

"' uJ ..J 0 J: 100 u

.l ::::> a: 85 "' "'

7 14 21 DAYS ON OIET !.

"-----~ ~~-~-~--------~ ~-~

The .effect o:f feeding cholesterol ana. oils separately (Experiment 14b) .. ----o- 10% cholesterol in sun:f'lower seed oil

(.2% of: diet) :A. 10% cholesterol in sunflower seed. oil

fed separately atter first seven days (see text)

• 20% sunf'l-ower seed oil control diet • fat free diet

The :fat-free and cholesterol-free ration produced

serum levels which were not l·ower ·then tho.se produced with a

similar diet containing 2 per cent of cholesterol in

Experiment 148. Although this comparison is not strictly

TABLE 18b - ! E;perimen t 1,Y:b)

MEAN PERCENTP.GE CHANGE IN SERIDv1 CHOLESTEROL COlTCEfi!TRATION OF GROUPS OF SIX RllTS JIFTER CHANGING FROM LABORATORY CROW TO EXPERIMENTAL DIETS CONTAINING 20 PER CENT OF THE LIPIDS LISTED

(STARTING V.ALUES = 100%)

DAYS ON DIET

GROUP DIETS 7 14 21

I SUn.f"lower seed oil plus 10% 134.8 .! ~o* 163 .. 8 :!: 8.9 148.1 + 8.4 cholesterol (2% of diet) (96.8) (97•2) (99.3)

II Sunflower seed oil plus 1~ 134.1 :!: 3·9 121.7 + 2.3 123.0 + 4.1 - -cholesterol fed separatel7 (98.5} (91.2) (95.0) after 7th day

III SUnflower seed oil + 7.7 105.3 + 6.8 110.8 + 3.9 109.7 - - -95.9) (95~9) (98.5)

IV Fat-f'ree 86.4 ± 5·9 (89.2)

+ 99.3 -(88.7)

3.6 + 100.5 - 5.2 (93.1)

Significance of differences (p - values)

I-II 0.9 o.oo1 o.o2 1-III 0.02 0.001 o.o1 I-IV o.oo1 0.001 0.001

II-III 0.02 o.o5 o.os II-IV o.oo1 0.001 o.o1

~II-IV - 0.03 0.4 0.1 '

E standard error of mean 0 Mean percentage change in weight

~

1\) 0'\ •

legitimate since the data belong to.two di:f'f'erent expe;riments,

these serum levels occupy the same quantitative poei tion in

rela.t.ion to the sunflo'\"ler .seed oil control curve in both cases.

The average weight losses are like\vise of the same order in

both groups. It is therefore concluded that in the absence

o:t dietaey fat. 2 per cent o:r dietary cholesterol could not

be shown. to influence the serum level.

c. Discussion and. Conclusions

In Section 17 it was shown that· dietacy cholesterol

in the pre.sence of egg triglyeerides has a mal"ked oholestero;...

genic property. The present two experiments have demonstrated

that egg trtglycerides can be sub.stituted by sunflower seed

oil without materially altering the eou~se of the expe:t-iment9

Ful'themore, they have demonstrated thet the presence .of

sun::f'lower seed. oil and presumably also other fat.s is a

necessary condition t:or this phenomena to tfl..ke place.. In

otller words, it .is reasonable to conclude that dieta!'y ft-tt

must be present for the absoi"pt1on of dietary cholesterol

by the rat• Thus the reported 1nei't nature of eholestePOl

in the diet of man (Section 16) has not been. eon:f'irmed in

the ease of rats.

Nearly 24 yea:rs ego, Cook (1936) fed. cholesterol

with ana without f'at to rats, measured the faecal output

.of' th1s substance and concluded that •••• f'choles terol is

absol"bed by rats only when f'at is present i..Tl the diet.n

No further disqussion of the subject ifl apparently required •.

128~

Unfortunately less conclusive results have been reported

by some investigators in more recent times,.

tn elose agreement with tne present findings are

those of swell., Flick, Field and Treadwell (1955) who ted

a ,combination ot eholeaterol, sod.ium taurocholate end olive

o1l to rats. In the absence ot both bile salt and o.il, the

addition of two pe:ra cent of cholestetool to the diet had a

negligible et:fect on the aerum cholesterol level ... The

supplementation of Cholesterol with one per cent of sodium

taurocholate alone, however,, caused an elevation which Vt&$

comparable to that produced by the simultaneous presence of'

all three substances in the diet. These authors suggested

that dietary fat serves a dual purpose~· 1 t atimulntes the

flow of bile necessary for cholesterol esterase activity

and it provides the :ratty acids necessary for cholesterol

esterification-.

In the same year, Pihl (1955) repol""ted his results

f'rom balance s:tudies with rats. He found eholesterol to

••••"be readily absorbed on a diet devoid of f'atau, admitted

however, that dietary fat stimulates cholesterol absorption

when it is present in large amounts relative to cholesterol.

av· using mostly the eololl'imetrie Sehoeriheime~Sperry method

i'ot> the "cholesteroltt dete.nnination in :faecal fat the saturated.

transformation products like dihydrooholesterol and oholestanol

(coprosterol) we!"e neglected. Thus Coleman (1956) found. that

cholesterol represented less·than 20 per cent of' tHe total

di.g!tomin-precipitable sterols in the faeces of·rats on a ...

fat free diet.

129.

In agreement with the present ~esuits;

A:f'tergood• Deuel and Alf.in•Slater (1957) r~pol'ted the lowest

se~ eholester>ola of' rats on fat free .d~e.t·s. . ~he addition

o:f' 12 pe:r;o cent of cottonseed oil .caused a rise in serum

·level from an average of 42 mg./100 ml to 64 mg./100 ml.

As has been mentioned in Seetion 16 the rabbit

absorbs dietary cholesterol With u.tmost ease.. EVen in this

an1m.al, however, ·steiner and. co-woi"kers (1959) observed. a

three•fold increase in serum concentration when o.s pe~ cent

o.f cholestel"ol in the ·diet fs supplemented by 12 per cent of .~

either saff'lower or ooconut oil,.

The eonclus&ons that have been tentatively dr>a.vm

trom E:xperiments 14a and 14b! can be ,sutmnarized again as

follows:-

1. Comparatively large doses .o:f dietary choles·terol

have a rnark.ed hypercholeeterolaetnie ef:raet 1f

given dissolVed :in fat.

2. In the absence of fat even large doses of' dietary

cholesterol are without influence on the serum

level of rats.

3. Dietary unsaturated fats represented by sunflower

seed oil produce higher serum cholesterol levels

than a fat free diet.

SU.bsta.ntia.l; if by no means unan1mous, support

for these statements have been quoted'f'rom the literature.

Very little support can be enlisted, however, f'or the

application of' most of these pt-ineiples to the htm1nn sphere.

It may ~easonably be aSked why so much attention

has been paid to the stu(iy of the conditions under which

dietary cholesterol is absorbed, as this 1n"V"est1gation is

concemed primarily with dietary triglyceride .fate.. An

answer to this question will be given in Part V o:f' this

thesis.·

PART V

DlSCU'SSION c AND OONCLUS!ONS

THE SOLUBILITY HYPO~HES!S

~mj.'ION. 12

DISCUS.SXON .. AND CO~USIONS. (EXPERIMEN'l'S 1. • .. 14,) I

It may be appl"opr1ate at this stage to pav.se fo~

reflection~ A considerable body of data has been. collected.

ltot>e than ,;G different l:t:p1d preparations have been tested ,

in 14 separate eXperiments. Defo:t-e attempting to draw any

.conclusions from these dat.a as a whole, however, the main

findings ~ su.mmari~ed again as follows:

eontl:'~ to the expectations to be derived from a

stUdy of the literature, the ~t has shown ttself' to be, in

the hands of the author1 an excellent experimental animal

tor the purpose for which it was used. The ear11e~ prelimin­

ary eonc1uaions arl'ived at in Part II under 'methods' were

fully ~u.st1fied-. 4lthough the magnitude of the response

of l'at serum cholestel"ol concentration to the two :represent­

ative fats~ i.e. coconut and Sunflower seed varied $lightl~

from experiment to eXpertment and from colony to colonY,

highly s1gn1t1cant diff'er-eneee Wt:;l"e found in all eases. !

Furthermore, when testing certain rat preparations ana using

onl:v' G animals pezo group, finer intermediary gradations between

the effects of coconut oil and Sunflower seed oil can be

obtained. vmieh can still be sho\m to be significantly differ­

ent from eithe%' of the high and low contl'ol fats or their

equivalents. fhe transient r-esponses obtained tn some earlier

experiments w~re due to inexpert experimentation •. In

one case with· fish oil and ~utter (Experiment 3), serum

~:Q.olesterol differences. wex-e kept up for at least ? months

and there is no rea.son to believe that. they ca.n.not be main­

tained indefinitely. Essentially similar results. were

obtained with ani~als of uncertain origin, bred in t~-~

different laboratox-ies. . .

~ffect of UnsaJ2onif:'iabl.e· Matter

Passing to actual results obtained from specific

tests, it was found in Part III that the unsaponiftable.

matter of Sunflower seed oil is partly responsible for·

the hyj;)ocholesteroiaemic ·effect of the latter. It follows

from this that no particular f'atty acl.d or grouping of · . . .

different acids can 'by itself a~count for all the properties

of this fat. It was shoWn., for example, that three different . . - .

oils with identical fatty acid compositions had' different

effects, and that two oils with very different iodine vafues

and' linoleic acid contents can have very similar effects.

It is concluded at this stage then, that the cholesterogenic

property o:r an oil depends on both the "sap." and "unsap.u,

i.e. on some property of the oil as a whole.

Effect of Cholesterol

The findings described in Part IV disclose that

under certain conditions dietary cholesterol has a profound

serum cholesterol.elevating effect. The significant fact

. emerges, ho\Vever, that this elevating etreot is pt>odttced

:only' w,ttb. oonzparativei;v larg~ doses of' ·cho1e$tero1 and only

when .fed' stmu1taneous't;r with,' and' dissolved in, larg¢ ~te. ot fat.'.·. zn· otlu~r· WCit-ds, an interesting ana pe!Pllaps ·highlY'

sS.gnificatit ··corineotfon has been este.b11sheii between ;(lholeste·rol

on the one hand· ana ta.·t on the other· ' . ·. . .. , . . . . . . ftu~~tttlated 'condl.t,i,~ns of ch~1~st·e~o1 Abso;t:Ptipn ,- - . ,- .-- -- -, . . . .. - ;··· -

the situation, then, :ts a.s %"ollows~ on the one ,

hand there extst fats devoid ot eholeatet>.ol such as eooonut

:eat which l'aise the settttm cholesterol· leva\ ~his .1s ~ howeverj

not .entirely dUe to ·a.ny peculiar fa.t'ty acid. composition. On

the other hand there seems to be little doubt tha.t lar~e ·.

amounts of ehole~tero1, when ad.mtnister'ea in dissolved f>omt

in appattentty. neu.t~l. o~ noti•l'aistns fatl.), p~bduc~ an

lfie'bl"easea.,se:Mlm eholeste~l· le'liel,;'

Can these 'tWO ·apparently (:)onfltct7i.ng aspects be

l"econciled. by a ·cormne>n explanation? 'o put it !11. othe:P words, ' '

are the . effects of dietary saponitiabte matter 11 ttnsaponifiable

matter and d.tetaey cholet?ter6l~ neoessacy manifestations of'

the same b!ologt<;:al phenomenon~ which'incidentall;V may not even ha-ve a ch$m:tcal baatat tf so 1 it :f.:s ·conce11table that

' . ' . . - '

further attempts at concentrating serom cholesterol lowel"ing

substance~ :t~ tr>ig].uceride oils may p~ove to be trottless •

. tn tact, such attempts have bee.n made by oth~ros (see Sections ' . ' . .

2 Gnd 3);.bttt gen~rally With .little SUQCeSS• ~llCUX>Sion$

into t}le field of physiology have so far 1leen de~iberatel:f '

As d!scuaaed in S~etion 18, :.it. is sign:tfioant that

larg~ do$es ot dieta.ey cholesterol are apparently- not appi:sec­

iabljr absot:bed. in .the -abs~nce ()f dietary. fats .•.. The p~eseno~

of fats. ma.y be n:ece$sary for the abso~tf.on of d.ieta.%7 .

cholesterol ana presumabl-y a1$o J9ndo'aenop-s o~.bili~ : .. -· ............ .

Chol~sterti1 t'rorn the intestinal ~ract,ii tt i$ also. re$son"""

a'bl,.e tQ 8.$$'Un1e that this .._,nolesterolf whatever !t~ or~gin,

must l>e dissolved. in tat in oraer to 'be et£eotf vely abeol'bed..

It the pretoequ:tst te to a.bsorpt.$.on !s t'ts state ot solution . in fat, the ~olubilit~ or· cholesterol in .tat.s would. detem1ne

' . the rate Qf absorpttort. 'the solttbi11 ty of c}lol$st~l'o1 may

be iiiftel?ent "n different f'ats• thus pe~hap.s ·accounting for!

different ael"Um cholesterol eoncentxaations p~od.ueed by the ·

latt¢t'~ ln ·.the next S¢et1on the determi11$tion o:f the

solU.bili t~ of Cholesterol 111 a V'lill:'iiet:v or tats at boat ·

temperature Will be de$cl"ibea•

Tb.e.t app~eoia'ble ·quantities ot end:og~noue ehol¢ste;roa are avatlab'le f'ot' re-abac.:u:'.Ption in. th$ inte·etine has \l$en demonstrat$d by Dan1el~son end aaetaf'fs$Ol1 ("l:.959) who foUnd that.gemn ~ee rats on a diet wtth completely f:lte:roi..d•free synthe-tic tat, exel:'et;e at least 2,1 mtS•' of cholester-ol dafl.y' S1nc.e this does not include thG presumed. r~abt:Jorbed atet-ol, .more endogenous e1to1e.stePol xna:r ol'tgl:nally have been attai1able in the it1testine. Scaled up, weight for! . weight tG the human seale this t-epresents a daily excretion of at 1eaat o.t5 sms• per d33.

.f

135,

SECTION 20

THE SOLUBILITY OF CiiOLESTEROL lN TRIGLYCERIDE OILS

(IN VITRO eamex-irnent)

§.el2er1inenta1

The deter.mination of the solubility of cholesterol

in triglyceride oils is essentially a simple matter. In practice,

however, a eerota:in amount of experimentation is :required. to

obtain reliable results. The apparatus and procedure eventually

used after a aeries of modifications is b:riefly described, ae

follows.

An a~arlum tank for.med the basis of a constant

temperature bath. A toluene - mercuey contact thermomete~ was

constructed to act as therm.o ... regulator. 1'h1s operated an

electrical relay which in turn regulated a conventional :tmmersion

heater. The wattage of the latter was made variable by an

external series resistance. A "quiCk heating uptt ci:rocu.it was

also provided. By these means and an elect:Pieal water stirrer

the temperature was kept constant at 37 ! 0.05°C •

Twenty gm. of oil a.nd 1.5 gm. of reagent grade

cholesterol were placed in o" X 1" Open test tubes and SUspended

in the constant water- bath,. .Agitation of the mixture was effected

by bubbling a steady stream of nitrogen through each tube. To

avoid cooling effects due to contact with the gas. the latter

was preheated to the required temperature by preliminary passage·

through glass coils in the bulk of the tank.

136.

Atter two houre• the excess cholesterol was allowed

to settle and the aupernatent oil sucked off by means of vacurum

in sintered glaaa Emich tilter sticks ot poro•1ty 3• Fig. 29

illustrates the whole apparatus du~ing the filtering operation.

fis. 29. Apparatus tor the determination o:r the eolub111t7 ot Cholesterol in triglyceride oils.

The dissolved cholesterol in the oil eaturated at

37°0 was estimated according to the method ot Abell et a1.(1952).

Since moat oils contain appreciable qaentities of tiebe~ann­

DUrahal'd oo10ttr-pl'04uoing subatenoee (e.g. unsaturated eydro­

carbona, sitosterols, carotenes, vitamin A etc.), a cholesterol

determination in the oil before saturation with the sterol served

* Experience has shown that saturation levels are reached in Jess than 15 minutea' agitation time. Por safety, a time ot two hours was allowed in routine testa.

TABI~E 19

THE SOLUBILITY OF CHOLESTEROL IN TRIGLYCERIDE F.ATS AND OILS AT 37°C IN GM/100 ML

Mean

Coconut fat 4.33 Butter fat {originally contains 0.18 gm./100 ml} 3.94 4.00 3.97 Coconut + "Artificial trnsap." Egg trigl.yceM.des Beef tallow Olive oil SUnflower seed oil, Sample I SUnflower seed oil, Sample II Sunflower seed oil• hydrogenated Arachis oil Cottonseed oil Cod liver oil Soya bean oil Com oil

3e48 3.40 3e39 3•44 3.28 3.32

3.81

3·15 3.71 3-59 3.48

3.50 3.46 3.36 3.40 3.26 3.29

3.22 3.12 3.14 3·13

Fish bo~ oil (sardina oscellata), 2.85 3.20 2.98 3.01

Coconut f'at + sitosterol Sample I Coconut fat + sitosterol Sample II Sunflower seed oil propane "overhead" fraction SUnflower seed oil propane "bottom" traction

Remarks

Expts. 1 - 14 Expt. 3 (incl. 0.18 gm./100 m1) Expt. 12, Section 14

Expt. 13, Section 17 Expt. 3, Sect ion 9

Expts. 1 - 14

I.V. 100, Expt. 16, Section 23 Expt. 3, Section 9

Expt. 3, Section 9

Coconut f'at saturated with ~-sitosterol at 37°0

Expts. 8 and 9, Section 13

4.5

4.0

..J ~. #

0 1 0

.......... ~ \-'

3.5

138.

COCONUT

BUTTER

COCONUT+ ARTIFICIAL UNSAP. EGG TRIGLYCERIDE BEEF TALLOW

OLIVE

SUNFLOWER ·· SEED AYOROG. SUNFLOWER {I.V. 100)

PEANUT ·COTTONSEED COO LIVER SOYA BEAN CORN

3.0 --r-- FISH (S.AFR. PILCHARD)

SOLUBILITY .OF CHOLESTEROL

IN TRIGLYCERIDE OILS AT 370C

as a blank. In a few cases the results were checked by the

micro-ferric chloride procedure described earlier, as well as

b.Y the more fundamental method of precipitation with digitonin.

Up to six solubility determinations were made simultaneously in

one batch.

The relative values of the solubility in d.i.:f'ferent

oils were more ~eproducible f'rom batch to batch t.han their

absolute values. For this reason, a representative of both

hyper- and hypoc;;holesterolaemic oils, :1.. e. eoco.nut oil. and

sunflower seed oil were carried through each batch as controls.

~his enabled the solubility determined in an unknown oil to be

corrected relative to coconut oil for which a mean value of

1-1 .• 32 gm./100 ml was accepted from a large number of determinations.

Resulte

'l'he solubility of cholesterol in a variety of oils

including some special oils used in previous rat experiments

are recol,'tded in Table 19 •. T~e reproducibility o.f the determinations

can'be estimated :f'rom the duplicate values obtained. with the same

oil in different batches. For visual ease of comparison., Table 19

has been reproduced in the :form of a graphical scale in Fig •. 30.

The discussion of' the significance of these.results as

a whole will be found in the next Section.

«'finnprog~ess 1n.b1ological rese~:rach ts de:P~l'ldant on the proper use ot l(ypo'th-esis. ". ·

. . .

E:Ven; a s~pex-fiotal 1nspeot!on of F'1gi :;o :in the

pl'ie'Vtous secticm ~evea:l.s a striking eotTelatton· hetw~en tJ:u~ · . '

eolubil:lty or ob.ol.esterol tn differ~rit · :tats· and. oils on the··

one hand >ana the serum Oholeate~ol concentration· pPodu~ ' . ' ' . . . .

'b~ the same tats and. oils en the othe:r. lthis c:l)rreiation

~OUld seem to 'be in QOnfortr:titf with the meOhanisrn Of

choleeteJ'Ol abeorptton as postulated In Section 19• \!Uhether

tl11$ correlation Ia » .ln fact, · a eonsequ~nce: o:f t:h$ abov~ ·· . . .

postulate~ 1f' tru~ .• or due to $on'le oth.¢r l"elat·ea mechanism

cannot be ee'ttl(!d at pt>esent, 'hut the to1lowing. eypothe:s1e

can nevertheless be stated tentatively at thia stage - · ' .

The p~ope:rt.sr of tlit.terent · dieta.ey · ta:ta whi.eh . d.eterm$.nai;l their eti'eot on th~ eer,;nn chole~te~o1 m23' l:le th~:tr cU.tterent oa.pael ty to' dt~solve· ®ole$tei:'ol,. · · . . . .

,.

!f tht~ statement can be demonstrated. 'to 'Be' 'true,,

not onl;v would the pl"Qblern f}et in seetion 1 .be sol '\ted, but· ' . '. '

.at tht same time thts ·solution -would e\iggeat a rreqhanisrn . . .

ot actton wh:tch 1s. 'both airn,ple ~d t'eago-nab~e. %n the

141.

remainder of thia thesis all efforts will therefore be

directed towa:rds a critical examination of' the validity

ot the above bypothesis,. Is the solubi11 ty. concept a

reasonable one, or d.o i.ts implications .clash with accepted

facts of cholesterol metabolism? I •

B. ponseguences of the SolubilitY:· !f.YJ2othes1s

Any hypothesis or theor.y, if valid• should not

only explain known facts but predict new ones. A number I

of both categories are listed below.

x. All dietary rats and oils have a me,r­cholesterolaemie effect relative to a. completely fat-free diet. It follows that the apparent lowering effect of same oils is·or a .relati~e nature only. This has been found to be the east;) in Experiment 14. Section 18, where a' typical cholesterol "loweringn fat like SUnflower seed oil produced higher serum cholesterol levels than a ~at-free diet. The . '

same has yet to be shown for fish oil•

II. The lowest.and $he,hishest.levels producible by fats can be defined. The lowest level is that due to a fat-free diet. On the other hand, there is an amount of' dietary cholesterol above which no fUrther absorption should take place when administered with a given quantity of tat. This amount is reached when th$ d1et~y fat in its passage through the intestine is saturated by cholesterol. That this predietion is true will be shown in Exper~ent 15 described in the next Section.

f, '

;

\

~e above argument can also be ):t~ver.sed a.s tQllowrs: th$~e ts ari amount ot <Uetar;v f'$,i

i;:

above; \Vhich no.tu.llther abitorption ot chol~~Ste~oi takf)s place When administeFed. With a given cw.antitt

, .or (endogenous) t:holf)sterol• t'h.ie amount is reaChed. When the s~pply o:t the lat·te:r is exhattf!ted~ Whether

· this s1 tuation is lik~lu to Sl*ise in: the J!'at fa unknown• stn'¢$ t.he tQtal endog~nQus choleatePol

· r!ttpply is .own• . '.. . . .

. I ,

Rle~~ ((hg1esteto~ is .not ea$1~ abeorbed when given in el7$tall1ne. form, .i.e,. out or ita natu.raa:t

' ha1:H.ta.t, even when fat is fed at the ·same ·tfJ.ne1 .

p~ov:ic:tea · the latte:rt .£·e k'ept out of ~ont~ct with t'b.e <)hol,estero1 as f'ato a.s p~es:fbleft Th~~s lis. t11ul'::ltrated.. bt ~xperiment 14 (a), Section 1S. ·

Relatively small p.tr,lO:t~nts ot'siet,~l :eholest.~r¢1 even ' .

if given in dJ,s.soltre:d tom in tats exe.~t a pero·ept• ible t:nfl.uence :on the se:t'Um choleste:rol . level on1y When these an'10un.ts fot'ill an appreciable pi-opo:rtion ot the total choleste::rdl pool ( 1., e• di~t8.X7 and endogenou!l ),. '»his is i1lust:'ated by ~pex-.:tment 4; Section 9; ~xpeJ:l'tment 1,;, seetto:n 17; ~ei>tment 141.

Section 18; ana Ex]?et'1rnent 15; Section 22•· The negltgibl.e 'effect of ~% o·f cholesterol in die.tary fat as oompared with the conaid:el#able ;$$teet of larger quantit:ie.a is :consist$nt tnroushout these experf,ments~ Th.t:: cont~ove:rsy whethe~ or not d!e.tary ~holest.er.o1 tn;tluences·the·serwn·cholesterol ts·apparentl;v x-et:!olved. ..

• • ' ; . ' . 1-.

' ' .

v~ '!'he pate f'f a'bso,~ti;ond of ,qho!e,sterq1 is also a ·tuncti,on of the solubility. iln the B.'C~ompanaing fat. ilf'o. data SX>e ·ava.:llable on 'rats but a htl!Itlan expe:r-tment (Expel'imen.t 16; Section 23) incttc;atee th~t th1a may

' .

'be so in the cas~ of math

.14l·

VI. · fhe cont.rotrt'U.~$;v of' , " ',sap. • · yersus. 9u.nsa;p. 9 .. is

resolved., both chtw,lcal fiiactiona of the fats . being necessarily irnp11cated tn the regul.at:f.Qn of the serom Chol~ste:rol level. l:t is seen :rot:s example tfurt the. redu~ed seJ:"'Unl oh.oleaterol level p:r;.;oduce.d. hsr 0.1ssolv1ng constituents ot SUnflower Set\ld oil ''unsap. u tn coconut fat (Expe~iment 12, Section 140) 1s reflected. by a rec1uce4 aolttbil.itY ot eholesterol. .1n this prep8J\at1on (cf• Fig~ so, Section 20). !these d1seolved. substances appa~~ntly

·have th~ p~opertu of' pa:rt1a11y blocking the solubiltty of choleste:rol. ::tn other words, the;y tnS1 compete with eholestero1 f'.ol:' the solvent powett ot fats. It 1e f'or example known to students of the Phase Rule that the solubility ot one $Ubstance .in another can be materially altered bu the p~es&no6' of comparatively smali amounts ot a third compound.- '.Phis phenomenon has in ta6t 'been used toto the detection of 1mpul'it1es ,(Bowcien~ 194.5),.

VII• Although :related to the abpve, the question of the se~ cholesterol ,reducing 2rozaer-t;v: pt /3 -sitoste~ol

. . ' ''' - ~~

mer>! t a .sepa~ate tt'eatment. Pollak (1953} su.gge s:ted. as a meoha.nism of action of [} •si to sterol the ·form­ation of ttmixed erz;stal:s" with cholesterol 1n, the

gastrc-lntestinal tract. This relattvel:r insoluble association comple}( ts thought to be ~sponsfblQ to~ the impat~ed abaoxoption ot ..axogenous and endogenous oholeste:Pol~ 'this ttmixed cl;\V'etal1'

hypothesi a is not incompatible w1th tlte solttbil:i tt concept although the latteJ;> can dispense with ft.

The n(l!gligibl~ eolubilitu of cholesterol. at .37°0 in eooonut oil. which had p~evlou$1Y b~en saturated. with slttosteroi. at that ternperatur$ (see Section 20) would lead one to expect this modified oil to have

/ '

ver,y low hypercholesterolaemic.properties. Whether this is actually the case with the natural experimental conditions used 1n the present rat tests is unknown but is. being studied.

VIII. The extraordinary choleeterogenic properties of dietn~ esg fats are eXplained 1n both their qualitative .and quantitative aspects.. In Experiment 13 1 t was shown that the cholesterol content of crude egg triglycerides is partly responsible for'high serum eholesterols.., Since the crude egg triglycerides contained 8.6% ot cholesterol, i.e. more than required to saturate the pUre tr1glyce:r1des (Fig .• 3o Section 20), the actual serum level observed is also the maximum producible under the conditions of the expertment; i.e. even higher quanti ti.es of cholesterol in egg lipids would not result in yet higher serum ehol­esterola. Provided then, that there is cholesterol present in the diet in excess of that soluble :tn accompanying fat, the precise theoretical maximum level attainable ia determined by the solubility as discussed under I.I above. .

It is thus conceivable, or indeed a necessar,y conclusion that eggs of other atlimals containing more choleste:r:-ol than is present in hens • eggs but at the same time also fats of lower solvent power would have lower cholesterogen1c properties than hens• eggs. This has, in fact, been shown in

the case of penguin eggs in .human experiments. (Bronte-Stewart, Welle and Wilkens, .196o). The triglyoerides ·rrom these eggs contain up to 18% of cholesterol, yet produce only a moderate rise in human serum cholesterol. 1'he correctness of the present explanation has yet to be tested by

a detetrm1nat1o:n of th~ aoiuoility oC ·Cli01~eterol . ·t.n peilgu:t:n:' eag· trigiyoei:tide·s Which have been :rr_eeii trc;;m a.ll unsapon1tiab1~ matte~~'

IX. ;'he taeo?ti,e:ttoretfon ot cholea'leX'ol. 1s likeli to 'be htghe~ on· diets ·o:r.low ~ol.ubility oil-s· than on h"gh solub!lltu. .pils ~ tha.t is in inverse ratio to the ease 6£ absorption ot 6holestetJo1+ This could con.:.. eeiva'b1y be'teated by expe:r1rtlentat!on with' germ fl'*e(l;l

·. .Z.ata.f The· faecal exe~tion .of cho1ell:~t·e:f.ol Will b~ more ·fully dl'sciussed tn Sectl'on 26• \

prove(j. tc be ~:tremel;v fe:ft1l.e in that 1t 1¢4. ~apab1e o1t

p:roducling reaso.na'b'le explari.Qtions ot maey fa-cta which hitherto

appea~ed e~t;rely uncon:n~oteci. It 1s,a1~o.oap~"Qle Qf prediC,ting

new facts, SOJne ot ·~hese p:r~ietions ba.ve. been t.estea. Bl'lti will

'be desc:rfbed.bel.ow. ~he relevant literatu:oe on..the cholesterol ' ' ,. . . ' ,• ' . : J: "

metabolism of: -pa.ta will be disoussed..to.gethe~ with that of .man ' ., t ' .

:tn section 26 w]len these new eo:ricepts wi11 be discussed. in

relation to the cbofeste:11ol m~tabo11sm as a whole' Although

offending against the. general. plan ot exposition •.. this joint

discussion ot·rat and man has been dotle,to avoi.d tixteeome and ' . ' ;;; ' . '~ -· . - ... .

146~

SECTION 22

THE SOLUBILITY OF CHOLESTEROL IN SUNFLO\VRR SEED OIL

,IN VIVO Exp~riment "": ,C E!J?eriment 12)

In Section 21B it was predicted that there 1s

a quantity of dietary cholesterol above which no further

absorption should take place, when administered with a

given quantity of fat. This maximum amount should be related

to its solubility in the accompanying fat. In other words

it should be possible to determine the approximate solubility

of cholesterol in fat in an in vivo expertment. ts this so?

There is here an excellent opportunity to test the validity

or otherwise of the solubility hypothesis.

EXperimental

A standard colonv of 24 rats was divided into !!I dietary groups of four rats each. All groups were offered

ad. lib. the standard experimental diet containing 20 per cent

ot sunflower seed oil. In addition to the above the different

groups received supplements of cholesterol of o, 2~ 4, 6 and

15 per cent ot the tat ted, i.e. from 0 to 3 per cent in the

total diet. As tar as this was possible, the sterol was

dissolved at 150°C in the sunflower seed oil before the latter

was incorporated in the remainder of the fat-free rations.

A single set (in duplicate) of serum cholesterol determinations

were made at the end of 12 days on the special diets.

I I I.

·.

147.

Results andDiscussion

DUe to the small number of animals within a graoup

(four) the results are presented without statistical treatment •

.All individual serum cholesterol values are therefore recorded

both at the beginning and at the end of' the exp~riment. As

on previous occasions, the details given in the table (Table 20)

are Stll'llmarieed in graphical form (FiS• 31).

~ :J a:

'/

, ., ,

, , , ~

, , , , ~ .. .

~ ... .

~ , ~ ~

""' , •

~ ~0~[ ____ _. ____ _. _____ ·~1----------~--------------~·-----0

E1s.. 31

2 4 6 10 IS

%CHOLESTEROL IN OIL (s.F.O~ OF DIET

The effect on the serum cholesterol of ' increasing quantities -of dietary cholesterol,

f-ed dissolved in 20% of sunflower seed oil. abscissa c per cent cholesterol in oil

(Experiment 15) ..

With a single exception (4% group) the means of all

serum cholesterol values lie remarkably close to·a smooth curve

148.

TABLE 20 - (Experiment 15)

SERUM CHOLESTEROL CONCErlTRATIONS IN MG./100 UL OF RATS . ON LP..BORATORY CHOW (A) AND AFTER 12 DAYS ON EXPERIMENTAL

S, s.o. DIETS (B) CONTAINING INCREASING .AMOUNTS OF DIETARY CHOLESTEROL IN 20 PERCENT SUNFLOVmR SEED OIL

PER CENT SERUM CHOLESTEROL

RAT CHOLESTEROL PERCENTAGE CHANGE NO. IN s. s.a. OF A B (~X 100) DIET

l!EAN

1 47 37 78.7 2 67 54 80.6 3 0 54 49 90.7 81.3

4 61 46 75.4

5 49 54 110.2 6 2 45 48 108.9 107.0 7 66 72 109.1 8 50 50 loo.o

9 56 64 114.3 10 4 62 67 108.0 112.6 11 50 56 112.0 12 46 53 115.2

13 51" '63 123.5 14 6 69 117 169.5 143.1 15 59 58 98.3 16 48 87 181.2

17 38 54 11+2.1 18 10 44 65 147.7 147.0 19 46 ·76 165.2 20 66 88 133.3

21 52 !£ * - -22 15 53 73 137.7 143.0 23 49 73 149.0 24 59 84 142.3

!f Sample spoilt

which ~t tirst rises steeply but l:n~gtns to 1'plateau" at

between :Cour and six per cent of cholesterol in the sunflower

seed ,oil of the diets.

!his s1m.ple exper1m.ent has conf'ii"med th~es predictio.ns

of both a qualitative and a quantitative :natU:~·

1. The phenomenon of. e. plateau.·

2, The height of the plateau. Th.ia ls at essentially the same level as that obtained with 10% ot cholesterol 1n.sunflower aeed. oil 1n EXperiments

· 14a. and l4b af'te~ 12 days • teedints•:

la .The approJCimate level ,of' dietary cholesterol a~ Which the plateau forms, The alight discrepancy· (3.,5% predictedt~ 4- 6% found) need. cause no , eonaern. iJ:ihis may hatte been du.e to incomplete saturation at marginal con<:entrations, to additional sC>l:Ubiliaing powe:Ps of'b11e acids or to ex:peribnental ert-or. Whateve~·other absorptionmechenisms ar~.at pley appear to pia;v a oompara.tivel.y tnihor role onlY~

Conclusions .. f't-om Par-t 'V

The abt1lty to plttn and prediot. the outcome df an . . experiment under conditions. as unfamilia%' a:s. those .obtaining

-.... at the present one, has served to gitt~ considel'abl.e str~ngth

to the ,iews expressed tn part V. of' this thesi'th 'rhe outlines

ot a true understanding o:r the central problem. ot thta

investigation a.t>e beginning to appea~.at last. For this reason

tbe final task of the application of the experience ged.nett ft"om

animals to man ea.n be attacked with confidence •

. be attempted in the last 'parl (Pait!t 'VI) .of this thesis• Without

the ~sta:blishment :of this ttna.l link, the ultimate 'Ye.lue of the

present labour,s would be severely limited, indeed.

P A.R T VI

',

SECTION 23

THE .ABSORPTION . OF DIEl'ARY CHOLESTEROL BY MAIV

(Exper-iment. 1 G)

"No lipid parameter is measured more f'l"equently or discussed as endlessly as is serum cholesterol ...

David Kritehevsky, 1959

!ntroduotton

The greatest obstacle to the application of the

solubility hypothesis to mah is the widely held beltef that

dieta~ cholesterol has little or no influence on the human

cholesterol level. If this is so; it would be di.fficult to

expla.1n why endogenous cholestel"ol should form an exception

to this rule. Beveridge .and his assoe3.ates (1959) .have

recently- summed up the history .of the subject by the remark

that ".-~.a .review of' the literature would lead one to the

conclusion tha.t this sterol in amounts up to several times

that of the usual daily intake has no demonstrable effect on

the plasma lipid l~vels in man.u

x:e the above eon,elusion is true, it would also have

to'be accepted that man differs from all hithei'to tested.

mammals in his reaction to dietary cholesterol. In the

absence of any special reasons :f'or occupying this unique

position, man's implied immunity to dietary cholesterol must

' .

151•

be l.'egarded With scm.e scepticism.~

· ·with ·the ·e~perien.Qe ga~ned and the views e1CpreJ;lsed

in theee pages as baekg~a., tt is not dJ.fticu.lt to venture

a guess as ·to a possible reason fort tlle. taflur"e to· de teet.· a ..

positive relationship between dietal"';Y' .and seriln! cholesterol.

'the ~easonmsy,b~ that in most :l"eported.experJments

the St~l."Ol· 'Wae fed U:r.'J.dGrt' conditions which made ~t 'd1ff:lC\t1t,

or imposeible tor this substance. to dissoive.in dietaey fat~ . . ' ' . .

In the following simple human experimen·t Cholesterol was ted

in it's natur-al. habitat, i,.e. d:ts.solved ·in te.~. . .

fbe authoi'!. waa fortunate in having been able .to ' make use ot an ~xistinswell ·()rganiaed elq)er1m~nta:1 maehineey

.to; human nutri~io:rml studies in the Department 'ot Medloline•

This. h~s been ·established to:r .. lip.f.d st-ud:tes by I>r•B ... Bronte­

Stewart and his assoeitites ln the eotirse of theit' o~ll.ginal . ' .- . t ' J .

studies (19S6) on the effect ot.dif'£ere:nt, dietal"y tats on the

serum cholesterol l.eve1 (see sections 1 and 2).

l!'he subject to~ the present experiment was a healthy

. mate }3antu 3S yeaz!'s .of age•. 1)-gl'in.g the ~xpe~iment he lived

in a metabolism w~rli and :roeQeiy.efll a ·nasal .di~t low in f'at , ... ;'

content (.a·- 12 gm~ datlyJ. Witl1 minor -variations~ .the basal '

diet 40nsisted ·Ot maize l'll~al; ptunp:ttin, Cas~lan, br-eaa and

sugat-' and except as indieated below was kept constant With

respect to ca.lo;t'tes (2500 daily) and protein (68 gme dat'J.u),

Du~1ng the expe:r1ment the v-olunteer slowly gained we.~gh:t

(113 l'bs to 119 lbs) and suffered no digestive d.isot'd.e~s.

ti~ldusup~1ement.!,

''the 11pid. supplements tali in nine ~U:c.ce~uJive

dietary period.s wex-e as .tollowee

1.t 'No .suppl~ents

2+ Lipids present in 10 egg yolks· fed. as :.V!Thole yolks~

3:~

4, 60 sm·sf ~nfl.OWf!!' seed oil (100 1. v, ) , no Oholeste~ol.

s, As foi:" 3 labo\Te

? .• 8,.

$}*

I

3 ~.~ etwst$lline eholestex-ol without ta.t, sprinld.eci ovel' food.

'' ,, < wo $U.pp:t~ent., •.

As .to~ J above,

No supplement.

Serum cholesterol eoneentrat1ons were estimated on alt$:ttna.te

d~s by the method of Abell et al•· (19.52).

'lhe .~esul ts repo:rtea.' in !able 20a and illuatra.ted

1n Fig._. 32 show that the high sel'U.m lette.ls created b;r the

ingestion of egg yolk wezte maintained by s:n equivalent amount

of cholesterol given diasol "ed :ln fat • Withdrawal of' the

cholesterol while f?Ontinuing the :rat supplement I~"esultea. ,in ' .

a marked d;rop which was z-ever&ed attar restoration of the

sterol to the diet.. The reverse prQCedure 9 i•e• the withdrawal

15.3-

of the sun:tlower seed oil; while continuing the cholesterol

snpplem.ent was aceom.pan1.ed by a most dramatic dtic>p of the

t4erum concentration to base level • . .

It the reaction or this single human subject to

dietary cholesterol. is a retlection of what happens in

the population at large there seems to be no basis tor the

statement referred to in Section 16,!1 namely that ••tn m?..n

the plasma cholesterol is largely independent of dietary

cholesterol."

fhe present results are thus at variance with the

views advaneed by most workers in the field. Keys and

associates (1956) fot' example have stated that ". •;; .in

a<iu1 t man the serum cholesterol l.eve'l is essentially

independent of the cholesterol intake o?er the whole range

o.f human diets~" Simila:r op1n1on.s have 'been expressed,

among others; .. by- Bronte-stewart et a1. (1956) ·~· Ahrens et al.

(1957) and Gordon (1959h

IJ.'a'b1e 20a/

TABLE~20a

THE EFFECT OF FEEDING CHOLESTEROL IN THE ABSENCE AND PRESENCE OF HYDROGENATED SUNFLOVIER SEED OIL TO MALE BANTU AGED 35 ( Expet'iment 16).

Dietaey Dul'ation Supplement Serum choles1erol (see text) ~(mg.LlOO ml per1.od of period

(days) z*

1 8 nil 136 2 12 Egg yolk 198 5 9 Chol.+fat 201 4 17 fat ,161_ 5 20 Chol .... f'at '201 6 10 Ohol. 140 7 13 nil 156 8 10 Chol.'iotat 188 9 12 nil 150

H Mean of last three t-egd1ngs of period a Last reading of period.

220

i .. -200 . * I U I :I 1 -ceo

~ ~ 160

::1 ~ 140

I <.J :l i 120 Ill

100

60GM. KSF.O. 3GM. CHOL.

3GM.CHOL. BASAL

I fBi

130 205 208 157 194 145 154 202 148

60GMI-\SI'O. 3GM.CHOL.

BASAL

1$ 20 2$ 30 5 10 IS 20 25 30 5 10 IS 20 25 30 5 10 IS 20 25 30

MAY (58) JUNE JULY AUGUST

1.55·

While the present studies were in progress

a new approach to the problem was likewise pioneered

by Beveridge and his group (1959a). They found that

the h;Vpercholesterola.emio properties of a volatile

molecularly distilled fraction from butter was largely

dtte to the cholesterol (3.83%) which had concentrated

in this fraction.. 'l'he eholesterol .... poor fmctions led

to relatively small increases in serum cholesterol levels,

but when supplemented with an amount of cholesterol

equivalent to that provided by the eholesterol-r1ch

fraction. increases of comparable magnitude were obtained.

Their explanation fora this effect is, ho'\ifeVer1 less clear,

for they suggest that the substance with which cholesterol

:reacts (wby'l) is a specifiC type ot triglyceridehaving

certain fatty acids attaChed at the al~h~; beta and alpha1 positions on the glycerol moiety •.

in an even motte I-ecent abstract (l959b) these

same workers reported the results of feeding increasing

quantities of cholesteroi (up to 1200 mg. per day); together

with a cholesterol•poor butter fraction, to mana At

ciietary cholesterol levels of above 4oo @th per 950 calories,

the serum concentration of the sterols p1ateaped. No

explanation is offered by these authors f'or this phenomenon.

Its significance is at once apparent if' the ttesults with

rats of Ex:pel'irnent 1.$, Section 22. are· reca:11eda-.

It is oonclude4 from this aeatton that the . - . . '

r&sponse ot mE!ll to,tl1etaey Oholesterol does not difter

matelt'1all1' from that of t'ats. \Vhen -rea !n its natu.~al

habitat- thiS BterOl :iS' e([llall;v Well abso~b~cl by' both

species. !'he Vlay has been cleared for an att~rnpt to ' • - ~ ' • ~ • < • - ' • - - - '

157.

SE01'ION 24

APPLICATipN . OF _THE. SOJrltmiLITX. JIYPOTHESIS TO MAN

tn section 9E it was cla.ime,d' that undel' the

condJ tiona created and used. tn this work, the se'Alm

Cholesterol of/rats reacts sim1~arlY to that of humans ' '

to dletaey tat tX'eatment? :rn the previous seotion it

, v~a.s shown that dietary choles~erol, if· dissolved in

dietary tat, elevates human serum cholesterol. 'rhere

ist therefore• no reason wh;V the solubility concept

should not apply to man. Is this so~

, Jtortunately, several groups of' workers have . . . .

produced lists of a variety of oils togeth$r with thei:r . quantitative effects on the serum cholesterol concentration

of me.n. One of: the most carefUlly conducted investigations

of th:ls nature is that dtie to Ahrens and associates who

summarised their results in a, diagram l'eproduced in ti'ig. 3

section 2.

Using only oils for which choleetettol solubi11t;v . ..

figures have been determined to (late and including JW.i'ens •

data on menhaden oi1 (19S9a), this diagttam has here been

ra.arranged into a con,-entional graph. For th1s.purpose

groups o:r individual values f~r each oil were condensed

to means, and a linear :relationship was assumed. The

2Jesu1 t ·is shown .in . Pig~ 331 and 111ustrat~s tn~ <'.P!Ia.ton .· . .

eXpressed ~ Ahrens as reoentlv af:J ·1959 (b) that the tt~g~ee

of u.naatul'ation fs the propet-ty of oils.· which best CC>X'~e~ates

with a$t'\1ftt. cholesterol 1eveltl•

ln a oompat-a'ble g!'aph·; iodine value$ have been

.replaced. by the so1ubililites ct oholeatero1 in ol.ls at . . . ' ' .

3?0 0 (see Pig" 34)•. :It ills. e~iuent both by vtt:ru,al insp$ctlon '

and by comparison o·f the eoxaz:elat1cn cot:::ffi~tents •. that the

correlation cf solubilities.w!th. sei'tltn choleste:rols 1~

.supe:r:tor when qompared with that of ·t.oa.ine values~

;In constxn.tcting th~ second: g~aph it was at.isumed

that the samf:" oil~ of ~itter~~~ ortsi.fls nave .1aentica1

com1:rositions• ~ie1 of c61,11'se, .is not tl'Ut:l es.p$~i~1;r in .. I . ' , '

the case· of beet tallow and tish o~'l· ln the latte:r case, . '

menhaden oil. baa actuallY been r~placed. by the boey oil of

the related. so;u.th Afrie~ pilchard. (sa:rdina osceila.ta).

It~ !n addition~ ·the unavoidable lack or· pl'E!Oision of

th-e .$elmt'tl cb.oleste:i'til data is' taken into aeeount., an actu~l ' ' . • • I. ' ; _. - ' . . '

r .

~h.e 11 ~bnorma11u"' · ioli serum cho1esterol. levell'

·obtained with c~rn o:b. (see A.rmstronEt et ail,., l957J also

p:redtQtlon equation <tt Keysj Anderaon and; O.~and.Ei• 195S)

deserver special. mention~ · 'l'h:t's oil 1 according to the data

supplied. by ~ens, actually produ,ces slightly h1.S'he~ 'Values

than here pred:i()ted. ~om solub:U.1 ty values. · 'l'hese and

0

20

40

60

80 w 3100 <(

. I > 120

w z 140 0 0

I 180

I I

158a.

ecoco

PEANUT e

r=0.919

'!} =1137.,-217BX

e FISH

-20 0 20 40 60 SERUM CHOLESTEROL

EXPRESHD AS PERCENTAGE DIFFERENCE FROM

BASE LINE ESTABLISHED DURING INGESTION

OF CORN OIL

Fis.33. Relationship between iodine value and serum cholesterol concentration (adapted from Ahrens et al., 1957) ..

.Fiso34

_J

0 ·~

_J

~ w 1-

"' w _J

0 J: u u. 0

>-1-_J

([)

::::> _J

0 (/)

w .., < ... z w u a: w Q.

4.50

4.00

0 SERUM

r = 0.982

'j= 3.279+0.01943)(

20 40 60 CHOLESTEROL

Relationship between solubility of cholesterol in oil at :37oc and serum ·eholeste.rol concentration.

·•.·

1S9· ·.

othe~ rntnoj:5 Variat#,ons are, h<'rWeVer, easily \VithtJJ. .

range. ?f. eXpe~$Jnental errox-* ' • Whateve;r the sho~t~

comings ·o~. th(:i' u.ttdi!rliinS ·me¢hard.$ms ot this ~P+'J?~l.at~on

at-e,. the .(legree ot ~¢>r:~1ation (r, .·~. Q.9B2) .is too l'lish • - I • , I • •' • •· ' •

s. In y):vo:. sdiu)11it:X d~t;~nn1nf1t1.on ·ln M~ -- "" "' ·- - .

• A':t1. e:Jtpe~iment t"esembllns that ae.scribetl 1n

Section 22 has been reportect, by 1tartt1nen ·a.rui co~wo~ke"S

on mmh !n ·'b~a:nC'Je' eXpertments on 16 sub~~ete;, the·

appal'~nt absot'Ption of cholest'erol was . stutlied when .

l~ l 11 6 and 9 gm• o:f aholesterol wa.e· a8.d.ed in' the

diet miged . ws.'th J7 and. 50 gm~ of: a m:hd~ure of ammal

and vegetable tats.· ~bese workers eunmer'ized the!~

~eeult$ tn a (li.e\SI"I:lm "LVhich .iS reprodUCed unchanged in

:rig .• l!); ~ey in:terpret these aata a.s ''demonstrating

that th~ human 1ntest1ne has a l!nitted capacity roza absorption ot Q.ietary choleate:t?ol** • Uihe appa~$nt fa11

. in absorption on tngerrtion of 9 sm11 of <molefjjterol ttaiiy

they atttitl.ntte to an _.,a~tefaQt d:ue to ~Xper!mental. etorol"''"• ·

..

I.

160.

e SERIES I (3SGM FAT)

~ 0 SERIES II (35 GM FAT) 0

' ~ 2.5 X SERIES .m (SOGM FAT)

(.,')

z Q2.0 1-a. - - - -.- - ---- -- -0::

~ 1.5 m <.( , ..J , 01.0

Jtf/ ,

0:: LLI

m ..Jo.5 0 :::t u ,

" 0 I

2 3 4 5 CHOLESTEROL INTAKE

6 7 GMjDAY

Fig. 35: Capacity of the human intestine foF the absorption .of dietary cholesterol!

(Reproduced from Ka:rv1nen et a.l.. t 19.57)

9

These same resu1ts are here interpreted as

demonstrat,ing that amounts of dietary cholesterol. lin

excess of that soluble in accompanying fa:t, are not

abso!'bed., on the assumption that the abso·rbed cholesterol

was prrevious~ :present in saturated solution in :rat., the

data supplied by- Karvinen can be used to calculate the

mean solubility of cholesterol in fat to be o1oee to

4 e:m•/100 gm. This is tn close agreement with the probable

solubility value of the animal - vegetable oil mixture

used by Ka.rvinen, somewhere between 3•5 and 4 percent~

·' .

!'he reeul ts o£ Experiment 16 aesortbed in the

pl'eV!oua Section a.re now :f.nte~:t-~tea as foll~wa, The

steep rise in serum cholesterol on 'feeding egg yolk lipids

i.s due to the excessive !lmount. of cholesterol dissolved in

these lipids~ The same amount o:r cholesterol dissolved

1n hydt-ogenated S'Unflowea' seed. oil maintains this ~iae.

· On subsequent withdrawal of the· die taw do~9 of' cholesterol ,. ;.-•

one obsel"Vea a tal.lto a l.evel which is, hoWe'Ver, still

above the·basal line owing to the comparatively smaller

.amount ot 43ndogenous cholesterol., which is now absorbed

along With the fat.. !he amount of endogenous sterol from

both bile and intestinal juic(it available tor ~~absorption

has been estimated by Ka:rvinen et al, (1957) to be about

1 sm. daily• R.esuming the cholesterol diet with the

·SUnflower seed oil again refriil ts in an elevation of:

Cholesterol Which on this occasion however, l":f.Ses but slowly

due to the lower solubilit;r of' choleste:rol in SUnflower seed.

oil as oompat>ed with that in egg tx-iglyceridea. When n~xt

the f'at is w:t thdrawn but the oholeetet'Ol continued, neithel'

dietalP.V ~or endogenous cholesterol oan any longe:r be

absor'bed and the concentrtation promptly falls tt> basal level.

!he "enigma" {Gordon et al., 1958) of the cholesterogenio

property o~ eggs is apparently solved.

The effect of l3 -aitostel"ol ~ . ·.·· "_ .. :J r- . -- -.. . -..

!t is significant that appreciab+e eho1esterol

depressing effects with sitostel .. ol have been obtained 1n

man on!w With veZ7 large dosea of this aubstan.ce. Thue

Farquhar and Sokolow (1957 and 1958) fed as much as 18 sm• per dE\Y to human subjects to produce only moderate :ralls

in sel'ttm cholesterol 1eve1st trp to 45 gm., daily was' fed

b;r Best and co-worketts (1954) to humans to prod.u,ce maxirnu.m

e:ff'eets, while Jever:ldge et al;t, (l957a) fed similarly high

d.oaagea (7 e:m~) of ~~s1 to sterol in their earlier. experiments.,

In late:r etuiU.ee the same worker~;J (1957b and. 1958) recorded'

some success w1 th more natu:ral dose·s when the sterol was

given dissolved in oil.

One reason tot' this ma;v be that this ste:t?ol was ' .t

. '

!his reason, in othel., words, may be analogous to that

advanced in Section 2.;3 tor the alleged lack of effect of

d:tetaey cholef:fterol~ The solub1l.1ty bypotheeta predic:ta

tttinimal a~rum eholesterols with oi,ls saturated w1th.aitosterol.

Expe;tot.m.ental 'Verification would be complicated by the fact

that .a!itosterol 1$ abeox-bed to the -extE:,nt of ab011t ·10% by

rats» as has been damonstz-atecl by Oould (1955) with t~itiuni . '

labelled· ~·s1 tostEn:•ol •.. A fi~e of 4% or moJ:>e has· been

qttoted fo't! man by th~ ·same author one year lateta. Oonvent!~:ma1

ffJ.ethods for the determination ot serum eholes.terol can not

be ·app11ed since these methods are not speci:flc f"o:r cholesterol,

but in addition measure most unsaturated sterols

including sitosterol. This :f'act has been overlooked

bJ' many workers, including the writer.

E. The. et:f'ect of !\V:drosenated oils

The equal solubilities of oholestel'>ol in

natu:ral and hydrogenated (t.v. = 100) sunflower seed oi1 '

(see Fig, ;50; Section 20) is difficult to explain on the

basis of the repol'ts 'by Bronte---stewart et al. (1956) and

Ahrens et al. (1957) • These workers :round that the

hydrogenation of unsaturated oils leads to higher serum

cholesterol levels than those noted with the natural oils.

!t has been suggested that this effect may in part be due

to the production of isomeric trans•a.o14s during the

hydrogenation process.

There is no urtan1m1ty of opinion on this point.

'!*hus lleveri(}ge et al. (1958a and 1958b) repo:rt equal effects

with na.tu!'al and hy'drogenated (r.v. 69) oo!'n o11s;·\lhi1e

Gordon et al. (1958) states that they have "f!'equent~

observed that certain.••~••••saturated fats, such as

hydrogenated Sunflower seed oil (t.v. 74) do not raise

the serum choleste:rol u. Ahrens et al. · ( 1957). also l'epo:rt

a. ease in which a h.vdrogenated corn oil preparation had

just as great a bypopcholesterolaemfe effect as the natural

oil. The problem of the effect of the hydrogenation of oils

can thue be regarded.as still unsolved.. The d.etet'mination of

solub11tty values of oils aotuall;r used. in successful human

experiments has yet to be done.

164.

F. Effect of oils relative to a fat free diet

If the differential absorption of cholesterol

from the intestine with different oils is the mechanism

responsible for the solubility - serum cholesterol

relationship it would appear that the lowest possible serum

cholesterol obtainable by manipulation with dietary fats

is to feed no fats at all. This seems to be the case with

rats {Experiment 14 1 ·Section 18). An increase in serum

cholesterol of man relative to a fat free diet with so-

called serum cholesterol "depressing" o.ils has been reported

by a number of investigators notably Keys ( 1950 and 1952),

Hildreth et al. (1951) and Meyer and associates (1954).

As has been suggested by Sinclair (1958) these works belong

to the

" •••••••••• Proto-Keysian period When all fats in equal measure Raised cholesterol in serum Butter, sardines, walrus liver, ••

u . . . . . . . . . . Some recent evidence from human experience points

in the opposite direction, i.e. a ~cholesterolaemia is

observed following the addition of some oils to a diet low

in fat (Bronte-Stewart et al., 1956). This is in apparent

contradiction to the solubility concept in its present form.

It will be remembered, however, that the absorption. of sterols

from the intestine is obviously not the only mechanism

whereby cholesterol gets into the blood. That fluctuations

in hepatic synthesis can, for example, be induced by changes

.in tile r-ate of ab.sorption of cholesterol· from the intestine

Will .be di:S~"Sed in ·the n~i:t eectiont:t. A ftt);ithet' po$sibl~

e~1anat1on of the depression•beicw .... tat ... rree phenomenon

rill al.so be offered. whi.ch invoke$ the solu.bi1t ty .eonee-ptf

G. !he. ra~e. oi .ch!f!S~ *in ·seJ?Um .4~n~ent~ation

fhe l!itt:erence, in· sol:ttbill tu of eholeste~o1 in

va:r:ious oils seems a;t fir>st s:tght to.be too small to accoUnt

tov the 'Vef7 dit:f'erent ertect.$·t.hat·these·o11s can have ¢1\

the serum· cholesterol level • ~be ·following s im;ple caleulat ton

will show1 however!!· that this .ma:u not be so.

Su.ppo~:ring a. human subject co:nstut!ing .$0 sm.~ Sun:f'lowe~

eeed ·Oil ia113f has a steady ee~ ch<>lesterol coneentratton

of 200 mg./100 ml. At. what rate wi11 his se.t"Um db.olest$~.1

~ise on ~ohangitrig 'trom Sunflower seed oil to tbe same qu.ant:t ty · . .

ot co¢onut oil tn the 41et?

'Jnle ·following 'aa.d.itio·nal data ax-e givent ' ;. , '1 I '

. · :fotal s~rum. vo1:u.me · :. Total cil.'oulating ehol:estet'o1

D1Q.ogenous ~holestero'l. available to'1Jt, . .· . . :r.e-ab$orptton · (K;iWlnf)~ et a1,,t 1.957) Solub1llty of choi~sterol:

!n SUnflower seed oil _(actual J.4G) . in Oocon:ut o~i'l (actual 4.$:2)

..

,,ooo n1l

G gm. i' gm./day

. 3% 4%

AsS\tntl,ng aro'bitlfarl,ly that the endogenous cholesterol.

is re-absorbed. by coming in con tact W1 th, and , aaturoat :tng half'

the 'dietary .fat (i•e~ ~.5 gm., fat) tn the int.esttne,, one

166.

can say:

Rate of absorption:

during the ingestion of s.s.o. oil i gm./day during the ingestion of coconut oil ·t gm./day

Therefore, extra absorption on changing from sunflower seed oil to coconut oil i gm./day

Other factors re~aining constant, this is

equivalent to a daily increase in total circulating

cholesterol of 2:gs x 109 • 4.2% which, in the present

example, corresponds to an approximate rise of 60 mg./100 ml

per week. This is well within the range of observed fact.

(see e.g. Gordon et al•; 195?). This serum cholesterol

enriching process will continue until the regulating

mechanism of the liver presumably stops it by decreased

synthesis.

Needless to say, the above very approximate

calculation is rather arbitrary. It is merely intended

to illustrate that small differences in solubility could

conceivably be responsible for quantitative effects of the

same order of magnitude as found by practical experience.

Similar calculations could be invented to account

for the different rates of increase of serum concentration

when cholesterol is fed together with egg fat (comparatively

high solubility) or sunflower seed oil (comparatively low

solubility) as was found in Experiment 16.

'!'he concept of: the ~e .. absorption ot enaoerenoua

cholestePol ~om the intestinal t:ta¢t ts b;v ~o me$Jls a

n~w one. ·T·ha ~lated ente'ro-..hepatte c·;t~culation of b~le

· acitis ·is indeEk\ dea¢vibed :tn most textboo1.ts on phya1olog,y.

AS far: aa ;ts knom1, ~loot.' (1943} vvas the first to ap'P1Y'

th,is pri,.nc:tp1e to obo1ester:-o1. le p~lnt$d out that t.he . pai't pla;yaa bF the b:t'le in hpplying. ohoa.est~rol to the

blooa has genea:'ailu been o\te1'1ooked; and. the chotesterol

ot tll~ l:rU.e~ Jle~absovbt5:ci d.ur1ng :tat abaorptton, mey be

sutffcient to exp·lain the inc~ase in blood eholeste:rol

after fe~d:tng tat~ '!'he essential r()le .Qf' ·:fat :tn the

a'bsor-ptton ot choleetel."t)1 has ali'Sad.y been d.t.Sc:n1El$~d. in . . .

S·(;)ct ion 23.

:tn 1949 OU.bn~r and. Urtger>leider refez. to 'hili~

cholesterol as but "'pne pl\ase. in the peysiologie ci:ttcu.l.at!on

· of cholesterol. (!ntl!stllne-.. blood - liver - intestine},

in its ~n1~ as an ,adnu.nct in tat transpol't~" \! •• Two yea~s

lat&·r H!ld~etl;.,. Mellillkoff; Jilai• an<i Hildreth (1951).

obee:ried art 'inorrease in se!'tttn cholesterol in thttee human . . ' ............. . .. ·· - - ' -. . , ' .. _- .

t:Jttbjecta att.er- vegetable :tat feeding and suggested the

same me~anism as an explanation for: these "t!ndlnga.,

"Its ·(biliary eholeatE:rd).) :t'~•a.bsol:'lption, togethet> w1tb the

abso:t"ption. of' 1nser;~t&d ohol.esterol., ,app~a~s to d~pE::t1d upon

the $fmttltaneoulil ab$o~tion of fa.t.t;v acids• tt ma;v well 'be

t]lat this fatty acid·,. oho1estel"o1 abso~tion t-elat!onahip •••

shares in the regulation of serum chole:steJ:"Ol concentration"~

A case has been made for i~testinal absorption

processes being the mechanism underlying the solubility -

serum cholesterol level correlation. lfhere is, however,

no reason to believe why similar principles should not be

operative in the body whe~ever fat and cholesterol is

transported across a membrane from one tissue to·another,

such as from the blood stream to tbe liver. cells. The

alteration ot the partition coefficient of cholesterol

between two lipid phases or lipid and aqueous phases

due to a change in the lipid composition at either side

of an interface, is indeed a phenomenon which might explain

the movement, deposition or removal of this sterol at

different sites of the body by simple physical concepts.

This view is strengthened by recent work of Gould, Jones

and Wissler ( 1959). They employed the injection of titium-·

labelled cholesterol to demonstrate the equilibration of

blood cholesterol with various tissues in the human body,

including atherosclerotic lesions.

SECTipN 25.

:;t'HE nATE OF J{!FFUSION. _ OF OHOtES~EOOL

FROM SERUM . TO . DIFFERENT FATS .

A. f!he 1ntravenous in~ection of t:a:L

There is an interesting expe:rtmental fact which

requires eluo1da.tion and that is the successful depression

of the serum cholesterol level in man by tbe intravenous

injection of a.n emu.lsion of' cottonseed oil by Gordon (1957).

An example of such an e:xper!ment 1s illustrated in Fig. 36.

DIETARY F'AT q/ooy

BODY WEIGHT 00UI1dS

SERUM-CHOLES.TEROL mg%

FAECAL FAT q.fdoy

200t 100

0 ~

145 [

135

120

110

100

go

80

70

·:[ 0 5 10 15 20 25 1

DAYS

Pig. 361 Effect on the serum cholesterol ot the intravenous tnjeetion of an emulsion of cottonseed oil (Gordon, 1957).. . ·

$1noe the injection of £at directl.Y·1nto t.he

· blood:at~~eam obviously. ·l)y~pe.sst;!s the normal intestinal ~ . . . -- '

a:bsorpt!on 'Procees thE! reau.ltfng depl?esslon of th~ serum

chol·ester>o.l appea~s to b~. in direct. contradil¢t.io:n to the

vict~W"$. ~d:VanQeB.. in thes~ pa.e;es!i · fo find a :loophole for·

eaeape from .tli.ts dtsttf~bing sit"Q,at±on i.e, the ·object of

'th~ last _e;)tpeplrP,ent,. · . . '

a. tthe. dif,tusiotl, of cholesterol tl'om serum · · · }o.d.i.fterent *'ats •. q()i1xpex>.:lment. ,:t:z.)._._·.

... . '-' '-- -- ; - - - .. ·- - '.- ---.-'

!lte~ e:Xker'tkentf:ll pl'*OC:~du~e. was as follows •

~o to ml ot b.mna.n serum contained ln a. G•• x 1n boiling

· · tube .t~ added: '10 mi· •of · th~ triEtlyeel.'.1d.e oil~· iJ!he seC't.Welu

· stopp~:red. tube' is •Shaken vi;gozioualy~ The· resulting t;i.ne

·eni't11sion iS theb: :ineubateci at $7°0 in .a. ·Qonatant · tel1'Jperatur-e

bath .for· two bottrs.~ During th:t s time tlie tu,be ~s shaken

periodieall.y by hand. 'lt'Vh(l)ru:~ve':r the emulsion threatens· to

break up. tmnediately after rem::ival f'~m tne bath the

em1si~n· ·ts ·t~anste~ed t.o a c'en.t:vifuge ttibe ·and spun at

high speed. (7;000 r.p.m .•. ) fo't' ;;o minutesi · Samples ar-e then

.cat'efull;Y withd~awn trom the npperw oil;v· la.yer and. the -serum.

lay"~~ below .f'ot• eholester()l d.eterminations ...

·"The th:ree oils used here, name.1y ¢oconu~,oi~,

. olJ:ve oil. and :Ci:sh. oil have been chosen :f'r.om the ce.ntre and . . >'. • - - '' - - . . - ' • - . - T-. - - ' . -~

both e.itreme ends o:t th~ .solubility mcal.e obtained ,.in

S"-hit" on 2"' •. :~-.. - - ~ . liJ

. .· ·. ~-

. ~The· :~e~sUlts, pt this pllot exper~ent are sutmHlrised.

in 'table :21 ana show clearly that some 'ahole.sterol has been

171.

TABLE 21

!.E!J?eriment 17)

DiffUsion of cholesterol from serum to triglyceride oils

Coconut oil Before After Difference Average contact contact Transfer

tube 1 (0) 84 98 +14 (S) 256 237 -19

17.7 tube 2 (0) 84 100 +16

(S) 256 234 -22

Olive oil

tube 1 (0) 181 193 +12 '-

(S) 256 241 -15 12.5

tube 2 (0) 181 193 +12 (S) 256 245 -11

Fish oil

tube 1 (C)) 533 544 +11 (S) 256 21.14 -12

tube 2 (0) 533 10.5

547 +14 (S) 256 251 - 4

Cholesterol concentration in mg./100 ml (FeCl~- colour) in oil (0) and serum (S) before and after intima~e contact of e~al volumes for 2 hours at 37oc.

. trans:tewed from the sel"Um pha.ae to the oil phase; p:tteaumably

by· tlitftts!on. !hey also show that a high solubility oil

(coconut o:t1). absorbs rnore cholesterol than a low solubility

<>il under the same conii1tions, With the olive otl somewhe~

in between, Uncler the conditions of tb1s e~er1ment the amount

of transfer of cholesterol is aamittadly sma.ll, but can be

more than dOU.bled by extending the time ot contact tro:m two

to four hour$ a~ bas been shown 1n anothe:r- pilot experiment

with Sunflower seed oil. The optimum conditions to~ the ~ate

of dittus!on. are at present und.ei? g3tu.dy. Eff;orts are also

made to. improve the precitdo:n of the method•

~he above ve~ stmple experiment shows that

trig1yoe.ride oils can be U$ed to "extract" cholesterol from

se:rum in much the same way as conventional fat solvents like

diethv1 ether .• Moreovex-. it shows that coconut oil is a

better solvent than, tor ~;xample, fish oil. The z-ate and

extent ort~ansfer or diffusion of cholesterol from the • .. ;. tl

se:rum to the tr1glyc;e:ritre d.~op1ets, can be v-isualised as

depending on the time o£ eont!lot, 'the aur:race area of contact,

the ooncent~t 1on in serum and oil, and the partition

coeffie1ent of cholesterol between these two phases; in

othex- wo~ds, on the :t-elative solubility ot this sterol in

the two ,phases. !he preci.ee state in which cholesterol is present 1n· the' serum is .t*elevant but will not be discussed

futtthel" than remembePing that ttt is slightly soluble J,n the

aqneout? layer.

It is conceivable that the mechanism by '

which intravenously in3eeted cottonseed oil low:ers t.he

serum ~holesterol concentration is based on a stmilar

prrinciple• !he injected oil merely dissolves and extracts

the sterol from the serum and posaibl.v other sit.•s and

transpo~s 1 t to the liver or some other part of the body.

It may behave like chylomierons in this respect_. Thus,

the eu.ceessfu.l in vivo removal or aortic atheroma of'

rabbits by. repeate·d infusions of phospholipid by Beyers

(1958) is here interpreted as a simple. ••extre.etion" process.

Frazer (1955) is·of the opinion that the

" •••• eventual partition of the lipid molecules between

the oil end water phase may affect their distribution in

th~ body after absorptionJ tt On this viewt 1nt:ravenou~ly

injected coconut oil with ite higher solvent capacity for

cholesterol, W()uld be expected to be an even more efficient

h.Y'Pocholesterolaemic agent then cottonseed oil. These

phenomena could be pictured as resulting from anothel;l>

manifestation of the. solubility theoey "in reverse" in

the blood stl'eam.

'n,le mechanism diecu.ssed above might be capable of

throwing light on yet another hitherto unexplained expel"imente.l

faot. This is the lowering of the serum cholesterol of

human subjects already consuming 100 gm. of a high solubility

fat such as dripping or 1\vdrogenated coconut fat by the

fUrther addition o:r 100 gm. of sunflower seed oil daily to

their diet (Bronte-SteWa.rt et al., 1956; Gordon et al.~ 1957).

What happens in this oase m~ be as :fo1lowsi -

'!'he first. 100 sm• of coconut fat 1n the diet is

suffiCient to cause the re .... absol*ption of all a1!ailab1e

endogenous eho1este~1 from the intestine• J:xt~a dietary

fat, ln excess of this amount Will then 'be absorbed into

the blood .sttteam free .from cholesterol and the;refore with un1fllila1 z.ed sol vent capac! ty. 1'he.ee tt'igl;vcer:tdes therefore

behave like intravenously injected oil1 thus reversing the

proe~ss and. lowering the serum cholesterol again. It

would be intel'"eating to :repeat the ekpelfiments :referred.

to above using coeonut tat instead of Sunflower seed ae the second additional dietary supplementt~ An $'Yen g~eater

.reversing eftect might thus be obtained. No reeot'd.s of

carefllll:t conducted studies c011ld~ in tact) be found of

the ttelat1onah1p between \'&eying amounts of the same fat

in the tU.et and the serum cholesterol level~

1'15.

SEC'l'lON 2G

~HOLES'l'!BOL METABOL:t~M . AS AF.F.E!J.TED .. laY

DIFFERENT . DIETARY' .. FAt'S

fbe con~apt. of tb& d.iffe·rent 1a1 absol!'Ptton of

endog~nous clloleat·erol du~ to dif'f'e:rent clietaey. tats mst

t'ema,in i<l1e speou.1at!on if it C)a,nnot be shown to dov~tail

into wtdett established aapeot$ of cholesterol metabolism~

Thts td.ll be attempted b; de$Ct"ib1ng 1n more aetail, the

cix>cul.ation of (Jholesterol (intestine·•blcoi!i""" live~-.

bile - intestine) as outlined in section au. In many

instances, s.!~eady t!"odtlen g~ound ia thus tte~covered-

fhis. is worthwhile it '-t toeveals new and wide~ rela:tio:n.shiP$•

~o d.iag:raml! ~e shown, whieh t1ep1et the state of

aff'a.12;1s When either high ot- low solub1l1 ty o1ls are fed.

«these a!ie.!tepresent&d reapectivel..;r by .co<!onttt and. com oil." '•

Abs¢~tion through the ~a.p11lar1es into ·th$ portal

ctrouia.t1on has been omitt.ed from. th~ di~• Neithel"

D,eut~1 tats (tra.zel!'• 19SSJ tlo.r cl'ioleat~rol (1Ugg.s; Friedman

and BeyEafH, 19$1)' f.oi.low th;ts ~outet

'i'here is goocl evld.enee to assume that cU.etaxw <

ohoiet!lterol-free t:ats ar-e able to 1nf'1uencte the serum .' -, ' . -- - ..

i

cholestal"ol for an .inde:tintte period of titne ~n ratt3

(~lt!>et'~ent :;- se~t1on 9) ·~nd :man .(<J<>Pdon and B:ro~- 1958).

The meta.bol.ism ot the traction of cholesterol responsible

for these et.:reets, therefoJ'e, :reaches a ~tate t>f dynamic

176.

--- -llo

DIETARY

CORN OIL BILE {tow solubility)

SERUM CONCENTRATION

t lymphatics.

INT-ESTINE EXCRETION

INT

SYNTHESIS

2 ---- ~

LIVER/:

// <!

v DIE. TARY

PRIMARY REGULATING

MECHANISM

SERUM I BLOOD CONCENTR

r-

~ COCONUT OIL t BILE (high , solubility)

__, , .. I.

... ' ' •

AT ION

ESTINE '-. \ ../ ; > EXC RET ION

' ... "'

ABSORPTION

Fig. 31 The circulation of cholesterol.

~qui11br1urn ana the l,'a.te of synthesit;J must equal the !'"at~

of' ~xoretion of cholest$:rol and 1 ts ¢nii .... produets. The

hit?;he~ absorption ~te of cholesterol d.u.e to ~oconut fat

_as compat'ed w$:th that due to· co~ oil intplies a lower

. f-ate of exc:taetlon. 'i'b.is, .in turn., 1s necessa·r-ily link'd

w.i th an . equally lower rate or synthesit:~. lt this were not

so, the body would in tim~ be either complete'-y a:raineti

of- cholestero.l or· pt>ogreasively ert~iched .with this substance. , """~'

l.Uffe~ent dietat"y fats Will therefore int'luenoe not only

absoX'!>tion and excretion but w111 alsG at.fec.t the rate of

hepatio eyntbesi$ in a characteristic way,. · :~hese three

~spects will now be treated. in mOPe detail fn relation to

existinsz' .. f':lxper1menta1 ev'-d.ence.

~. !ntesttn$la.bs~PJ!t1on

DiXJec·t proof of di:tfer~noea in the ra.tes .of ·

resorption of' endogrenou.s cholesterol due to saturated qr ·

unsatul"ated ·nietaz>Y f'ats woulii be dfffieult .tQ .qbta.fn

eJtpe:r1merttally.. -•tA'bsorpt1onn as rneasu~ed. by· a change 1n

se:Mtm concent~at1on tn$Y be misleading; as this change mar 'be 4,ue tce· othez. causes.. :.rh:e ctetermination of cholee~erol

in dhylomicrons in cb;Vl¢ Qf toats :fed with and Wi thaut olive

o$1 and ehole~terol by Bragdon {'1959) described :in Section 18

..m~ht profitably 'be exteniied. to other oils. A t~ao~~ method

might conceivablY' be devised for thla purpose. tl'lhat .amounts

to indirect· pl'oof ot·ttitfettential absorption will b~ f."twn1shed

under r;,. below.o

178.

Lin, Karv1nen and Ivy (1955), working with rate,

at-e responsible for the only report that could be found in

the records of an attempt to ascribe differences of absorption

of' q.iet~ cholesterol to differences of solubility oi' this

sterol in dietary lipids. The following solubilities are

recorded.

% solubility Te~erature % absorption ·Of cholesterol c

Oleic acid 22 38 74 Tallow .'·4.,6 39 59 Corn oil 2.7 38 64 Triolein 2.7 .39 49

Although oleic aoid caused significantly higher absorption

as compared with corn oil, it was not as different as the

.solubilities. For this reason the above investigators did

not consider the solubility of cholesterol in fat an tmportant

factor.

According to Frazer (1955) fatty acids, as distinct

from trlgl;vcerides, are absorbed in the aqUeous phase, through

the portal raute. This mechanism is likelY' to be t•adically

di:f'fel'ent. If this is eo, the in vit~o solubility of

cholesterol in pure oleic acid has little bearing on the

processes at play in capilla~ absorption, and the concentration

in the systemic circulation is unlikely to be directly affected.

IJ.'he simil.arity in behaviour o£ cholesterol and

neutral fat during intestinal absorption impressed Fraze~ (1955)

who suggested that cholesterol might be transported across

~he · inte~:rtinal wall in· solution :tn fat. This view ariae$

out ot .. his pat't~tion by_poth..,sis, acco~aing. tQ whi® . . .

ncomplete h:v<b"o~ais ot gly-cer:tdes to ·:tatty ao.S.ds and· glJree~l ' • ' • • '• • . ,l

t.s not an t>l;J11gatow step fn the process of fat a'bs6~pt1tm.~

.· t!pids d~ ... ncrt ~ve · to be. b.rottght into wat~r•so1·u.ble forrn .

b&t()r~. they. ean ~e ):*fl:znovea. ~~m the $mall inteetinat lumen"i!

· ·Glover and :Morton (1~58) ctti tfcieed lr'toaze:r'1 a ~oncept

ot tra:n$poxst by a:trnple solu.t~on ~s being tco'uneomplicateci

since it does not aecount for the martt~d seleotitrity of th$ . . I

intesttnel tn .abso.r'bing some eterois ana ~ejecting otliere•

*l!heee authol'ls nQ not -euggeat, however, how sueh a seleetfon

mechanism could be visualiaea mo~e easalsr as taltfng piaca

in the aqueous phase •

~b.$ l'e1at1~e eaae of esterification .ot t:holeatero1

w! th ditf'~r~ent fatty acids ot> the i-elative abso:r'ba.blli ty of

the et;rtera h~fil been suggested aa being responsible for the

di:tterent. ~tes ¢!' absorption~ e~s· L:tn et a1. (19,SS). fhis

view cannot easily be dismissed.~. but 1 t is .not supported ba­

:tecent wo~k by Swell anti as,socia:te~ '(1959) who believe that

~h<>leaterol· ie abao,J!bed in the f't'ee state~ This ag.rS.es

with the obaervai:?ions made by a gr-oup of: 1n1r~~tigators led

'by OangtU~y (1959) who .ted ft?~e cholesterol; its acetate and. . - - ' ' .

palnti tat~ ·1n a rrd.nimwn quMt:J.ty of o.1l to ~atsa From 60 to

90 minutes late~, the same .amount ot cho1es'Gerol, p:reaom:tnant1y

in the f!oee $ta.tE~J was :fOttnd 1n all eases in th~ mucosal. dells'~ ·

fl'le eJ.~ination ot cholesterol f':rom the bodsr takes

plaee as unchanged eholesterc)i, its bacteroial transformation

180.

products in the intestine such as coprosterol (coprostanal)

and finally as bile acids.

various authorities have assigned different

tmportance to one or the other groups of compounds as

catabolic end-products. These differences of opinion need

be of no concern here, since emphasis is placed on variations

in total end-products onlY. According to the mechanism

outlined at the beginning of this section, decreased abso~ption

results directly in increased excretion of cholesterol (and

probably its hydrogenation products). This is more or leas

obvious. It also leads to increased liver synthesis, however,

If it is further assumed that the same ratio o:r the total

cholesterol s,vnthesized in the liver is at all times converted

to bile acids, increased excretion of the latter substances

results.

To swmmarize; dietary change from coconut to corn

oil may be accompanied by increased excretion of cholesterol

and its end-products and bile acids, and vice versa. -Extrapolating from this argument, the highest excretion would

be expected with a fat free diet.

E;Per~ental evidence from rats is scanty and

inconclusive. Lin; Karvinen and Ivy (1956) found that

" •••••• tallow slightly decreased the endogenous elimination

of cholesterol, whereas corn oil and triolein definitely

increased it." !n a later study (1957)t these investigators

unfortuna.tely worked with free acids which probably follow a

different absorption path. Wilson and Siperstein (1959a)

injected rate consuming either corn oil ol" lard Vlith cholesterol

181.

-4-c14 and found no dif'.fer.ence in excretion products. No~ did these workers (Wilson and Siperstein, l959b) detect

an1 influence .on the concentration of these substances in

the bile. Since intestinal absorption - the pr-imary regulating

mechanism~ was by-passed, these results are not surprising.

Fo~tunately more data are avai1able from .human

experiments t Clordon, Lewis, Eales and })rock (1957a.b) were ,

the t:trst t() show. that the substitution of hydrogenated coconu.t

oil by Sunflower seed oil in the diet is accompanied by sn

ine.rease 1n the taecal excretion of 'both bile acids and neutral

ste.rols. The average increased excretion in four such expel'"iments

with 100 gm. of :rat .in the diet was 350 mg. daily.- Although

the Li~bermann-Burchard-posi tive sterols measured we·rs not

an accurate reflection of the sterol ex~rretion, this figure

is of the same order as that calculated. from the change

.in ~bsorption of endogenous cholesterol.. in similar eircromstances

(see Section 24 G).

!n a modification,of the ~xpe:rimental conditions the

extra addition of 75 gm. of' sunt'lowe:r ·seed .oil to the CJ.iet of

subjects already consuming the same amount of coconut fat caused

an increased excretion 1rt both bile acids and neutral sterols. ' .

This was accompanied by a. fall in the ee~ eholes'f?erol level •.

These experiments have been inte.rpr>eted a.s de~onstrat1ng the

special prope.:rtie,s of' sunflower seed oil as a promo~er of chol­

esterol catabolism. No suggestion was made, however, as to how

this might happen. A different interpretation is possible.

An explanation has been,suggeated in Section 2.5 t:or

182.

the decrease in serum cholesterol level following the extra

addition of large quantities of !B[ fat. The extra fat,

comparatively free from endogenous cholesterol, once in the

blood stream, dissolves out the cholesterol from serum and

transports it to the liver, until the serum cholesterol drops

to a new equilibrium value. During this transitional period

then, extra cholesterol is transported to the liver \rlthout a

change in the rate of absorption. Extra excretion might

reasonably be supposed to follow. The important point made

here, however, is that extra coconut oil in the diet might

have had an even greater effect. Unfortunately, no control

experiment was conducted. The comparatively low excretion

of sterols and bile acids on a low-tat diet remains unexplained.

Conditions resembling more closely to the steady

equilibrium state under consideration here obtained when

Haust and Beveridge (1958) recorded the following bile acid

excretion in man during the last four d~s of an eight d~ -experiment:

Butter Corn Fat free

{60% of cal.) tt

tt

0.72! 0.36 gm./4 days 1.00 ! 0.47 " 1.11 ! 0.52 "

Although eonfir.ming a comparably higher excretion with a low

solubility oil, the highest excretion was observed on a fat-free

diet :

Increased faecal sterols with corn oil has likewise

been reported by Hellman et a1. (1957) and more recently by

Rosenfeld and Hellman (1959), whereas Antonia {1959) found

more faecal sterols and bile acids with Sunflower seed oil than

with butter in the diet.

tt is c.oncluded that there .is good evidence tor

increased excretion of' total catabolic Emd•prod.ucts with

low, ascompared with high, solubility oils in.the diet. In

addition, one report exists Which recorda maximum exexoetion

on a fat-free diet.

The link between the hepatic synthesis of eholesterol

and its solubility in dietary :rats is .remote:; and perhaps tor

thi$ tteason a most fascinating one, Esta'bliE>hed more firmly,

this link would fUrnish ~othe~ indirect proof of the different­

ial resopt>tion of endogenous cholesterol due ·to different :f'ats.

Effect of die~a~eholeste.ro~

There is a good deal of evidence that dietary ~holest•

erol suppresses the synthesls of thi,s substance in the liver.

'!'he elassical experiment o:f' Gould ana Taylor {1950) With dogs

and rabbits demonstrated that dietary cholesterol suppreae~d

hepatic synthesis to a few pel' cent of the control rate. These

workers used the conversion ot o1~ labelied ac~tate into

cholesterol. as an indea of synthesis in both liver slices and

in vivo experiments. Similar conclusions were arrived at b:V

'1'-omk:lns and Chaikoff' (1952} who worked on rat live%' slices.

av feeding a diet containing $% eholesterr;>l, hepatic synthesis

was almost completely suppressed.

Both Alfin~ Slater et al.. (l9S2) ana. Franz et a1.

(1954) l'eportea .a higher liver ebolesterol concentration on

cholesterol f~d rats than :ln controls. This higher liver

1S1.t..

cholesterol was associated with a lo'W'f)r synthesis o.f this

sterol• Mo:rris et al. (1957) working With l*ats found that

increasing the tlieteey intake of cholestel"ol containing

cholesterol -4-a14 from o.05%·to 2% of the diet, -decreased

the synthetic component of serum cholesterol from 67 - 80

per cent to 10 - 26 per cent. ,

The suppttession of hepatic synthesis by dietar-y

cholesterol can thus be taken as en established fact in some

animals but has yet to be Shown in man., ltepatio cholesterol

synthesis then, is regulated at least in pa!'t by' the rate of

absorption of this sterol from the intestine.· Aceor!Ung

to the solub111t;v hypothesis, high and low solubility oils

(e.g. coconut and com} cause different rates of absol'ption

of dietar,y or endogenous cholesterol. Other things being e~al,

it follows tha.t dietat7 high solubility oils suppress liver

synthesis and vice versa. Support for this vie\v oomes from a

number of sources.

Effect ofh1Sh and low solubi~1ty oiltt.,

over 20 years ago, Eckstein (1938) repo~ted a larger

sterol synthesis in rats consuming corn oil than 1n those on a

comparable diet of coconut ratfl 'this sterol balance study,

howeveP~ gives no account Of a possible interference by

excreted bile acids and its interpretation is therefore open

to criticism.

More recently Avigan and Steinberg (19!58) usibg

~odern tracer techniques studied the incorporation of 1-c14 acetate into the unsaponifiable matter of the liver of rats

consuming e1the:r coconut or corn oil and :found a higher

incorporatto~ with the latter oil,. Similar conclusions

werce arrived at 'by Merril (1958) who reported increased

ineorporati~on of sodium acetate 1-~ 14 into oholesterol.

with dietaey linoleic acid• in spite of its lowering action

on the serum cholesterol.

·· Further supporting evidence. was presented by

.Wood and hti.gicovsk;v' in 1958. These a'1tho~s f$d unsaturated.

oila and fatty acids to rats and ~port ''a stin1Ulating effect"

on the inoo~oration of o14 acetate :in~o cholesterol, both

in. vivo .. and in liver homogenates.. lil®al amounts of .saturated.

material such a.s coconut oil and. lauric' acid. had the oppoai te

.e:f:rect. Only three days • feeding was required to demonstrate

these phenomena. Finally, Wilson and Sipe:rste1n (1959b)

tailed to di.Sti"nguie~ com· from lard as affect::i.ng the :tncorpoi­

ation of acetate into cholesterol. in livez; slices.. Th~ 'Wiha.ppy

choice of lard as an e.xpe1 .. imenta1 fat~ however, 'becomes apparent

on inspection of Fig~ .3, Section 2• Its serum regulating

pl;'ope:rty is not conspicuously diff'erent i'rom that ot corn oil.

Sunrrnarizi!!S thie brief' review; one can say that there

'is substantial experimental evidence that unsaturated oi1s have

a g;t?eater .stim;lllating eff'eet on hepatic cholesterol synthesis

than saturated fats. The parallelism 'between the deprer.u:d.:ng

effect on liver eyntlle.s1.s of dietary cholesterol on the one

hand and the .relatively greater depressing effect of high as

compared with low solubility oils on the ethel', is impressive ..

. tt .is suggested that these two phenomena are 'based ·

186 9

on the aame primacy mechanism, namely the ~ate of intestinal

absorption of cholesterol, both dietary and ertdogenotta.

Another independent link has thus been established between

li'\"el' synthesis and absorption. While not tu:rn1t3hlns a

fonnal proof ot differential absorption as influenced by

ditferent dietary fats, this link provides strong supporting

evidence.

E, Conclusion

*ll:u,re can be little doubt that the picture drawn

here of the metaboliC inter-relationships ot cholesterol

as affected by different dietary tats and the solubility

principle;; is both fragmentary and grosslY over-simplified,

Much of the picture is based on controversial e·vidence~

Even the broad outlines; if true at allt require fu~theF

confirmation. If accepted, however, the folloWing conclusions

ean be drawnt

The solubility hypothesis does not obviously contradict an;v established principles of physiology or cholesterol metabolism. !t ma;v• in factt be used to ~ita' knowledge on the subject and explain appa~ently disconnected phenomena on a common basis.

A brief review of the recent literature has shown

that choleste:ttol-:f'ree dietary fats of d1f'fe~ent composition

have charaeterist:tc e:f'f'eets on the excretion, serum concentration

and liver synthes.1a of cholesterol. An attempt has been made

to demonstrate that these effects are inter-related and

187.

ul timate1y caused by one pr1nlaey regulating mechanism -

the rate of intestinal ·absorption of cholesterol. !h:ts

in tu:rn 1s believed to depend on the presenc(!: :tn the

intestine of endogenous cholesterol and its solubility

in accompany-ins tat,.

18A.

SUMMARY

The effect of different dietary fats on the serum

cholesterol concentration has been discussed. In a aeries

of experiments, conditions have been ol'eated under which

rats can be induced to imitate humans in this respect.

Using the rat as an experimental animal, the hypo­

oholesterolaemic property of dietar,y sunflower seed oil

has been investigated by the chemical and physical fraction­

ation of this o.il. By feeding the saponifiable and unsap­

onifiable matter of the sunflower seed oil, it was found

that the latter fraction was partly responsible for the

serum regulating property of this oil.

This finding was contir.med by altering the composition

of the unsaponifiable matter by the physical fractionation

process of liquid propane segregation. Several fractions of

oils With similar fatty acid compositions but differing in

both quality and quantity of the unsaponifiable matter were

found to have different effects on the serwwn cholesterol level.

In a further series of experiments • the various known

components of the unsaponifiable matter were next fed as pure

compounds, dissolved in coconut f'at. Sitosterol had little

effect, while squalene had a slight cholesterol lowering

effect. Combinations of these with ~-carotene and ~-tocopherol

produced a larger and more significant eff'eot.

189,e , - .

Although th~ unsaponttiable matte~ and acme of its

components were s~own to have serum. lowering p:roperties; it

was not possible to demonstrate that this traction was

responsible .for the action of the oil aa a whole~ From this

it was concluded that it is probably impossible to isolate

h:i.ghly potent sel?tlm cholesterol lowering aub~tances ftoom'this

oil.

In fUrther ·experiments to elucidate the meehan:tsms at

play* the ~peroholesterolaemle properties of ,ben•tl: egg 11pids

were studied and traced to the CholesteX>o1 content of the ;yolk.

When this sterol. was ted alonet however, no incr-ease in serum

Cl\Oleaterol level;OOCUrre(l unless a.dminiaterei in solution . - . . J .

,;:

Attempts to e~l.a:tn the above phenomeb.~ led to.tlie

postulate that cholesterol ts absorbed only when in sol't+tion

1n tat• A b;vpothes1s \Vas neltt proposed aeeordtng to whiCh

the amount of cho1esterol absorbed. is a tu.nction of' 1 ts·

solubility in dietary fat. :rn the absence ot chol.est€)rpl. 1 · . ,, .. ;,

fats would exert their effect on the serum eholesterol ,'t;)y'.

causing the r~abeox-pt:i.on ot endogenous or bf1iaey ehole$·terol.

!o test this hypothesis the solu'b11i ty of cholesterol

in a variety of fats and oils at 37°0 was. determined .1n Vitro.,

and found to correlate With the serum cholesterol. level::·.·

produced when ·these Oils were fed to ratse Thus the high

serum cltolesterol level produced. by coconut fat •. ~ol' exemp1e1 ~·,

was reflected in a C!lomparattve high solu.bil.i ty in this tat.

The differ>ent cholesteroJ..-:raisins pro;pe~>ties ot tats o:r1g1nally

tree from cholesterol were thus explained. The hypocholesterol­

aemic effect of the unsaponitiable matter of sunflower seed

oil was likeWise accounted for.

To teat a prediction from the solubility hypothesis

it was demonstrated. exper1mentall;r·that there is a certain

amount of cholesterol• when fed With a given quantity of tat,

above which no fttPther rise in the serum cholesterol is

observed.. This amount of' cholesterol was shown to be related

to its solubility in accompanying :fat.

The above principles were next applied to the human

sphere. F}$om a single experiment it was obsetb'Ved that dietary

cholesterol markedly raises the serum cholesterol of man

provided it is administered in solution 1n fat. A good.

correlation was- also obtained bet\veen the solubility of:

cholesterol 1n diff'e:t>ent tats and theil:' published quantitative

sel'Wtl cholesterol elevating effects on humans ..

tn a finol chapter it was proposed that the influence

of different dietary fats and oils on the excretion and

hepatic synthesis of cholesterol can likewise be related to

their solVent power f'ott dietary or endogenous cholesterol.

it :ts a pleasure. to record m.v thanks and

apprecia~ion to the many persons who hav_e helped m.e

with this s_tudy.

To Professor J .P\ Brock_; Head of' the Department

of lled1cine, I am indebted t~r creating the conditions

which have made this work_ possible• !U.s gepe~au.s help I

and encouragement at all stages of this project has •. ' • ; - • J

ensuJ>ed that ·any !Sho_rtoom:tngs of this 'research are due

to neither laCk of time nor facilities. For this t ov.te

him much and :r welcome this opportun1ty of thanking him.

Professor H. zwarenstein I thank for the ' .

intere$t he has taken in this work and tol'- his sympath~tic . ' ' ' . ' - , . encouragement. His advice, .especially on the ptws1ologica.l·

aspects of the work, has been invaluable.

From the Council for Scientific and Indu.strie.l

Research I received f'1nancial. SUpport th~ough being on

thei:r staff and also by thei!' generous pztovieion of research

grants.

Dr. B11 S:ronte-Stewart is responsible for arousing 'i.,

ntr interest in this field of .reu;earoh. ! am indebted tohiltt

for innumerable valuable suggestions duFing the man.v stimulating

discussions he1d in his enthusiastic resea:rch group. I was ' ' ' . . '

al.so fortunate to hav& been allowed to make use of the

' '

facilities of the metabolism ward at Groote Schuur Hospital

which he originally organized for lipid research.

Mr. H. de Wit was responsible for the management

of the rat colonies and the cholesterol detenminationa. His

responsive, patient and moat conscientious assistance through­

out this work is gratefUll7 acknowledged.

To the following persons I am also indebted for

stimulating advice or practical participation: Mrs. G.O.Young;

Miss L.P. Pmsl1e, Mr. B. Roberts,. Mr. V.M.Wells, Staff-Nurse

G. Schoonraaa, Dr. M.E.Fewster, Dr. J.Lovelock, Dr. S.R.Lipsky,

Dr. H. Gordon and Professor P.G. Holliman.

Thanks are also due to the management and s taft' of

Marine Oil Refiners of Africa, Ltd. Apart from making their

liquid propane segregation unit available for experimentation,

they have made many donations of special oils and have given

freely of tpeir laborator,y facilities.

Finally, I wish to record my thanks to

Mrs. o.M.Cartwright who pointed out many errors and contributed

constructive criticisms du~ing the typing stage of this thesis.

ooooooo

193.

The e:f'teet ot cUetaey lipid fl"a~ttonf:l on the s&rum ' -oholeate:l-ol ~f th~ rsa~~.

(Abt:rt·~act),. s,. Afr~ M~et. ~-I .i!t as. 1~58. ' . ' ' .

A pl'oposaa mechEU"1iam .tor the serwn cholesterol regul­ating e:r.teet. ot d.ietazw fat a and. oil·s1> . · . . (Abstract)·;,; 9.. Afr. Med~ 3 f • Jl, ·1076, 1959 ..

~he-~t as an exper1m$ntal animal tor the investigation of the etteot ot ·dieta1:1y fats on the serum Ollolesterol le-v~l~. . . (A'bstre.ot). llfP1• )~eet1na (1959). Nutrttioit·soe~ of southem Africa {Prooee8 . .1ngs in prass:J 1960).,

' ) ' .

Vlith Dr.; ft., Go:rdon end PX"ot • .t. F;; BPOCk! ' ' " c ·, •• • •' ' ' '

Serom-cholestsrol levels a.rterconauming eggs with Increased oont\!,tlnt of unsaturoated lip'tds. - "· , taneet., 'tij 244; 1958• · · · · · ·

The eff'eet of 'Vartous dieta.ry factors .on the serwn­eholes.terol level and· on the fec~i1 fat· ciori.tent,& (Abst'raet'). s. Af!r~ -;.~ed.. 3-., .22, 549, 19SS ..

With DXti .• :s.:Bil"onte~stewart and Mr. v.M. wells: Factors responsible tn dietary :f'ats :tc)r the changes

in serum ehblestei~.ol levels in man~ Jtutrttion Soc •. of' Southam Aff\.ica (Proceeli1nga .in pt"esel

With Miss t .. A., Emsliec A' possibl.a m.eehanism of the effect of cU.fteerent dtet~ry

· . fats on the .seP'ilnl Ohol~sterol le'Vel .. 8'!1 Ltp:td. Research (submitted.).. · .

With M~. H. de Wit: A. tn!oro-method ·tor th~ · deterraination. ot toteil serum

cholesterol oonoentl?ation in 942 ml of blood. s~· Afr.. ;r. Lab. ~l.tri•. Med., ( su'bnii tted.)

Other papers in'p~eparatfon

. NOTE: Halt•tone· photographs in th;ts thesis were taken by the author~ ·

REFF...RENCES

Aaes-Jprgensen, E., teppik, E~E~ and He.ves, H~w; (1958) J. NUtrition, 66, 245. -

Abell,

Essential fatty acid deficiency. II. In adult rats.

L•L•t LevY,, Betty B., Brodie, B•B• and Kendall, F.E. (195~) J. Biol. Chem.; 122• 357• A e1mplif1ed.method for the estimation of total choleat..:.. erol in serum and demonstration of its specificity.

Aftergood, L., Deuel, H.J. Jr. and Alfin•Slater, R.B. (1957) J. Nutrition, 62, 129. · -The comparative effects of cottonseed oil and lard·in cholesterol levels in the tissues of rats.

Ahmad, B. (1937) B1ochem. J., ~~ 1195. The fate of carotene after absoJ>i,tion in the animal organism.

Ahrens, E.H. Jr., Hirsch, J., Insull, W.R., Tsaltas, T.T., Bla.matrand, R. and Peterson, M.L• (1957) Lancet, .!• 943. The influence of dietary fate on serum-lipid levels in man.

Ahrens, E.H. Jr., Ineull, w. 1 Hirsch, J., Stoffel; w., Peterson, M.L., Farqunar, J.w., Miller, T. and Thomasson, H.J. (l959a) Lancet, !• 115. · The effect on human se~lipids of a dietary fat, highly unsaturated, but poor in essential fatty acids.

Ahrens, E.H.Jr. (1959b) Lancet, ,!1 315. Letter. SUpply and dietary use of fat.

Alfin-Slatert R.B., Wells, A.F., Aftergood, L. and Deuel, H.J. Jr. · (1954J• Circulation Research, g, 471•

The effect of plant sterols on the cholesterol levels in the rat.

Alfin-Slatert R.B., schotz, H.c., Shimoda, F. and Deuel, H.J. (l952J J. Biol. Chern. 122• 311. Effect of low tat and high tat diets on the synthesis o:t: cholesterol in rats.·

American Oil Chemists' Society (1953). Official and tentative methods. Section CD7/48.

Jr.

.. 195 .. ·

' Antcnis, A·o (1959h . . .

Pepso,nal COITll'l')Unioat:L_oli~t, .

Armstrong• . W~Di 11 'van PilSwri, 3•'~· :Keys,. A~· •. Grand.Ei~ F.;, .A-nderson~ J. T~, ana. .Tob:tan, t~ (1957)

· Prod• soc:~ E:x.ptl·,. Bioi • .Mea.·. 26• .302. · · .A.:lte~at:ton ot serum .cholesterol. bY. a:tataey tats,

' '

AV!gant- :r, and .Steinberg~ ]j. (1958).:. Proc_. Sode Exp., Bl.ol;. and lVfed. 2Z.; 8l4e ..

Ef'f'eets ot saturated and unsaturated fats on ·Cholesterol metabolism in the rate

Bankrott, rt .. {l957h . '

·Int~oduetionto niostatistics ltoe'bet' ·;r... Harpe~, New fot"lt• . ' .

Barenther, A•. (1923}• Ohem•. Umschau11 lQ.1 ll i <>

A: new occurrence ot ce~yl eerotate, ~ ;:- ' t • j • -~ ,

Behe:r., W&T• t B~er* G:n~ ''and Anthony, \W.st.. (1957)'1 Circulation· Res•. !• 202,. · · ·

Best;

· Etteet of dihydrooholestelr'ol .. ana ~ .... sito~terol . on cholesterol athexooaclerosis tn·mbb1ts ..

f 1'- f ' f ~

M.N. 8 1J'I.:tncan, o.a ... Van Loon, E.J,. and Wathen, J.D. (1954)+ Circulation, !9.• 201., towering of serum oholester~l by the admfniatra.tion

. of plant". ste~ol.

M.N. 9 Van LcH.ln,, E.oJilq; Watlten; J-. and Segei"' 11 A;;Je (1954)., Am. :~.. Med~ 16, 601~ · . Oompalt"ison of sennn cholesterol methods (Abst:t>act),

MoN~ and· Duncan, C.H. (l997h Circulation, !€t 861 .. (Abstract >• Factors influencing the hypocholesterol ... emi:¢ '" e'ffect of sitosterol~

Best-·o.,a.~ Lucas, C.Ce 9 Patterson, J.M-. and ltid.outp J.R,. (1956). Cane J .. Biochem. and l?hysiol. ~- 613• Effects of different dietary tats and eholille on be;patlc and serum lipids of rats,

Be,-aridge~ J .MiioR•, Connell~ VIi-F. and MB:.Yer, ·G. A. (1956) Can~ J J/1 Bioehem. and rh.v'siol. J!t, 441• · . Dietaey £actors af.f'eeting the level of' plasma cholesterol in humans: role of f'attJ

196. :Beveridge, .:r.M~_R~q Connell, w.F. and Mayel"., G.A.- (l957a)

Can. J • Biochem. Physiol., .2,i, 257., nteta.ey f.at affecting the level of plasma cholesterol in humans.

Beveridgep J'c~MaR., Connell, W..F., Mayer, G.A. and Haust~ H.A. (19!S7b)" Cireulationt .!2.t~ 49lt ·· · · Further-assessment ot the.role.of' sltosterol inaceount .. 1ng for the plasma depressant action of eorn otl.

BevePidge~., J .,M.R~, Connell, We. F. and Mayer, GoA• (l9.58a) •. C~n. J •. B-t~chem. Pbysiol. P 35, 89,5. · · Plant sterols, degree of unaaturation and h.'vpoeholestel'­olemie aetion o:r·eerta1n fats.

:Beveridge, J'.M.,Re, Connell, W.F., Mayer, G.A., and Haust, HeLe (1958b) .. Fed. Proc • . g 11·, 470.. · :tnfluenee or eXperimental design on assessment of .hyperacho3.esterolem1e activity of' certain vegetable o1.ls ..

Be'V'eri4ge; _ J.M.R,., Oonnell-; W.F., Haust, H~t. an£f Mayer, c,A • . · . {19 . .59a)• ·oan •. J. Biochem. Ph.Vsiol., lZ.t 57:,~- ·

Dieta%7 cholesterol and plasma cholesterol leveis in man.

Be~eritlge, J;.M.R.:, C~nnell 1 W.F~ * Mayer~ GeAj$ a~d H~st» HeLe . · . , ~l959b).-; C~rculation, g,Q.1 970· . · .

~esponse of man to dietary cholester«:>L,

Biggs; M.w; j Friedman, M. and. ·srers, s.o ... (l951h . Pr>o¢• Soc~ ~ptl, Btol. and. Med., 78j ·?41. · .

Intestina1 lyrn:phatic transport of absorbed clJ.oleste:rol.·

iuoo:r, w. R~ ( ~943) B:toehemistey of the Patt~ Acids. Reinhold PUblishing Corp.

· New York, ·

Bowden, S.T. (1945)• The Phase RUle and Phsse Reactions Po io~ Macihillan ·ana:·· co.· ;London. - ·

l3ragd.on; J.H .. (1959). Annit N.Y.Acad. Scf•·' ,U(14)84S:• Chylomi~rons and lipid transpoX"t.-

Bronte-stewal't,-. s •. ~ Keys, A. end Brock, J.F. (l955) Lancet!) 11, 1103. ·-Serum-cholesterol, d1et:t and coronary heart diaeaseei An ~nter-raeial survey in the Cape Peninsula.

Bronte-stewart, :B.; Antonia, A~, Eales, t. 8nd Brock.~ .J,')Fe~ (1956h Lancet, !.1 521Q · Effects of feeding different fats on serum-eholeote~ol level.

197.

Brqnte-ste\va:rt, B., Wells, v.M. and Wilkens, J.A. (196q) To be published.

Byers~ s.o. (1958) Amer., J. Clin. Nutr.-, §., 638. 'l'he origins of plasma cholesterol.

· Channen, H.J • (1926) Biochem. J ,. , .lQ.; 400 .. The biological significance of the unsaponifiable matter of oils .. r. E~ertments with the unsaturated hydrocarbon

sqUalene.

COlman, D.H. and McPhee, A.F. (1956). Amer. J. Clin. Path. 26, 181.. . - .

An ;improved method for determination of total serum cholesterol.

Oo;teman, D.L., Wells, W.W. and Baumann, C.A. (l956)~ · Arch. Biochem. Bip.phys., 6o, 412.

Intestinal sterols. I!. Determination of coprostanol. and certain related sterols.

Coleman,, I. anti Beveridge, J.M.-Rt (1959) Nature. ~' l04l. Effect ot dietary fat and extent of blood sampling on the level of plasma cholesterol in the rat.

' .

Cook, R.P. (1936) Biochem. J., .2Q., 1630.

Cook,

Cook,

Cholesterol feeding and fat metabolism.

R.P. and Thomson, R.O. (1951) . Quart., J. Exptl.. Peysiol., l€.' 61. fhe absorption of f'at and of cholesterol in the rant; guinea pig and rnbb.:tt.

R.P., Edwards, D.c. and Riddell, c. (1956). lUoehem. J.; 62, 225. Cholesterol metabolism. 7• Cholesterol absorption and excretion in man.

cook, R.~. (1958) Cholesterol, B1oohemietr,y and Pstholo~ Aeademio Press Inc.' New York. ·

Cru:t.ckshankt E.M. (19.34) Biochem. J., 28; 965 .. -Studies in fat metabo11.sm in the fowl.

Danielsson, H. and Qustafsson, B. (1959) Arch. B1oohem. B1ophys., ~~ 482. On serum-cholesterol levels and neutral fecal sterols in germ-free rats. Bile acids and steroids.

I

198t~

:f.liller, E,Rc. 1 WOods, Bit~t and. Barvey~ O.A~ (19!$7) Oil-CU.letion1 l§.; !)05.. . . Ef'feot ot degr:ee of unsatu:retion of veg¢tabl:&. l'f.:pid upon the ·plasma and hepatic cholesterol content in the rat. ·

Eckstetn, · R. · (1938) (f • n1.o1.1· · .qhetn~ • 122• 99• St~rol metabolism in ;voung. white ,rats; effee·t ot saponifiable lipids.ana.~egree of l1tlsaturat1on ot lipids on sterol metabolism ·· ot whtte ratE! ..

:rf~qUhe.r, JtW~e and Sokolow., M• (l9S7) OlrculatiOiltt 1£~ 494<i (Abstraot) A eo¥tpar~eon of ~be effecf.s of ~-sitosterol and Srafflowel:' oil on sertm1 lipids of hi.mlans* . .

i'a~quha:t:~, · J .s w. and Sokolow,· M •.. (l-958} ··a:trcula.tion.) rz., 890 Response of seru.m lipi<t$ and 11pop~oteins ot man to ~·sitosterol. :arid $afflowe~ o:i.l~t ,

:rewster1 M.E. and Y()ung, a.o. (1959) P~rsonal conlml.tnication Egg tzsig:t;vceride:J corrtposition · ·

Fitel.son;, J• (1943) .J<JU.t'e Asaoc. Offic., Agrio~ Chenh, .26t 506. The oceu~renee of squalene in natu~al f~ts

Forbes, J •. Ct 1 Watlington• 0.0<4.; Vfe.sse%l11an, A,J.~ Camp, ;P . .tP•·t · rttcke:rt w. T:t and PErttel"'eon. o •. M. (l957) ·. ·

P~oc~ So$f exp. Biol,· Med•• 2!1t .S4f5., . Effect of high $peed oetitri:fusat1ol1 on diet~ibutton · ·

· · ot serum cb,olesteroi. · · · · ·

Prant~~ a:.n,, schneider, t!. s ... and. JU.nkelm.an, ·~'. T. (19S4) lf. .B1o1, Ohern, ~ 206, 465"'. . , . . . · sttpp~ss:t.on ot Hepatic cholesterol llt3"htheats in the rat b3' 4ho1eate~ol feeaing. ·

. .

'3:0a~er.9 s.c •. (1,955) Nature, .ua• 491. M&chan)ism·ot: intestinal absovption o~ fat.,.

' • ' I

Fttnch, J.-P.- i N:teisent 1£•. and· Dam, H.o. (1960) · . Brit~ J. Nutr., 14, 1. . . · · · . · ·

'Jnle eff.ect of' varic11:s ·tii.etaey fatst ·~epe~tally butter and aome margarines on blood cholElsterol tn ratt;l ..

FU'ftist; v" and twig~_. D, (1954). aoaria.. lf~:. cltn. tab• Invest., §.. Go• . ~ ·. . . . Evaluation or an ~det:to acid •·su,1phu~ic acta. method foJ:> (J.'llalttattve d.et~nntnation of chole!1terol ·ttn se:rum

199.

Ganguly, J., ll'ishnanrurt~, s. and Mahadevan, s. (1959). Biochem. J., 71 1 755. The transport of c~otenoids, 'V'itar,nin A and cholesterol across the intestines of rats and chickens.

Glover, J. and Morton, R.A. (1958). Brit. med. Bull., lJ!; 226. The absorption and metabolism of steroids.

Gordon, H. (1957) Personal corpmunicat1on.

Go·rdon; H., LeWis, 13., Eales, L· and Brock, J.F. (1957a) ·Nature, 180, 923. Effect of different dieta~ fats on the fecal end­products of cholesterol metabolism,

GOrdon; H., Lewis• B., Eales, t. and BroCk, J.F. (l957b) Lancet, ii; 1299. Dietary fat and Cholesterol metabolism. Fecal elimination of bile acids and other- lipids.

Gordon, H., Wilkens,, J. and Brock, J.F. {1958).; Lancet, 11• 244~ Seram-cholesterol levels after oon~1ng eggs with increased content of unsaturated lipids.

Gordon, H. (1958) , Disser~ation~ Un1ve:rsity of Cape '!ovm. The :regnlation of the serum cholesterol level in man.

Gordon- B. and !rock, J.F. (1958). s.. Afr • Ked. J" , J.g,, 397. studies on the regulation of the se:rum.-cholesterol level in man,

Gordon, a. (1959), . Postgrad. Med. J.; J2, 186. The regulation of the human serum-cholesterol level.

Gordon, s., Stolzenberg, J. and Ceklenick, W.P. (1959} Am. J. Physiol. , !21.1 671. . Effects of cholestei'ol and -sitosterol in the·gerbil.

Gould, R.G. and Taylor; c. B. (1950). Fed. Pt-oc., ,2:, 179. Effect of dietar.1 cholesterol on hepatic Cholesterol synthesis.

Gould, R.G. (1955) Trans. N.Y. Acad. Sci. Ser. II. Vol. 18, 129. -Absorbability of beta-sitosterol.

-~

Gould,

200.

R.G., Jones, R.J. and Wissler, R.w. (1959) Circulation, gg, 967. Lability of cholesterol in human atherosclerotic plaques.

Grande, F.G., Anderson, J.T. and Keys, A. (1958) Proc. Soc. Exp. Biol. and Med., ~. 436. serum cholesterol in man and the unsaponifiable fraction in corn oil in the diet.

Greenberg, s.u., Calbert, C.E., savage, E.E. and Deuel, H.J.Jr. (1950) J. Nutrition, ~' 473.

Groen,

The effect of fat level of the diet 1n general nutrition. VI. The interrelation of linoleate and linolenate in supplying the essential fatty acid requirements in the rat.

J., T~1ong, B.K., Kamm. ingo, C.E. and Willebrands, A.F. (1952) Voeding, ll• 556. · The influence of nutrition, individuality and some other factors including various forms of stress, on the se~ cholesterol.

Grunbaum, B.J., Ge~r,v, J.R., Grande, F.,Anderson, J.T. and Glick, D. (1957). Proc. Soc. Exp. Biol. and Med. ~. 613. Effect of dietar.y lipid on rat se~ and liver cholesterol and tissue mast cells.

Qubner, R. and Ungerleider, H.E. (1949) Amer. J. Med., 6, 6o. -

Heust,

Arteriosclerosis. A statement of the problem.

H.L. and BeVeridge, J.~1.R. (1958) Arch, niochem. and Biophys., ~' 367. Effect of var.ying type and quantity of dietacy fat on the fecal excretion of bile acids in humans subsisting on formula diets.

Hegsted, D.JJ., Got sis, A., stare, F.J. and worcester, J. (1959) Am. J. Cl1n. NUtr., z, 5. Interrelations between kind and amount of dieto.ry fnt and dietary cholesterol in experimental hypercholester­olemia.

Hellman, L., Rosenfeld, R.s., Insull, W. and Ahrens, E.H. (!957) J. Clin. Invest., ~~ 898. Intestinal excretion of cholesterol: a mechanism for regulation o~ plasma levels.

201 ..

Herb • s .• F\; and Riemenschneider., R. w. { 195.3) .Anal~· Chem: •• , .2!$, .. ·952,. ·

• ., < • ,,

A'spectr6photometric method for det~~ining poly-u.n·saturated· acids, · . . · . . ·

Her~.ande~~~ M.H., .Pet~:r.sont .n~w~ 9 qhaikoff, t~Le and. · .Dau1n~n• W.,G, {1953}. .. . · · · . . . 'P~oc• ·soc~ ·~xper• Biol.t and ·Med• .~ .B:}i :49,8.· ,

· Abso:rption .ot Cholesterol 401'4 in ·i'ate fed mixed· soybean st~rols and p ~sitostero~. . " · ·

Herrmann,,' R.G., (1959) ~cit-cula.'tion Res. 7·, 2'24 ' ' '~

· Eff'eot of t~U:rine, g:l.yc~ne,· B~ fjitci~t.er~l on a·erum. and tis.sue cholesterol 1n rdt and rabl)i t ...

Hilditch,, T. P~ · and Barker·,, c~ (1950) . . J.Oil and·Co1our Oheniists• Assoo.1i J.lJ 6"

. . ·The component glycerides of drying' oils 1. Linoleic rieh oils. ·

Hilditch~ T .• P. (1956) The Chem&cel . Constitution of 1Natura1~ Fat§ Chapman and Hii!l :P 'London•

I '

Hil~reth, E~Dl\t> t Mellinkotf' ~· s.it~'! Blair, I G~ W~t and Hildreth, D.M. ·(1951). Circu.lation .J.; 64lo · The ef'f'ect ot vegeta.bl.e ... tat tnges t.ion on human serum cholesterol concentration.

tiau~ Joll•. and Chow, e. F. (1957) Fed .• Prooe 16, 63, (abstract) .• Vitamin B12 defid1ency and eypereholeate:rolemieo

Jonesj R~J., -Reiss, o.K., Huffman, s. (1956) ~~be• Soc. Expo Sioli, and Med. • .2.2,1 88.., Oorn oil and hypercholesterolemic response in the eholesterol .... f'ed chick.

Xa:Mr1nen, E.; Lin, '1\M. f4"1d IV'Yt .f\.oCo. (1957) Jt; Applie~ .Phya~olo !!. 143, 57~-

Capac! ty of' l\uman intestine to absorb e~ogenOl1S cholesterol.

l<eys'# Air;. Mickelsen~ Ot.>, Miller, E. V.O. and Chapman;; c-B• (1950) Science, 112; 79& The relation !ln men between cholesterol levels in the diet and in the blood•

Keys• A. (1952)... Qir.culat,:ton, i~ ;1l!h Hwnan atherosclerosis and. the diet.

202 ...

Keys; A.n.·.Andexoeon., J.T •. t.Mlck. ·else·n· o ••.. Ad.elso. n, s .. F. and Fidanza,, F.; (l956J• J~ Nutrition 2,2.; 39 .. Diet and se.rum ehole.sterol in man. taCk ot ef£e¢t ot 'dietaey ·eh()lest~rol. . . · · ·

~ ~ . ~ . .,

1\eys• Ae 1 Andersonil· J.T. and.' Gx-andej' F.e (l957). · · Laneet, 11,; 959 .. · . .

,.~ ' .

. . Ptoet11ciiion of serum-cholesterol responses of man to changes an· fat'S in .:the diet•

' . '

A,;~ AndeX'son, J.T 11 and Grande, F.: (1958} . . . P~oe~ Soc, ~E:u,.lrnt Blol-o and M¢th .~ 2fb 3S7<l Effect ()n aer'al11 cholesterol in man of mono•ene fatty acid (Qleic a~id) in ttte (liet~ .

Keys, A•* Andei'son,.toT~ .. and.Gran.(l~;·F. (1959) · &nert~ :t" clin& Nu.tr~ z, · (4), 444,. · ·serum eholesterol response in man to ·oral ingestion ·of ~rachidonic acid• · ' ! •

. , ; , • . ; • i < ! I ~ If i

Xinsell,.· · t .• w~. i. P. art.f'idg. e, J .• jj. B¢!1 .. 1ns~ L• Ai,. M.as ... en, s .•. and . · ·. Mich1le s .• G. n~ (19.52}•, J4 clin.t Endpc~. and Metab.

' 12 ... 909. ,.,.....,,~, ,, I. Dietary modifi eat. :ion of · serum ·cholesterol and phospholtpi·d· levels-. · ·

Xinaellj. L~W• and., $1n.f4a1,r,· HeMo (1957) . Lancet.- i.o 883•

.. t ... ...,.,.., .. .. 1.' •• ~ t

]!tats and Disease ·

lUnaell·,·. L.w., ·mteh···· ·aels .. , ,(ijn.-·!.1··· ~. rtek~:Vt n .. w.· ·ana Splitte·~,. s. (1958) Lancet, .!i S.,j1..1.41 · •

· Es$ential fatty acidSt lipid metabolism and. a.ther.osclerosiSit· .

·.:tans<;\ont.. R•O; .•... a.nd Bl·o·ch~.·tr •. (19. 53)' J• Biol.: Chem~ • soo,. 135~ ·. · .

., ' ' ·~, •. , ,? ; -.f• ..

The utili2atton ·of squalene in the bio$,Uthests ot cholea~erol.." ·.

tange~ w., . (1!?50). 11" Am, Oil Ohem~sts·• Soc•, 27, 414. Ohole~tero1, phytot:rterol~ and tocopherol content of food ·products and animal tissues. ·

t.r.f.t~. Karvinen;· m. and JVY-~ A.c. (19,$5) .Anu J., Ph..Vs1o1li • .· l8J,, 815. Relation. ot dietaey ·.tat . to the abso~ptton ~d elimination .of axogenoua ann ~ndogenou.s.eholesterol.

T.M. b JCarvinen, E.~ t'\1'3t, A. C., (1956h. Am._ 3.-. Ptwstol. g 182~ 17,. .· . Effect of .~ertain fatty acida on excretion of end.ogenO'!ls oholestetrol. ·

203.

Matt1kow, it~ {194S), (1950). J. Am. 011 Chemists• Soc. ~. 200: ibid, 21; 11 .. Ret~ning fatty oils with liqnid propane.

Mayer.,. G. A., Connell, W.F., De Wolfe, Margaret S., and Beveridge* J.M.R. (1954). J., Olin. NUtr., _a, 316. · Diet and plasma cholesterol leve:Ls~

McBuire, J.s. and Lipsky, s.R. {1955) J. Olin. Invest •.. ; ..2!!• 704. Th~ effects of squalene on the inco~oration of acetate into plasma cholesterol in man.

Merrill, J.M. (1958). Fed. P:roc., ll• 111. Effect of linoleic acid on incorpo~ation of radiocarbon into cholesterol. -. .

Mertens, Bl1sabeth, Albers, c. (1953). Happe-se;rl. z. Peys1ol. Ohern. ~. 244. Uber e1ne Mod1f1kat1on der Cholesterinbestimmung tm Blut-·serum nach Schonheimer und Sperey.

Messinger. W.J.t Porosowska, T. and Steele, J,M. (1950). Arch! Int, Med., 86, 189. -Effect of feeding egg yolk nnd cholesterol on eerttm cholesterol levels.

Morris, M.D., Ohaikoff, I.L., Felts. ;r.M,, Abrsham, s. and Fansah,. N.D. (1957). J. B1ol. Chem., 224, 1039. The origin of serum cholesterol in the rat. Diet versus s~nthe$is.

Nath,. N. • W.iener,. R. 1 Harper, A. E. and Elveh3em; C. A. (1959) .. J. Nutr., §!, 2~9. Diet and cholesteremia. I. Development of a diet for the study of nutritional factors affecting cholesterolemia in the rat. . .

Nath, N• and Harpet':t A.E. (1959). Am. J. Physiol., l9Zll 102. Ettect of ~ .... sitosterol on serum cholesterol concentration in the rat. ·

Oke~- R. and. Stewart, D. (1933). J. Biol4> Ohern., 2,2, 717~ t>iet and blood cholesterol in normal women.

Okey, R,. and LYman, M.M. (1957). J. Nu.tr. 61, 523., Dietary fat and cholesterol metabolism.

o.key.· ·, lfu.. 'th, LelYfilanli Nt.M~ Sa:toris, Anne '<f•, E1nse1e Betty end Hatn, w. (1959 J · · . · · · · · ·

:fl!etiabolism., a J 2410 ' ' .' ,4111111111) • ' '

Dietal"Y fat· and <::holes terol metaboliSm• .. i!lff'ecta. of unsaturat:ton of' dietat-y f'a.ts on liver and sert1m lipide, '.· ~ ;, " ~ .

Olson, R~E·' ~ablone~i I J ~tRIIi ana ·Talo~, E;, (1957} Fedt Pl'(lCit , !it 395,. .. Eff'eot of dietary fat, p:i.'iote1n and choline upon serum .lipic}s $l'ld :L~poproteins of the .~at. · ·

. '

Passino, H:.J'. (1'949)... Xnd,. Eng,. Chem., !,tl, 280~ !he SOLEXOL ·process ..

Pearson, s·~ Stem, s. and. McGavaek• 'l'·t.H,· (195,l) . · Amal,• · ~emii'; 22;: 81;3o ·. · ~ ·. .

A :rapid aeeu.rate method tor the determination of total cboles%erol in s~rum• ·

Peterson, l>~,W~ (1951). ~roc.:e.' soo• E;gpe~ •. D1ol• and Med.~, Z~~ 143~ :$ff'ect of soybean sterols· 1n the diet on. plasn1a and l.iver choleetezool in ehieka. ·

?eterson.? n.w,- t Niphol~ 1 S,:•Wj 'Jr.,; and Shneou:r, E;eA6 {1952) J~ Nutriti<m., !fl• 57• ·. $t)tne relationships among diet~y ste:rolst plasma and 11'\l'e~ chQl·e$tero1 levels atld athsrosoleJ>oaia in chioks. . .

P:ihl; ~A- (l~S5) Acta ~hy~1a,1., scand•., J!u ,18:;. The E!ff'ect of dietary :tat on the intestinal cholesterol abito:rpt1on and on th61 cholesterol. metabolism tn the ltver of zaats6 . · · .

POllak, o.J·. (l9.53a) Oil"culation I~ 696. Successful. prevention ot experimental b.;vp'e:rcholesterern:la ruid cholesterol atherosel.f$t'OS:1S in the 1'-abbit.

Poll.akt Oc.J• (195.;b) . Circttla_tion,. !• 702. Redu¢t1on of blood Cholesterol in man~

PollaltJi o.J. (1957) Ci:r-oulation M.t 494. Rabb1ta on egg diets.

Politman~ ·o.w.o, La~, E~Y· and -13~o, D~ (1956)"" · Pro.c... Soe~ Exp~ Biol. and Med•.f 2!• 321 •..

Eftect of dietary oarbohyd.~ate on expet-imerttally induced b,yp~:rcholesterolemia and bNPerbetalipoprote1nemia ~ ~ats.,

205 ..

Rhodes, D.N. and Lea, C.H. (1957) Biochern. J:•, .~ (3), 5~6. On the composition of hen's egg phospholipids.

Rosenfeld, R.s. and ltellman, t. (1959) Circulation; gg,, 968. . Fece:i: steroid analysd.s: in man

Rosenman, R.ll. ,~ Beyers, S~O. t. Friedman, M. (l954a). C1x-culat1on Res. _g, loO.

Effect of soybean sterols on the absorption of cholesterol by the rat.,

Rosenman, R.H., Beyers, s.o_. and· Friedznan, M. (l954b).

Sachs,

Circulation Res. 21 4.5• -rhe effect of dihydrocholeeterol on the absoPption of·cholesterol by the rat.

B.A., Danielson, E •. and Sperber, R. (1959) Cir<mletion, 20, 762. ttYPocholesterolemie eff'eqts of·N • (1: -methltl•21 3-d1-p-chlorophenyl propyl) • male runic acid in hypeP!ipemie and notmol1pemic man.

SChettler, G~ (1948) B1ocham. z. J121 349. Studien uber den Cbolester1nstoftwechsel der Mans. Die Beeinflussung des Blut- ttnd organcholeste:rins durch verschiedene Fette und Ole.

Sesk1nd, C .. R, , Schroeder; M.T., Rasmussen. R • .A. and Wissler, R.w. (1959)~ . Proc. Soc. Exp. Biol. and l~ed., 100., 631.

' ............. Serum lipid levels in l'ats fed vegetable oils w1 th and. w1 thout cholest.erol.

Shantz, E.M. (1949). Ann. Rev. l!ioehem.:; 1{!, 410; Unpublished data. cited. b~ P.L. Harris.

Shapiro, s.L. and Freedman, L.. run. J. Plcy'stol., 1 181, 441. Effect.of' essential unsaturated fatty acids and methionine on hypercholesteremia.

Sinclair, H. (1958). Amer. J. 011n• Nutr., §,- 584• Hiawatha t s Lipid .•

Sipettstein, M.D., Nichols, c.w. and Cba1koff, I.L. (195.3) Ci~cu.la1;ion, 1• 37 • · · Prevention of plasma cholesterol elevation 1n the cholesterol•fed bird by the administration of d:.lhydrocholesterol,

206.

Sm1t, Z.M. and Vieym.eyer, A.s. (1957)• S.A.. :Ji of Lab• and Olin. Medt., lt 321• A oompa:rative study of two methods f'or the determination or serum cholesterol.

Steiner, A, Varsosj A. and Samuel, P. (1959). Qit-cmlation Res. z, 448. Effect ot saturated end unsaturated fats on the eoncentrat:ton of serum cholesterol and ·experimental atherosclerosis~

Stubbs, A.L., (1951) · . , .· . '.rbe SoutH African Industrial Chemist. Januat"Y !t 1951.

'The propane segregation of glycezoide oils.

Swell, L. Flick, D.F. Pield~t· H• and f~ead\vel.l, C.,R. (1955) Am. J. Physiol., 180, 124. . . -. '

Role of fat and .fatt:r.acid in absorption of dietary cholesterol .. · · ·

swell, L~, Trout. E .. c., Hopper, R., :Field, H• and: Treadwell~ o. R. ( 19.59) :~o Anth N.Y. A cad. Set, • .zg,~ 813. 'he meehanism of cholesterol absorption.

Thorbjarna.rson. T. and Drummond; J.c, (1935). Analystj §.2., 23. Ooeu::rrence of an unsaturated bydrocax-bon in Olive oil.

Tomkins, G~tRt and Ohaikotf', I.L. (1952) J. D:tol. (:hem., 1961 569. Cholesterol synthesis by liver. 1. Influence of tasting and. of diet~

'i'sujimoto• m. (1920). J. tnd, Eng .. Ohem., 121 63, Sq\lalene; a highly unsaturated h.Vdrocarbon in shark-liver oil.

Turner, T.J. and Eales, L. (1957) . · Scand• J,. Olin. and Lab,. lnveate . .21 No• 2. QUantitative detenntnation ·of cholesterol in serum W1th P• toluene snlphonic acid.

Weeks, G.,G., (1957) Proc.- N'tltre Soc,, 16t 66. 1he assessment of' the diets of' laboratory animals,

Vleitzel, G .. , Schon, H., ae:y, F. and 'Buddecke, E., (1956) HOppe-Se:Vler Zei tsahr• Phys. Ch~• 1 ~. 247,. Fett1osl1che Vitrunine und Athe:roskleros&

207.

Wilkens, J.A. {1958). s •. Afr. Med. J., Jl• 85. The effect of dietary lipid fractions on the serum cholesterol of the rat.

Wilkens, J.A. (1959). s. Afr. Med. J., .:u_, 1076. A proposed mechanism for the serum cholesterol regulating effect ot dietar.y fats and oils.

Williams, G.E.o. and Thomas, G. (1957). Laneet, i• 428 .. Sunflower-seed oil and serum-lipids.

Wilson, J.D .• and Siperste1n, M.D. (1959a). Am. J. Phys1ol. , 196, 596. Effect ot saturated and unsaturated fats on f~~al excl'etion of end p:roduota of cholesterol -4-C 4 metabolism in the rat.

Wilson, J.D. and Siperete1~·0(1959b). Am. J. Physiol., 12§, 599. Effect of saturated and unsaturated fats on hepatic s.rnthesis and biliary excretion of cholesterol in the rat.

Wood, J.D. and M1g1ooveky, B.B. (1958}. can. J. B1ochem. and Physiol., 2£, 433·

Zak,

The effect of dietary oils and fatty acids on cholesterol metabolism in the rat.

B., Dickenman, R.c.t White, E.G •• Burnett, H. and Cherney, P.J. (1954J• Am. J. Clin. Path.,~~ 1307. Rapid estimation of free and total cholesterol.

Zlatkis, A., Zak, B. and Boyle, P .• J. (1953). J. Lab. and Olin. Med., ~, 486. A new method for the direct determination of serum choleste%'01.

SUMMARY . OF . THESIS

entitled

THE EFFECT OF. DIETPRY . LIPIDS

ON TnE

SERUM O»OLESTEROL CONCENTRATION OF THE RAT

presented fot' the Degree of

DOCTOR OF PHILOSOPHY

1n the

l'a.culty .o.f Science University of Cape Town

J.A. WILKENS, B.So. (Hons.)

June, 1960

SUMMARY

The effect ot different dietary fats on the serum

cholesterol concentration bas been discussed. In a series

of experiments. conditions have been created under which

rats can be induced to imitate humans 1n this respect.

Using the rat as an experimental animal; the b.ypo­

cholesterolaem1c propevt:v- of dietary sunflower .seed oil has

been investigated by the chemical and physical fractionation

of this o11. By feeding the saponifiable and unsaponif.iable

matter of the suntlower seed oil, it was found that the

latter fraction was partly responsible tor the serum regulating

'property of this oil.

This finding vJae confirmed by a1 tering the eomposi tion

of the unsapon1fiable ~atter by the physical fractionation

process ·o:r liquid propane segttegation. several fractions of

oils with similar fatty ac1d.compoait1o:ns but dif't'ering in

both quality and quantity of' the unsspon:l.fiab.le matter wet>e '

:found to have different effects on the serum chol~sterol level.

In a £u.~ther se~ies of' experiments, the various known

components of t.he unsapon1f'1~ble matter were next f'ed as pure

compounds. dissolved.· in coconut fat.~ Sitosterol had little

effect, while squalene had a slight cholesterol lowe~ing

effect. Combinations of th~ee with ~-carotene and \)~..-tocopherol

produced a larger ana. more stgnificant ~ff'eet~

Although the unsaponi:f'±able matter and some of its

components we:re shown to ha'Ve aerwn lowex-ing propel'ties~ it ·. .

·was not possible to demonstrate that this fr'~ction was

respo.nsil?le _ fo:r the action of the oil as a whole., trom this

it was conclud~d that it 1s pl'obably 1m;possible to isolate

highly potent serum cholesterol lov.re!ling substances from this

oil.

. t:n 1\trthet" experiments to elucidate the mechanisms at

play., the h;9'percholestePolaem1c pztopatat1es of hen* s egg 1ipids . .

were. etud:ted and traced to the cholesterol content of the yol,k • . ·. .. "

When this .sterol was fed alone.:, however, no increase in serum.

cholesterol level oecur~ed unless administexoed in solution

in fat.

.Attempts to explain the above phenomena led to the

postulate that cholesterol is ab.eorbed only when in solution . .

in t'at. A hypothesis was next propoa.ed according to which

the amount of: choleste:t'ol. abso~bed is· a tunction of' its

solUb·ility. in dietary fa.t,. · l'n the absence of cholesterol~

rats would. exert their effect on the serum cholee tet~ol by

·causing. the re .... absorption of endogenous ol! b1lia~y cholesterol. . . 'ro teet ,this hypothesis the soll;tbility ot: cholesterol,

in a variety of fats and oils at :57°c was de·ierm1ned 1n v!tPo, . .

and found to correlate with the serurn cholesterol- level p:t"oduced

when.these oils wer>$ fed to rats. Thus the.high se~ cholesterol •

comparative high solubility in this fat. The'different

choleeterol-~aiaing properties of tats originally free

from cholesterol were thus explained. The bypocholesterol•

aemic effect of the unsaponifiable matter of sunflower seed

oil was likewise accounted for.

?o test a prediction from the solubility hypothesis

it was demonstrated experimentally that there is a certain

amount of cholesterol. when ted with a given quantity of fat,

above wh1dh no further rise in the aePUm cholesterol is

observed. This amount of cholesterol was shown to be related

to its solubility in accompanying fat.

The above principles were next applied to the human

sphere. From a single experiment it was observed that dietary

cholesterol markedly raises the serum cholesterol of man

provided it is administered in solution. in fat. A good

correl~t1on was also obtained between the solubility of

cholesterol in different fats and their publif?hed quantitative

serum cholesterol elevating effects on humans.

In a final chapter it was proposed that the influence

of different dietar.y fats and oils on the excretion and

hepatic synthesis of cholesterol can likew:i.se be related to

their solvent power for dietar-y or endogenous cholesterol.