FISHERIES RESEARCH BOARD OF CANADA

Translation Series No. 2669

Biological studies on the nutritional value of edib_le,fats.Part III. Influènce of selected.fats on themorphological changes in various organs

by Swiatoslaw Ziemlanski, Teresa Opuszynska,and Stefan Krus

original title: Badania biologiczne wartosci zywieniowej

tluszczow jadalnych. Czesc-III. Wplyw wybranych

tluszczow na zmiany morfologiczne roznych narzadow

From: Roczniki Panstwowego Zakladu Hygieny (Annals of the

State Hygiene Institute), (6) . 685-694, 1972.

Translated by the Translation Bureau.(^.MJK)Multilingual Services Division

Departme nt of the Secretary of State of ,Canada

Department of-the Environment.Fisheries Research Board of Canada

Halifax Laboratorÿ'

Halifax, N. •S .

197.3

16 pages typescript

•

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L'ORI GINAL

685-694 YEAR

ANNÉE

1972

ISSUE NO. NUMÉRO

6

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16

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Swiatoslaw Ziemlanski, Teresa Opuszynska, Stefan Krus TITLE IN ENGLISH — TITRE ANGLAIS

Biological Studies on the nutritional value of edible fats. Part III. Influence of selected fats on the morphological changes in various organs.

TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTERES ROMAINS)

Badania biologiczne wartosci zywieniowej tluszczow jadalnych. Czesc III. Wplyw wybranych tluszczow na zmiany morfologiczne roznych narzadow.

REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS.

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Roczniki Panstwowego Zakladu Hygieny

REFERENCE IN ENGLISH — RÉFÉRENCE EN ANGLAIS

Annals of the State Hygiene Institute

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143913 Polish MJK 'JUL .2 3 1973

Roczniki Panstwowego Zakladu Hygieny ga l 1972, Nr.6. pp. 685-69+ (Annals of the State Hygiene Institute)

Swiatoslaw 2iemlanski, Teresa Opuszynska, Stefan Krus

Btolongal studies on the nutritional value of edible fats. Part III. Influence of selected fats on the mornhologi_n_e

chanm§ in various gnarans.

From the Institute of physiology and biochemIstry of nutrition in Warsaw. Manager: Assist. Prof. S. Ziemlanskl M.D.

From the DeDartment of pathological anatomy of the Institute of Biostrucure, Academy of Medicine in Warsaw. Manager e Assist , Prof. St. Krus MD.

Authors , English abstract

•

0 In 300 Wistar rats in long-term experiments (7 and 12 months) the effects ofe,, ;

MB, MC and MD) of different composition were studied (Table I). The conteiit .c*

' of erucic acid in these margarines ranged from 18% (MA) to 33 010 (MB), in liquid'

rapeseed oil it was 47.6% and in „oma" fat it was 37.1%. The animais received À.

ad libitum experimental diets containing 15% by weight of one of these fats. One

group of rats was kept on granulated laboratory choi.v „Bacutil" containing only

7% kcal of fat. Gross and histological examinations of the liver, kidneys, spleen, adrenals,

testicles, pancreas, myocardium and small intestine i.vere carried out. The degree

of fatty degeneration of these organs i.vas studied, staining specimens with oil red

0, fat red 7B, and sudan III. The degree of fatty degeneration of the liver was

determined using Thaler's staging system. No pathological changes were found

• in the organs examined, with the exception of the heart. In the myocardium of

;

animais receiving liquid or hardened rapeseed oil and experimental margarines with higher contents of erucic acid in diet focal infiltrations with histiocytes and fibrosis were found. These changes increased in intensity after 12 months of the

experiment (Table IV), e.g. in the group receiving liquid rapessed oil after 7 months

; on diet in 8 of 9 rats (88.9%) cardiac changes were found, and after 12 months ,

changes developed in 17 of 19 (89.5%) rats in this group Such changes are an evidence of microfocal myocardial necrosis. •

• •.•••2 305-200-10-31 *Translatorts note: the number in the right margin is the

page number of the original text.

0 A . e 0

t;•4; _ , rapeseed oil — liquid as well as hardened (orna) and experimental 'margarines (Mig',

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In long term experiments with rats of the variety

Wistar, investigations were made into the effect on the

occurrence of morphological changes in various organs, produced

by applying liquid and solidified rape oil and experimental

margarines of different raw material composition and different

contents of erucic acid. Occurrence of focal histiocytic

infiltrations in the heart muscle and its focal fibroses

was established.

In the first and second part of this work (1,2),

general and methodical principles of the investigations

conducted-and the results of some physiological-biochemical

and histo-chemical experiments, as well as changes of fractions

of fats in the blood serum and the content of fatty acids and

their trans-isomers in the lipids of the tissue of selected

organs were presented. The purpose of this third part of the

paper is to give the results of macroscopic and microscopic

anatomo-pathological investigations of various •organs , and

tissues of animals to which rape seed oil liquid or hydrogenated

(fat "Orna") and also experimental margarines containing

various quantities of rape seed oil were administered_in their

diet.

As follows from the results of investigations

by many authors (3 1 4,5 2 6,7 1 8,9,10,11,123,1 )+,15,16,17,18),

rape seed oil administered to various experimental animals in

their diet brings about a number of functional and structural

changes in determined organs, in particular_in the heart muscle.

3

Investigations of Roine et al. of 1960 (6),

confirmed 7 years later by Rocguelin and Cluzan (8) and

subsequently by Abdel:Latif and Vles (.7.0, 11l 12) have shown

that administering major quantities of rape seed oi1, to

animals in the diet causes pathological changes in the heart

muscle. These changes are characterized by the occurrence

of microfocal histiocytic infiltrations and fibroses.

Disorders of this kind are evidence of the forming of micro-

focal necrosis of the heart muscle.

The changes described are preceded by a fatty 686

degeneration of the heart muscle. The intensity of the fatty

degeneration is particularly great in very young rats. Fatty

degeneration of the heart was found also in ducklings, guinea

pigs, harnlsters and rabbits (10, 11, 12).

Other anatomical disorders due to consumption

of larger quantities of rape seed oil in the diet are: transient

fatty degeneration of skeleton muscles in young rats, occurrence

of vacuolation of the heart muscle, hydropericardium and

occurrence of cirrhosis of the liver in young ducklings,

splenomegaly in guinea pigs and widening of kidney ducts in

rats. These changes occur when the content of rape seed oil

amounts to about 50-6Jô of the total caloricity of the diet.

A lower content of rape seed oil in the diet

does not result in such drastic deviations from the normal

condition in experimental animals, nevertheless changes in

the heart appear after application of not more than 3011'o' kcal

f rom rape seed oil in the diet. 1L

. . . . . T

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20

Methods of investigation

A detailed description of the methods of

investigation was presented in the first part of this paper

(1,2). Long-term investigations of 7 and 12 months were

conducted with Wistar rats of our own breeding.

Four kinds (A, B e C, D) of experimental margarines

of different raw material composition (Table 1) and different

content of fatty acids were used for the investigations.

Parallel, year-long studies were conducted with liquid rape

seed oil and 6 months' experiments with solidified rape seed

oil, i.e. the fat ''Oman . The content of erucic acid in the

margarines investigated varied from 18% (MA) to 33% (MB). In

liquid rape seed oil, the content of erucic acid was 47.6%

whereas in the fat "Oman it was 37.1%.

TABLE 1. Composition of experimental margarines

Sunflower Rape seed oil Fish oil Type of marga- oil solidified Palm oil hydrogen-

rifle % high- low- e 10 ated

e % of erucic acid erucic erucic /0

____ .

••••■•■•■

Margarine A(18.4%) 30 Margarine B(33.0%) 30 Margarine C(20.3%) 30 Margarine D(22.9%) 30

The animais received ad libitum experimental diets

containing 15% by weight of fat from margarines, liquid rape seed

oil, the "Oman fat, or butter as control fat. One group of

animais obtained only the treeding granulated diet of the

5

"Bacutil" Co containing 7% kcal from fat.

The determination of the degree of fatty

degeneration of the organs studied was performed by dyeing

with a 0.5% solution of Oil Red 0 in 60% triethylphosphate.

The time of dyeing the scraps was 10 min. at room temperature.

The fat-Red 7B and Sudan III also were used. The degree of

fatty degeneration of the liver was determined by the scale

of Thaler (19).

According to Thaler, three degrees of fatty

degeneration of the liver are distinguished: I - scattered

fattening $ with small drops of fat appearing in separate

foci of cells on the background of the unchanged parenchyma;

II - zone fattening, occupying a part of the lobules or whole

lobules of the liver; III - diffusive fattening of the whole

liver parenchyma.

The total sum of fatty degeneration of all the

animals of the given experimental group was divided over the

number of the specimens examined,thus obtaining the index of

fatty degeneration of the liver.

Macroscopic and microscopic anatomo-pathological

investigations included the following organs: liver, kidneys,

spleen, supra-renal glands, testicles, pancreas, heart muscle

and the small intestine.

The microscopic preparations for inspection

were made following the commonly accepted method of dyeing

paraffin strips with hematoxylin and eosin.

6

6

Results of investigations.

The analysis of the weight of the selected

organs expressed as percentage of the body weight of the rat

indicates differences between groups of animals receiving

margarines and rape seed oil in the diet, in comparison with

the control group receiving butter. Changes in the weight

of the heart deserve particular attention. In the group MA.,

MB and that of liquid rape seed oil, after both seven and

twelve months of experimenting the average values of weight

of the heart were much higher as compared to the butter-fed control

group.

After 12 months, the mean weight of the heart in

the group MA. was 1.64 g(% of body weight -0.33 ), in group

MB- 1.71 g(% of body weight - 0.36), in the group of rape seed

oil - 1.62 g(% of body weight - 0.32) and in the butter-fed

group 1.49g (4 of body weight - 0.31).

The weight of the kidneys also increased in the

groups of animals MA, MB and in those receiving rape seed oil.

In the group MA - 3.67 g(% of body weight - 0.78); in the

group MB - 3.50 g(% of body weight - 0.75); in the group of

rape seed oil - 3.72 g(f of body weight - 0.75) and in the

butter-fed group - 3.27 g(% of body weight - 0.68). The

weight of testicles showed distinct increase in the groups of

animals receiving margarines A and B, after both 7 and 12 months.

These changes were not found in the group fed rape seed oil.

The average weight of testicles in the group MA was 3.58 g

.....7

- 7 -

(% of body weight - 0.76), in the group MB - 3.52 g(x of body

weight - 0.74) and correspondingly in the butter-fed group -

3.12 g(f of body weight - 0.65). In the groups MD and '10mat°;

underdevelopment of the spleen was established.

Miscroscopic anatomo-natho^,,o1icai inves ons

Deviations from the normal condition in the

microscopic structure of the kidneys, spleen, supra-adrenal

glands, pancreas and in the skeleton muscles have not been

established. I,Sicroscopic inspections of the liver (with the

exception -of cirrhotic changes in one rat of the group M)

did not show any deviations from the normal state.

Investigation of fatty degeneration of the liver

with application of the Oil Red 0, f-at Red 7B and Sudan III

have proven that all the types of margarines investigated 688

caused fatty degeneration changes in the liver; their intensity

depends on both the type of fat administered in the diet and

the length of duration of the experiment.

A,fter 7 months of administering the experimental

diets in the groups MA, T14B and "Oma", the fatty degeneration

of the liver was minimum and, in the majority of the animals

studied, remained within the limits of values obtained for

bred rats (MA-0.53, 14B - 0.93 and "0inat' - 0.96). Although the

extension of feeding the diet to 12 months increased the

0 0 0 0 . 8

7 - miesiey months /2 -miideithe

• (5 Keer CD

N/ granicach grupy kon!colnei within eontrol group Q il s/op/en stiuszczenta 0 and I degree of fatty degener.

El If slopier; sllaszczenia II degree of degeneration El lit III " ti•

Manlaryna A (1)

Margaryna 8 0 Margaryna C

Margaryna D eD

-8_

degree of liver fattening in animals of the groups MA and MB,

the values obtained were still the lowestdn comparison with

the other types of the fats studied (1.46 and 1.33).

The margarines C and D, just like butter,

caused after 7 months of experiments somewhat greater changes

of fatty degeneration which became intensified after 12 months

of administering the diet. This intensifidation of the changes

in proportion to the length of the experiment becomes manifest

with particular distinction in the group MD (1.33 after 7

months, 2.4 after 12 months).

The greatest changes, after both 7 and 12 months

of.experimenting, were determined in the group of animals

obtaining liquid rape seed oil in the diet (index 2.5 and 2.1).

A comparison of the established changes of *fatty degeneration

are shown graphically in Fig.'.

(1)4edogeelle) el) „DNA"

Fig.l. Fatty dggeneration of the liver in experimental animals.

.....10

These data are also illustrated in Tables II •

‹.:.nd III.

Microscopic inspections of the heart muscle

have shown distinct deviations from the normal condition. In

the heart, the occurrence of focal changes having the character

of granulomas, i.e. histiocytic infiltrations and focal

fibroses, in animais fed various kinds of experlmental

margarines was established. The greatest changes were determ-

ined in the group fed liquid rape seed oilmd in the group MB

containing in the diet the largest quantities of erucic acid.

These changes can be interpreted as conditions of inflammatory

and necrotic character.

Table IV and examples of photos of microscopic

preparations (Fig.2,3,4) illustrate the deviations from the

normal state found in the heart muscle of the animals studied.

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Dlarbaryna. A

?liaigitryna B

1lititirgaryneti C

Margaryna D

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Ra De 5 ce »d

Table II

FattY degeneration o° the liverTi$a^n° irien^al animals after 7 and 12 months Z

^ 71 of ex^erimen ^sKSluszczonie w&troby u zwiorzqt doswitidczalnych po 7 i 12 miesiqcach doS^viadczcnia rn

number of

i1o§é szczurc5w;.,rats

1

15

15

14

15

15

9

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O

m

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14

7

9

14

I1

9

5

14

10

7 miesi@cy 7 months

degree of fattystopion stluszczonia

w^;trob_yde~eneration of live

5

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2

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8

1

8

9

1

HH

2

2

6

1,73

0,53

0,93

1,G3

1,33

1,50

12 months

nt.zir,jJc'r ofilosc szczurGw

rats

8

15

15

15

15

15

19

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rn• c OC •f ^ ^ n

- • X' P ryt- ::^ f!) ^Ià 'Îi C>

15

14

12

15

15

19

12 micsiI^cy

degree of fat t^jstopicii stluszczenia

wq.trobyâer,ene).^?tilOn of live

9

8

7

3

5

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10

11

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8

8

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2,0

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2,10

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after

Number of

Animals Group

47% 7% 28% 67% 60% 31% 67%

14- 15

15 15

15

29 15 9 78%

Margarine A Margarine B Margarine C Margarine D "Oma" * Butter Rape seed oil

53% 80%

10010

15 87% 19 74%

after Total number of animals.

months number of animals vith changes.

- 11 -

TABLE III 6q0 % of established cases of II and III degree of fatty degeneration of the liver in rats receiving various fats in their diet.

jfls

% of fatty degeneration of II and III degree

after 12 months

Number % of fatty of degeneration

Animals of II and 113 degree

INIMMY1101•••••■•••••••■a"......1

* data for the group of animals receiving in their diet the fat "Oma" were obtained after 6 months.

TABLE IV Frequency of occurrence of granulomas in the heart of rats obtaining fats at the level of 30% kcal in the diet.

Group

Breeding Butter Margarine A Margarine B Margarine C Margarine D Rape seed oil "Orna"

15

14 0 2 (14.3%)

15 1 (6.7g) 15

14 7 (46.7%) 4 (28. 6% )

15 1 (6. 7%)

9 8 (88.9%)

29 22 (75.»9%)

after 12 months

Total N imber of number animals Of with animals. chunges.

24 0

24 2 (8.3%)

25 8(32. C%)

22 11 (50 - 0%)

24 11(45.8%)

23 11 (47. )

19 17(89.5%) not investigated

The data obtained indicate that the greatest

changes occurred in the group of animals receiiiing liquid and

solidified rape seed oil. After 7 months of experiments, in

12

- 12 -

as many as 8 out of 9 animals fed 15% by weight of rape seed

oil in the diet, occurrence in the heart muscle of numerous

small granulomas , 3.. e. 88.91/iI4 was noted-; in the group of

animals receiving the fat 110ma'l, in 22 animals (75.9%) of

29 animals examined, changes of varying intensi-ty in the

heart having the character of granulomas were established.

In the group of animals obtaining margarine B, the same

changes were found in 7 out of 15 rats examined, i. e. 46. 7°0.

In the remaining groups of animals these changes occurred to

a lesser degree; in the group IAC in 7 out of 14 rats i. e.

28.6%*, in the groups ?-ID and MA one case among 15 animals

of each group was determined i. e. 6.7%.

*Translator's notei 28.^:, not clear to me.

.....3.3

- 13 -

6Q1

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^^^^^•^ ^^+âi.f^^D^^^w',^n•_♦ i.°'^-°.t} o-::t:ee+ ♦, i=i^\x^^:i^ '^ t^ ,.4ar ^^.i.:F^h-7.'^7^:^65c^.'•:i i

Ryc. 4.

Fig.2. Heart muscle. Dark sections of the fibers in the uppertop of the picture are proof of necrosis or necrobiosis ofthe muscle. Histiocytes are accumulated in the vicinity.H.E. magn. 300 x.

Fig.3. Granuloma composed of histiocytes among muscle fibres.H. E. magn. 300x.

Fig.4. Fibrosis of the heart muscle (centre of picture),remains of histiocyte infiltration (centre of pictureon left). H.E. magn. 30Gx.

The histopathological picture becomes somewhat

intensified after 12 months of administering experimental

margarines or rape seed oil. The greatest intensification of

the changes ( 89.5%) was noted in the group of animals obtaining

rape seec' oil. These data are illustrated in Table IV.

In the control group, i.e. the breed;ng group,

pathological changes were not observed after experiments

0 0 0 0 . 1 4

-

lasting 7 and 12 months.

In the group of animals obtaining butter, 2 cases

of occurrence of small granulomas for 14 rats examined (i.e.

were established after 7 months, and 2 cases for 24

rats (i.e. 8.3%) after 12 months.

In many animais of all the groups studied,

occurrence of changes in the mucous membrane of the intestine

was noted; these consisted in shortening and widening of the

infiltration of acid-absorbing lymphocytes and

granulocytes in the stroma of the villi, and fibrosis of the

villi. These changes became intensified after 12 months of

experiments. This problem requires further investigation, in 692

order to clarify whether the changes found in rats occur with

age, or whether the quantity and quality of the fat and the

kind of diet have a direct influence on the occurrence of this

kind of disorders.

Discussion of the results

It follows from the macroscopic and micro-

scopic anatomo-pathological examinations of various organs of

rats, to which experimental margarines of various composition

as well as liquid and solidified rape seed oil /7ere administered

in the diet, that the most substantial deviations from the

normal conditions were established in the heart muscle. There

is a strict relationship between the occurrence of morphological

changes in this organ and the content of erucic acid in the diet. *Translator's note: page 693 - Russian abstract - omitted.

English abstract - see first page of translation.

.15

- .11

The occurrence in the heart muscle of focal

necrotic changes should be connected with the impossibility

of a regular metabolic conversion of the erucic acid.

Investigations of many authors (3,6,10,11,12,17,18) and our

own (unpublished data) indicate the occurrence of fatty

degeneration of the heart and accumulation of erucic acid

in this organ. These changes appear in the first place in

young animals. It is worth noting that Beare-Rogers (18)

achieved fatty degeneration of the heart muscle in young

animals also after application of larger quantities of fish

fat, the so called herring oilj in the diet. This fat is

characteristic by its large content of monounsaturated

long-chain fatty acids. However the fatty degeneration of

the heart never reaches as high intensity as after administering

rape seed oil in the diet.

The appearance in the heart muscle of micro-

focal granulomas, similar to those which occur after applic-

ation of rape seed oil in the diet, was determined by us in

our investigations after administering to the animals in the

diet a major quantity of the theobroma oil-like fat used in

some countries as a substitute for cocoa butter in the

manufacture of chocolate products.

One can conclude hence that microfocal necrotic

changes of the heart muscle may occur as a result of inferior

metabolic conversion of certain fats and oils. The mechanism

of these changes is the subject of further investigations.

. 16

-16-

Lit erature

(For translated items see below)

• - - . • • • - - • - - _________ 1. e., .0pus2yziska T., Bulltak-Jachymezyk B., Okolska G., Bartlet H.,

Olszewska Buclzenska-Topoloieslca J.: Badania biologiczne wartogci iywienio- wej tluszczôw jadalnych. CzOé I. Roczniki PZI-1, 22, 4, 1972. — 2. zierniafiski e., Bullzak,-Jachyniczyk. B•, Kucharczyk B., Rusiecka M., Opuszyziska T., Budzwisk- -Topolowska J.: Badania biologiczne 1.vartoci iywieniowej tluszczôw jadalnych.

• CzeU II. Roczniki PZFI, 22, 5, 1972. — 3. Bear J., Jensen B.: J. Nutr., 1947, 03, 339 • — 4. Thornasson H. J.: J. Nutr., 1955, 56, 455. — 5. Thornasson H. J.: J. Nutr., 1955, 56, 455. -- 6. Roine P., Uksila E., Teif "M., Rapo/a J.: Z. Ernahr. Wiss., 1960, .1, 118. — 7. Thornasson H. J., Gottenbos J., van Pijpen P. L., Vies R. 0.: Zesz. Probl. Post. N. RoIn., 1970, z. 91, 381. — 8. Rocquelin G., Cluzan Zcsz. Probl. Post. N. RoIn., 1970, z. 91, 403. — 9. .Ttocqzâire G., Chtzan R.: Ann. Biol. Anim. Bioch. Biophys., 1968, 8, 395. — 10. ex/el/a/if A. M. M.;. Vles R. 0.: Nutr. and IVIetabol., 1970, 12, 285.

11. Abdellatif A. M. M., Vies R. 0.: Nutr. and Metabol., 1970, 12, 305. — 12. Abdellatif A. M. M., Vies R. 0.: Nutr. and Nletabol., 1971, 13, 65. — 13.. Zion/b-e/a Zesz. Probl. Polst. N. Roln., 1970, z. 91, 420. — 14. Ziemlediski Opuszyi-ska T., Rusieeka M.: Zesz. Probl. Post. N. Roln., 1970, z. 91, 425. — 15. Nikonorunz

. M., FIomrowski S.: Roczn. Wojsk. Inst. Hig. Epid., 1965, 4, 39. — 16. Honirowski S.: J. Nutr. Dietet., 1969, 6, 22. — 17. Walker B. L., Zall S. P., Slinger S. J., Bayley H. S.: Pr‘ocedings International Rapeseed Conference Ste. Adéle, Quebec, Canada- September 20-23, 1970, str. 377. — 18. Beare-Rogers J. L.i Procedings International Repeseed Conference. Ste. Adéle, Quebec Canada, September 20-23, 1970, str. 450. — 19. Thaler II.: Gastroenterologia, suppl. 1961, 95, 2-16.

• 14.V.1972 r.

Warszawa, ul. Powsitiska 63.

Translated items:

Ziemlanski et al. Biological studies of the nutritional value

of edible fats Part I. Annals of the State Hygiene Institute 22, 4, 1972.

2. Ziemlanski et al. Biological studies of the nutritional

value of edible fats. Part II. Annals of the State Uygiene Institute 22, 5, 1972.

1 .