n

FISHERIES AND MARINE SERVICE

Translation Series No. 3876

Metabolic toxicological studies on rats fed high rapseed and peanut oil content diets for 90 days

by E. Lanzola, C. Roggi, A. Montani and M. Allegrini

Original title: Ricerche di tossicologia metabolica sul ratto mantenuto per 90 giorni con mangime a base di oui di colza e di arachide

From: g Sanita Pubblica 31(5-6): 121-137, 1975

Translated by the Translation Bureau ( MG) Multilingual Services Division

Department of the Secretary of State of Canada

Department of the Environment Fisheries and Marine Service

Halifax Laboratory Halifax, N.S.

1 976

18 pages typescript

„

DEPARTMENT OF THE SECRETARY OF STATE

• TRANSLATION BUREAU

MULTILINGUAL SERVICES

-

CANADA

DIVISION MULTILINGUES

SECRÉTARIAT D'ÉTAT

BUREAU DES TRADUCTIONS

DIVISION DES SERVICES

TRANSLATED FROM - TRADUCTION DE INTO EN

ITALIAN ENGLISH

DATE OF PUBLICATION DATE DE PUBLICATION

PAGE NUMBERS IN ORIGINAL

NUMÉROS DES PAGES DANS LORI GINAL

PUBLISHER - ÉDITEUR

PLACE OF PUBLICATION LIEU DE PUBLICATION

ITALY

VOLUME

31

ISSUE NO. NUMÉRO

5-6

121-137 NUMBER OF TYPED PAGES

NOMBRE DE PAGES DACTYLOGRAPHIÉES

18

YEAR ANNÉE

1975

AUTHOR - AUTEUR

E. LANZOLA, C. ROGGI, A. MONTANT, M. ALLEGRINI

TITLE IN ENGLISH - TITRE ANGLAIS

METABOLIC TOXICOLOGICAL STUDIES ON RATS FED HIGH RAPSEED AND PEANUT OIL CONTENT DIETS FOR 90 DAYS

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

RICERCHE DI TOSSICOLOGIA METABOLICA SUL RATTO MANTENUTO PER 90 GIORNI CON MANGIME A BASE DI OLII DI COLZA E DI ARACHIDE

REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÉRES ROMAINS.

IGIENE E SANITA' PUBBLICA

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PERSON REQUESTING A.T. REID DEMANDÉ PAR

BRANCH OR DIVISION FISHERIES & MARINE

YOUR NUMBER VOTRE DOSSIER NC:i

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Inforrns::on DATE OF REQUEST

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DEPARTMENT OF THE SECRETARY OF STATE

TRANSLATION BUREAU .

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BUREAU DES TRADUCTIONS

DIVISION DES SERVICES

MULTILINGUES

MULTILINGUAL SERVICES

DiVISION

Summ.ev — The authors have pointed out the effects of diets with a high content of rareseed-oil and peanut-oil, i.e. in the presence or absence of ei ncic acid, on rat heart mitochondria respiration in a medium-term experiment.

Wistar aleino rats (50% males and 50% females) were fed on the desired diet for a period of 90 days, after this period the rats were sacri-ficed and on the sarcosomes prepared according to the method of Chance and Hagihara, the oxygen uptake was measured with the polarographic n:ethod.

The results show that there are no difference with regard to both sexes and, con:rary to the results quoted in references, that erucic acid is rot the only factor responsible for the lower respiratory activity, since the values cbtained usig peanut-oil are quite similar to those obtained usig rupeseed-oil.

ZusAmmENTAsst:No — In einem zur Hâlfte durchgeführten Experiment wurde die Wirkung ciner Diiit mit hohem Gehalt an Rüb-und Erdnussig (cl.h. die Anwesenheit bezw. Abwesenheit von Eruziksâure) auf die kardiale I l■ fitochondrienrespiration nachgewiesen.

Albinoratten der Wistar-Rasse (50 mânnlich, 50% weiblich) wurden für die Daller von 90 Tagen mit einer Diiit nach bestimmten Format • erniihrt; nach cliesern Zeitraum wurden die Tiere .etütet und die Sark.oscrnen cntsprechend der Chance-und Hagihara-Methode prâpariert und der Sauer-stoffverbrauch mincis Polarographie bestimmt.

Die Ercebnisse zeigten keinerlci Unterschiede hinsichtlich Geschlecht und, entgegen den Schrifatunsanraben vire die Eruziks:iure nicht der allein verantwortliche Faltor fiir die gespirationshemmung, da die mit bciden Diâten erzieker: %Verte durchaus vcrgleichbar sind. .

CLIENT E S NO, DEPARTMENT . DIVISION/BRANCH CI'Y N0 DU CLIENT MINISTRE DIVISION/DIRECTION VILLE

Environment Fisheries and Marine Ottawa

BUREAU NO. LANGUAGE TRANSLATOR (INITIALS) NC) DU BUREAU LANGUE TRADUCTEUR (INITIALES)

1101522 Italian M.G.

TOXICOLOGICAL STUDIES ON RATS FED RAPSEED UNEDITED DM'T 4E4NUT OIL BASED FOOD DIETS FOR 90 DAYS (*)

0111

• iricun r1 n u1rnn • Lanzgla, C. Roggi, A. Montani, and M. Allegrini • • , .-eP

(*) This paper was presented at the VII National Meeting of the Italian Society of Human Nutrition. Riva del Garda, 25-28 September, 1974.

305.-200-10...51

• - Risumi. — On a mis en évidence l'effet de diètes à haut connu en

In ile de colza et d'arachide (c'est-à-dire avec ou sans acide érucique) sur la respiration cardiaque mitochondriale au cours d'une expérience à terme moyen.

Des rats albinos de la souche Wistar, dont la moitié étaient rr..11es et l'autre moitié femmes, ont été alimentés pendant 90 jours suivant des entes formulées; après cette période les -animaux ont été tués et on a préparé les sarcosomes suivant la méthode de Chance et Hagihara et la consommation d'oxygène a été déterminée par la méthode polarographique.

Les résultats ne montrent pas des différences en ce qui concerne le sexe et, au contraire des résultats rapportés dans la littérature, l'acide érucique ne serait pas :e seul facteur responsable de l'inhibition respiratoire car les valeurs obtenues au moven des deux diètes sont tout à fait comparables.

i

INTRODUCTION

Several experiments have shown that the ingestion of rapeseed oil is

responsible for harmful effects on various functions of the animal organism:

in particular it has been shown that this oil, administered as the only source

of fats, in amounts equal to 10-20% of the diet ingested, causes a slow down

of growth in experimental animais which may be due to the decreased consumption

of food caused in turn by the fact that digestion and absorption occur more

slowly (1-2-3-4-5-6).

Besides the increase of erucic acid in the deposited fats (7-8), a

negative effect of rapeseed oil on the reproduction of rats (10-11) and the

onset of hypothyroidism and hypercholesterolemia have also been observed.

Other negative effects described involve some endocrine glands,

particularly the gonads and the adrenal glands (4-12-13).

There are also several observations involving effects on the heart

(5-14-15-16).

In the rat the administration of 60% of the daily calories as oils

containing erucic acid, causes cardiac lipidosis followed by foci of histocyte

infiltration and lastly fibrosis (17).

2

3

According to Houtsmuller et al. '(18) erucic acid in the diet of rats

causes a decrease of aobut 50% in the respiratory capacity of the mitochondria

isolated from the heart muscle.

It has also been recently shown by Turchetto et al. (19) that the bio-

chemical lesion is accompanied by functional damage that affects the contractile

ability of the skeletal muscles causing lowered resistance to physical stress.

The toxicity of rapeseed oil is of great practical concern since

rapeseed oil is one of the edible oils most used in the human diet in Italy.

Rapeseed and mleseed oil, in fact, accounted for about 50% of.all seed oils

available on the market in 1973.

The purpose of this investigation, which has been carried out with the

help of a grant from the Ministry of Health, has been to demonstrate the effect

of rapeseed oil, and at the same time of peanut oil, on mitochrondrial respira-

tion, though a medium-term experiment, following OMS recommendations for food

toxicity tests, and using a period of 90 days for the rat.

MATERIAL AND METHODS

In this research, Wistar albino rats were used, 50% of which were male

and 50% female, immediatley after weaning.

The rats, on an average weight of 90-100 g, were divided into 10 lots

(5 containing males and 5 females) of 20 rats each.

Two of the ten lots were used as controls.

The diet administered to the rats was the one suggested by Randoin and

Causeret, modified by Bonetti, and consisting of: wheat, maize, powdered barley,

milk casein, skim piilk powder, wheat germ, dry yeast, calcium lactate, sodium

chloride.

4

An identical diet was administered to the two control lots, one male

and one female. Peanut or rapeseed oil was added to the diet of the remaining

lots in amounts equal to 20% or 30% of the total calories.

Thus, four different diets were administered, each diet to two dif-

ferent lots of rats: one male and one female. •

The raw lipids made up 3.6% of tbe basic dry weight: following the

addition of peanut and rapeseed . oil, the percentage of the lipids in the total

calories of the final diet rose to about 27% after the addition of 20% and to

about 36% after the addition of 20% of these oils respectively.

At the end of the 90 days, the rats were weighed and sacrificed by

decapitation; their hearts were removed. Five hearts at a time were pooled;

a portion was kept for gaschromatographic determinations, while from the rest,

sarcosomoes were prepared according to the method of Chance and Hagihara (20).

The oxygen consumption of this preparation was measured by polarography, using

a Clarke electrode.

The substrates tested were: 5mM glutamic acid, 5 mM f3-hydroxybutyric acid,

2.5 mM succinic acid and a 5 mM a-keotglutaric acid.

The incubation medium showed the following composition expressed as final

molar concentrations of the incubation medium:

0.25 mannitol

0.01 M KC1

0.01 M tris-HC1 buffer

0.005 M K1-K2

phosphate buffer

0.02 mM EDTA

260 pM ADP

sarcosomes 0.100 ml (about 2 mg of protein).

The final volume was 3 ml, pH 7.2 and the temperature 22°C.

The protease, ADP and succinic acid were from the Sigma Chemical Co.;

EDTA, glutamic acid, a-ketoglutaric acid and D-mannitol from Fluka A.G.;

S-hydroxybutyric acid from Schuchardt.

All the other reagents were products of Merck A.G.

Fatty acids were determined by gas chromatography, after esterification,

according to the method of Mason (21).

The protein content was determined by the biuret method.

RESULTS AND DISCUSSION

It must be noticed that weighing did not reveal differences in growth

between rats treated with peanut oil and rapeseed oil in the two different dosages.

Tables 1, 2 and 3 show respectively the percentage values of fatty acids

in the heart, kidney and liver of rats treated with the previously mentioned diets.

It may be seen that erucic acid (22:1) is present in moderate amounts

in those rats which received diets containing rapeseed oil.

These values are comparable to those reported in the literature (22) and

show that, in practice, there is no accumulation of erucic acid in the myocardial

tissue.

It is well knownthat the consumption of oxygen by living tissue, which

occurs through a process of energy conservation called oxidative phosphorylation,

requires the presence of a very specific structural organiztion of cytochromes,

flavoproteins, pyridine coenzymes and enzymes which phosphorylate ADP. It has

already been shown that enzymatic complex is located at the level of the mito-

chrondria and specifically within the cristae of the inner membrane (23) (oxysome

of Chance).

18,4 8,7

31,3 21,7

8,0 1,9

1,0 2,0

16:0 18:0 18:1

. 18:2 20:4 22:1 22:5 22:6

17,9 15,3 16,7 27,9 15,7

1,9 2,3

18,8 125 18,5 35,3 12,6

1,1 2,1

16,3 11,0 22,3 18,8 11,1

1,7 1,4 3,6

19;0 9,7

26,7 23,1

• 10,5 1,3 1,8 1,7

23,5 145 23,5 26,8 1%9

3,6

3 ! '

Acidi . Arr1-4 rassi chide

20%

/%1aschi 1

Ara- 5 chide 2110' .

7 .‘raJ3

Colza . chide

Jv, 0 20%

Femmine 2 10

Colza 20%

11 Colza 30%

6 Colza 20%

Ara-9 chide

19,8 7,2

2%5 4,7 2,9 %8 1,4

15,3 10,5 20,5 34,6 11,1

1,0

1,9

• •

;d

6

o

TABLE 1

Percentage of fatty acids in the heart

I Femmine 2 ,3 Maschi ,

Acidi Ara- Ara- 6-

Colza Colza Ara-8 Ara-9 Colza 0 Colza grassi ['chide 5 chide 20, , 30„ chide chide 20% 20% 30% i° ° 20% 30% ' 30% .

16:0 11,7 12,9 14,4 9,4 13,1 11,3 12,1 11,4 18:0 21,1 19,8 22,9 . 21,6 21,0 20,3 19,9 19,7 18:1 8,2 6,8 8,3 9,9 7,5 6,2 5,7 12,3 18:2 27,5 27,0 23,9 27,8 23,2 25,1 15,6 26,8 20:4 17,7 19,6 10,1 17.4 17,9 18,2 10,7 16,0 22:1 -- -- 1,3 1,4 -- -- 1,1 1,1 22:5 20 30 1,1 1,9 2,0 2,4 lj 1,8 22:6 9 6 10,5 5,0 6,1 11,4 1 0,3 4,9 7,7

Key: 1-Males; 2-Females; 3-Fatty acids; 4-Peanut; 5-Peanut; 6-Rapeseed; 7-Rapeseed; 8- Peanut; 9-Peanut; 10-Rapeseed; 11-Rapeseed.

TABLE 2

Percentage of fatty acids in the kidney

Key: 1-Males; 2-Females; 3-Fatty acids; 4-Peanut; 5-Peanut; 6-Rapeseed; 7-Rapeseed; 8-Peanut; 9-Peanut; 10-Rapeseed; 11-Rapeseed.

7

TABLE 3

Percentage of fatty acids in the liver

3 Maschi 1 Femmine 2

Acidi Ara-4 Ara-5 6 ^L

AraB Ara-9 10 ilC l

grassi chideo•

chideo.

Colza20

o'

Coo

30chide

o^chide Colza

o,20^

o zaoi

30io20 , 0 30 ,0 ;20 /0

030 /o 0

16:0 18,6 15,6 17.6 18,3 18,8 13.7 16,7 17,118:0 16,9 20,2 17,5 16,0 19,9 15,9 18,6 20,218:1 10,0 7,6 17,6 16,6 8,0 11,0 12,8 13,918:2 27,9 27,7 19,6 21,5 25,9 33,3 15,8 16,620:4 18,1 20,2 17,1 17,0 -18,3 17,2 21,2 17,722:1 - - 0,3 0,2 - - 0,2 0,322:5 1,0 1,2 1,6 1,4 1,8 1,6 1,6 1,422:6 6,1 5,4 4,5 4,3 7,1 6,2 9,8 9,3

Key: 1-Males; 2-Females; 3-Fatty acids; 4-Péanut; 5-Peanut; 6-Rapeseed;

7-Rapeseed; 8-Peanut; 9-Peanut; 10-Rapeseed; 11-Rapeseed.

During the first seconds of incubation the sarcosomes show some res-

piratory activity which, however,_sharply decreases after a few minutes. The

oxygen consumption characteristic of this first phase occurs at the expense

of a small amount of endogenous substrate which is rapidly oxidized because of

the presence of ADP in the incubation medium.

Sarcosomes under these conditions, that is in the presence of endo-

genous substrate, phosphate and ADP, are in a typical stage 2, according to

Chance and Baltscheffsky (24) who investigated the spectroscopic effects of

ADP on the respiratory pigments of rat heart sarcosomes. In fact, cytochrome

b undergoes a process of oxidation which reaches a maximum when all endogenous

substrate is used up.

We should point out for the sake of clarity, that stage 1 occurs by

definition when the sacrosomes are diluted in the incubation medium in the

8

presence of inorganic phosphate but in the absènce of ADP. In this case the

cytochromes are partially reduced.

When the graph shows the oxidation of the endogenous substrate and

therefore a very low respiratory activity, phenomenon which generally occurs

between the fifth and the eightliminute from the onset of respiration, 10 pl

of a solution of 5 mM glutamic acid as substrate is added to the incubation

medium by means of a microsyringe equipped with a flexible teflon needle.

After a few seconds the oxygen consumption undergoes a net increase (from 5

to 8 times) which persists until the ADP still present in the suspension is

completely used up.

Under tfiese conditions, that is in the presence of inorganic phosphate,

substrate and a phosphate acceptor (ADP), the sarcosomes are in stage 3 or

active stage or phosphorylation stage.

It is easily seen that in stage 3 the conditions characteristic of

physiological respiration occur, that is an oxygen consumption closely linked

with the conversion of ADP to ATP; or in other words, an oxygen consumption

détermined by the concentration of some nucleotides. Spectroscopic measurements

have shown that, during the active stage, the reduction of cytochrome b, which

begins with the addition of the substrate and results from the reduction of NAD

and flavoproteins, ceases and reaches equilibrium (steady state). When the ADP

is used up, its reduction starts again and reaches a maximum; the sarcosomes are

then in stage 4.

This stage is in fact defined as that phase of oxygen consumption which

occurs in the presence of inorganic phosphate and substrate but in the absence

of ADP. Generally the oxygen consumption in stage 4 is considerably lower (about

7 times) than that occurring in stage 3.

•

For purposes of this research, we immediately added the substrate or

the substrates to the preparations of sarcosomes so that stage 4 (20-23-25)

began at once.

Stage 3 was then induced by adding ADP, so that stage 4 and stage 3

alternated and the respiration could be directly controlled.

The ratio between rate of respiration during stage 3 and stage 4 is

indicated as respiratory control (RC) and indicates the degree of "coupling"

between phosphorylation and oxidation in the mitochondrial preparations used.

The results are reported in Table 4.

To better demonstrate the inhibitory effect of the diets containing

rapeseed and peanut oil on respiration of heart mitochondria, the polatographic

graphs obtained using as substrate 5 mM 0-hydroxybutyric acid are shown in

figure 1, 2, 3, 4 and 5.

This substrate has been chosen as an example because it is the one

which best illustrates the inhibition.

It may be seen that the graphs relative to the peanut (fig. 2 and 3)

and rapeseed (figs. 4 and 5) diets are almost superimposable, independently of

the amounts of oil administered; a net decrease in the consumption of oxygen

with respect to the control during stage 3 is also evident.

Fig. 6 shows the specific respiratory efficiencies during stage 3

that is during active prosphorylation, compared to the specific respiratory

activity of the control rats taken as 100% for purposes of summarizing the

results obtained. As has been noted, the respiratory activity of rats fed

diets rich in rapeseed and peanut oils is significantly lower than that of

controls, especially when S-hydroxybutyric acid is used as a substrate. In

this sense, no appreciable difference can be found between the two oils.

9

The results obtained would suggest that the inhibitory effect cannot

be attributed to erucic acid, which is present only in small amounts in the

myocardium of the rats treated with rapeseed oil, while it is absent from the

myocardium of rats treated with peanut oil, but on the contrary must be attri-

buted to other factors of uncertain nature.

In this regard, further research is under way to determine whether

the effect obtained with rapeseed and peanut oils also occurs with other oils,

where used in the rats diet in similar percentages, and to attempt to identify

the factors responsible for the observed inhibition.

ACKNOWLEDGEMENT

This work was made possible by a grant from the Ministry of Health.

The diets were prepared by the Piccioni Laboratory in Brescia.

1 0

TABLE 4

Rareseed qi

anut 20e Peanut 10% Rateseed 20 0j

Substrate i Stage Pe

le

Control5 ' - - '

.. m F M le M F le, F ---'

3 0;737 -10,1. -18,4 -29,7 -23,5 • -24,9 . -14,9 -29,0 -35,3 Glutamic acid 4 0, 1 3,5 - 3.,7 - 1,5 -12,6 -16,3 -13,3 + 4,4 - 8,1. -32,6

CR 5,46 - 6,4.. -17,0 -19;8 - 8,8 -12 1 1 -18,9 -23,1 - 4,0

3 0,745 -42,9 -41,3 - 158,6 -57,4 -49,6 -49,6 -54,8 -53,1 P-hydroxybutyric 4 0,138 -19,6 -13,8 -;,9 -25,4 -16,7 -13,8 - 9,4 -25,4- acid Ci 5,43 -29,8. -32,0 -41,9 -43,8 -38,5 . -41,9 -49,7 -37,4

........ .._ 3 1,187 -26,4 -27,8 -37,3 -31,9 -23,2 -19,8 -13,7 -2 7, 8 Succinic acid 4 0,349 -18,9 -10,6, -12,0 -24,1• - 9,7 - 7,4 - 6,0 -21,8

CR 3,39 -8,8 -20,9 -28,6 - 4,7 -15,0 -12,9 -10,6 - 7,7

3 0,789' -10,6 -28,9 -24,6 -33,3 -18;1 - 8,9 - 4,4 -12,4 . u-ketoglutaric 4 0,150 4- 6,0 +11,3 - 0,7 - 2,0 + 5,3 +12;7 4. 9,3 - 8,0

L acid cit 5,26 -15,9 -35,7 -23,4 -31,7 -20,5 -19,0 -12,3 - 4,9

The control values are expressed as specific activity, le. as 111.102/sec/mg protein.

All other values are expressed as percent variation (+ or -) with respect to the controls.

12

/3-OH 5 mM

Normal diet I 100

90

80

70

60

60

40

30

20

I.

lmin

10 Fig. . 1

13

/3- OH 5 naM

Peanut oil diet (20%)

Stage 3

Stage 4

1 min

rei.0' 2 ,..,..

— Jo

90

80

70

60

50

40

30

20

10

14

1001

90

80

70

60

50

40

i 30

1 20

10

/3-OH 5 m1VI

Peanut oil diet (30%)

Stage 4 '

C'ig. 3

Stage 3

Imin^------^-

15

Fig,. 5

/3 - OH 5 mEg

rapeseed oil diet (30 %) Stage 3

100

90

80 '

70

60

50

40

30

20

10

16

F- • 100

80

60

40

! 20

Glut /3-OH Suce «-KG

Fig.6

17

The values are expressed as specific activity (pM02 /sec/mg protein) during stage 3 of respiration. The value obtained with the control rats are taken as 1007 As there are no relevant differences, the values obtained with male and female rats were averaged.

Ua rats fed a diet (calories) of 20% peanut oil

rats fed a diet (calories) of 30% peanut oil

in rats fed a diet (calories) of 20% rapeseed oil

rats fed a diet (calories) of 30% rapeseed oil

The substrates concentration is given in "Materials & Methods".

BIBLIOGRAPHY

. - - - • -

1) VON BEZN 11: A., VON BEZNAK M. e H.AJDU L , Ernfihrung 8, 209, 1943. 2) vo N E LER B., VON EULF.R H. e LINDERMAN G., Arkiv Kemi, Mineral.

Geol.. BL6, 1, 1948. • 3) C.ARROLL K.K., J. Biol. Chem. 200, 287, 1953. . 4) CARROLL K.K. e NOBLE R.L., Can. J. Biochem. Physiol. 34, 981, 1956.

5) ROCQUELIN G. e CLU ZAN R., Ann. Biol. Anim. Biochim. Biophvs 8, 395, 1967.

6) DEt:EL H.J., HALLMAN L. C LEONARD A., J. Nutr. 20, 215, 1940. 7) WAGNER H., SEELING E. C BÈRNILARD K., Z. Physiol. Chem. 306, 96, 1956. 8) BERNHARD K., LANDLAR F. C WAGNER H., Z. Erniihrungsw. /, 48, 1960. 9) BEARE J.L., Can. J. Biochem. Physiol.• 39, 1855, 1961.

10) KENNEDY T.M. c PuavEs H.D., Brit. J. Exper. Path. 22, 241, 1941. 11) CARROLL K.K. e NOBLE R.L., Can. J. Biochem. Physiol. 35, 1093, 1957.

12) BEARE J.L., MURRAY T.K., Grice H.C. e Campbell J.A., Can. J. Biochem. Physiol. 37, 613, 1959.

13) &WINS JC POLOWSKA J. e ZIENILANSKI S., Ann. Nutr..Alim. 27, 249, 1973. 14) EDF.I.LAT:F .A.M.M. c VI-FS R.O., Nutrit. Metal). 12, 285, 1970.

15) TitomAssos H.J., J. Nutt-. 57, 17, 1955. 16) Rolm- P., UESILA R., TEIR H. e RAPOLA J., Z. Erniihrungsw. /, 118, 1960.

i 17) AEDELLATIF A.M.M., Nutrit. Rev., 30, 2, 1972. 18) Hou'ES I 1: ELEA STR L. I JK C. B. C VAN DER BEEK A., Biochcm.

Biophys. Acta 218, 564, 1970.

19) TURCIIETTO E., CABRINI L., IARANESI M. e BARZANTI V., Boll. Soc. It. Biol. Sper., 98, 1201, 1972,

20) CHANCE B. e HActitARA B., Proc. V Int. Congr. Biochcrn., Moscow 1961, cd. da E. Slater, Pergamon Press., New York 5", 3, 1963.

21) MAsoN M.E. e WALLER G.R., Anal. Chem. 36, 583, 1964. 22) MARANEst M., BIAGI P.L. e TURCHETTO E., Boll. Soc. It. Biol. Sper.,

98, 1203, 1972. - 23) CHANCE B. «Control Of Energy Metabolism», Academic Press., New

York c Lendra pag. 418, 1965. 24) CHANCE B. C BALTSCHEFFSEY M., Biochem. J., 68, 283, 1958. 25) MoRATrt R., MoNTANI A. e RUFF() A., Quad. SClavo Diagn. 8, 80, 1972.

18