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Behaviour of lipoperoxidation in rat liver during orotic acid treatment
Life Sciences Vol. 10, Part II, pp . 99-111, 1971 . Printed in Great Britain
Pergamon Press
BEHAVIOUR OF LIPOFEROXIDATION IN RAT LIVER DURING OROTIC ACID TREATN.EIv^P N~ .V . Torrielli, N:. U. Dianzani and G . Ugazio Istituto di Patologia Generale dell'Università di Torino 10125 Torino - Italy (Received 28 September 1970 ; in final form 7 December 1970) Experimental evidence indicates that both protein synthesis( ) and assembly of very low density lipoproteins( 2 ) are impaired in the livers of rats fed on a diet containing orotic acid . This treatment has been reported to cause an increase in the liver's content of lipid soluble compounds reacting with thiobarbituric acid, referred to by Kinsella( 3 ) as malonyldialdehyde . The orotic acid induced fatty liver has been prevented by a,-tocopherol( 4 ) . These observations were offered as evidence for the involvement of lipoperoxidation in the pathogenesis of the observed liver fat accumulation( 3 ' 4) . Recent work in our laboratory has demonstrated that DPPD (N,N'-biphenyl-p-phenylendiamine), a lipid soluble free radical scavenger, prevents fatty liver in rats when the diet contains orotic acid( 5 ) . However , although a severe derangement of the morphologic organization ofendoplasmic reticulum occurs very early after orotic acid administration( 6 ), to date, no evidence other than that of Kinsella( 3 ' 4 ) is available indicating that peroxidative alteration of membrane lipids is involved .
Experiments reporteà here took into consideration lines of
work previously used in order to evaluate in vivo lipid peroxidation of liver cell particles(~' 8 ' 9 ) . Both the ultraviolet absorption spectrum of total lipids and the arachidonic acid concentration in phospholipids were determined in mitochondria and microsomes of rat liver during dietary administration of orotic acid . In
addition, the spontaneous lipoperoxidation of liver homogenates 99
j,ipoperaaidation in Rat Liver
100
Vol . 10, No .
2
vas studied in rats under steatogenic treatment either in the presence or in the absence of DPPD or adenine injection . MATERIALS AND METHODS In vitro non-enzymatic lipid peroxidation oP liver hom
mates .
Male rats, 90-100 g initial body weight, of the Wistar strain, were randomly allotted into Four groups . Control animals (group A) were offered a diet containing casein (20X), corn starch (70X),
cellulose (3X), corn oil (4X), minerals (2X) and vitamins (1X) . This diet vas devoid of any non-natural antioxidant (Piccioni, Brescia, Italy) . Experimental rats (groups B, C and D) received the same diet provided with 1 .5X orotic acid (B .D .H ., Poole, England) which replaced an equal amount of corn starch . Fifty mg DPPD (Schuchardt, Munich, Germany) per 100 g of body weight or 20 adenine sulphate (H .D .H ., Poole, England) per 100 g of body weight, prepared as previously reported(5), were daily injected into the peritoneal cavity of the rats belonging to the groups C and D, respectively . Animals from each group were sacrificed after 2, 4 and 8 days of treatment . Liver homogenates (6X v/v), prepared at 4°C in a Potter-Elvehjem apparatus with 0 .05 M phosphate buffer, ph 5 .6 or 7 .4, containing 0 .15 M LC1, were shaken in a metabolic incubator (Dubnoff, 37 °C) For up to 240 minutes . Malonyldialdehyde production vas determined on the trichloroacetic acid-soluble Fraction oP the homogenate after incubation and treatment with thiobarbituric acid( 10 ), Followed by XOH(17), IIltraviolet absorpti on spectrwn of lipids from mitochondria and microsomes . Male rats, 200-220 g initial body weight, oP the Wistar strain, were used in this series of experiments . The animals, divided into two groups, were fed on the before mentioned diet either devoid of or provided with orotic acid (1 .5X), respectively . They were starved for 16 hours and then sacrificed at 2, 4 or 8 days oP dietary treatment . Liver homogenates (10X v/v), prepared at 4°C in
Vol. 10, No . 2
Lipoperaaidation in Rat Liver
101
a Potter-Flvehjem apparatus, with 0 .25 M sucrose containing 3 mM EDTA (sodium salt ; Fisher Chem . Co ., Fair Lawn, N .J .), vas spun at 1,000 x g For 10 minutes in a Servall centrifuge . The decanted supernate was made up to the initial volume and then centrifuged at 10,000 x g for 25 minutes . The microsomal fraction vas harvested by centrifuging the second supernate at 78,500 x g for 80 minutes (Spinco preparative ultracentrifuge, rotor no . 30) .
Both the
10,000 x ~ and the 78,500 x ~ sediments were extracted according to the method of Folch, Lees and Sloane-Stanley( 11 ) . The washed lipids were dried down under an oxygen-free nitrogen stream, dissolved in cyclohexane (spectrophotometric grade: C . Erba, Milano, Italy) and scanned in the ultraviolet region with a Beckman spectrophotometer (model DH) .
The absorption spectrum vas
normalized on the basis of the lipid concentration (1 mg per ml) which was assayed with the potassium dichromate-sulfuric acid method of Chiang, Gessert and Lovry( 12 ) . Lipid Fractions of the cytoplasmic particles . The lipids extracted from both mitochondria and microsomes oP rat liver at the eighth day of orotic acid treatment were further fractionated . Triglycerides were separated by means of thin layer chromatography and estimated with the chromotropic acid method of Van Handel and Zilversmit( 13 ) . Neutral and polar lipid Fractions were eluted with chloroform and methanol, respectively, from silicic acid columns (~iallinckrodt Chem . Works, ST Louis, Mo) and then photometrically determined( 12 ) . Fatty acid pattern in mitochondrial and microsomal phospholipids . An aliquot of the lipids extracted from the cytoplasmic particles was fractionated on silica gel thin-layer plates . Phospholipids were scraped off and submitted to hydrolytic methyl ation( 14 ) . Fatty acid methyl esters were then separated with a gas liquid chromatography apparatus (Fractovap, model GV, C . Erba,
102
Lipoperoaidation in Rat Liver
Vol. 10, No . 2
hilano, Italy), according to a procedure as reported by Comporti, Bardino and Ugazio( 9 ) . Data are expressed as per-cent distribution of the fatty acid fractions . RSSULTS Treatment with orotic acid in vivo induces an increase in the spontaneous production of malonyldialdehyde by rat liver homogenato when incubation occurs in an acidic medium (pH 5 .6) (Figures 1, 1a, 1b) . This phenomenon is particularly evident 2 days after
p H 5.6
p H 7.4
FIG . 1 - Rate of in vitro malonyldialdehyde production by liver homogenate from rats fed on a diet containing 1 .5X orotic acid for 2 days .
Ordinate : optical density at 543 nm of the compounds reacting with thiobarbituric acid, produced by 45 mg-equiv liver, per ml of final volume .
Abscissa : incubation time, in hours .
Treatments : C : diet devoid of orotic acid ; OA : diet containing orotic acid ; OA-DPPD : diet containing orotic acid fed to animals daily treated with 50 mg DPPD per 100 g b .wt, intraperitoneally ; OA-Ad : diet containing orotic acid, fed to animals daily injected with 20 mg adenine sulphate per 100 g b .wt, intraperitoneally . Plotted points represent the values obtained with duplicate estimations on samples of 4 pooled livers .
Vol: 10, No . 2
Lipoperaa~idation in Rat Liver
pH
s.e
103
pH 7.4
FIG . 1a - Rate of in vitro malonyldialdehyde production by liver homogenate from rats Fed on a diet containing 1 .5X orotic acid For 4 days . For other details see Figure 1 .
C
OA
_
I
~~ 0 .1 0 0
OA+OrPO
OAtAd
FIG . 1b - Rate of in vitro malonyldialdehyde production by liver homogenate from rats fed on a diet containing 1 .5X orotic acid for 8 days . For other details see Figure 1 .
104
Lipoperoaidation in Rat Liver
Yol . 10, No . 2
orotic acid feeding and is prevented by the concomitant injection of either DPPD or adenine both of arhich are kno~m to inhibit the orotic acid induced triglyceride accumulation in the liver( 5 ) . Figures 2, 2a and 2b shov that no peroxidative alteration
~.s ~.o 45 ti. ,,., .._,\~ 0 N
FIG . 2 - Ultraviolet absorption spectrum of hepatic mitochondria) (A) and microsomal (B) lipids after 2 days of dietary treatment ~rith orotic acid (1 .5X) " Ordinate : optical density Abscissa : wavelenght (nm) .
(fi0 .1X) . 1 cm
Plotted points are means of either 4 treated (~~) or 4 control (~---0) rats .
occurs in the mitochondria) and microsomal lipids during the experimental period, as far as the shifting of the double bonds in the unsaturated lipids is concerned.
Vol . 10, No . Z
Lipoperoaddation in Rat Liver
105
r~ L5
A
LO
FIG . 2a - Ultraviolet absorption spectrum of hepatic mitochondrial (A) and microsomal (H) lipids after 4 days of dietary treatment with orotic acid (1 .5?~) " For other details see fig . 2 .
0 N L8
ns 0
_ . ., . : . t20
2"0
2~0
ä0
700 nm
L6
A
1A
0 .5
0 d,_______- .- .
.
.ti
For other details see fig . 2 .
~s
B
La
Q8
0
2L0 ' 220 '
FIG . 2b - Ultraviolet absorption spectrum of hepatic mitochondrial (A) and microsomal (B) lipids after 8 days of dietary treatment with orotic acid (1 .5i) "
280
280
300 n m
108
Lipoperaaidation in Rat Liver
vol . 10, No . 73
In addition, no significant change in the fatty acid pattern of the phospholipids of the cytoplasmic particles occurs within 8 days of orotic acid treatment ( Figures 3 and 4 ) .
100 C 1&0 C 16=1
c .Q
60
in
i0
v c v â
C 16~0 C 18~1 C 16i2 C 20~i
0
C
OA
2 days
C
OA
4 days
C
OA
8days
FIG . 3 - Fatty acid pattern in hepatic mitochondrial phospholipids of rats fed For 2, 4 and 8 days on a diet devoid of (C) or supplemented (OA) with orotic acid (1 .5x) . 100 60
- C 16~0
_ C 16t1 C tB0 60
'v u
i0 20
v
â
C18~1 C16~2
Ill il~~
C20~~
0 C OA 2 days
C OA 4days
C OA 8 days
FIG . 4 - Fatty acid pattern in hepatic microsomal phospholipids of rats fed For 2, 4 and 8 days on a diet devoid of (:) or supplemented (OA) with orotic acid (1 .570 .
Vol. 10, No . 2
Fatty acids : C16 :0 C16 :1 C18 :0 C18 :1 C18 :2 C20 :4
Lipoperaaddation in Rat Liver
paLnitic acid palmitoleic acid stearic acid oleic acid linoleic acid arachidonic acid
Values are the means From groups of 4 animals .
The results reported in Figure 5 demonstrate that the alterations in lipid content of mitxhondria and microsomes are accounted Por by an increase in triglycerides, while the polar lipids are not affected after 8 days oP-treatment .
e aays
h"CROSOMAL FRACTION
FIß .
MITOCFpNORIAL FRACTION
5
Hepatic concentration in mitochondrial and microsomal lipids of rats treated with orotic acid For 8 days . Treatments : C : diet devoid of orotic acid OA : diet containing orotic acid . Values represent the means t standard error (vertical bar) of 4 esperiments .
10 8
Lipoperaaddation in Rat Liver
Vol. 10, No . 2
DISCIISSION The present findings confirm our previous results shoving that spontaneous lipid peroxidation of liver homogenate in vitro is enhanced by dietary treatment with orotic acid(5) .
The increase
in malonyldialdehyde production occurs earlier than the detectable onset of triglyceride accumulation (2 days) . The administration of either DPPD or adenine prevents both the changes in lipid peroxidation and fat infiltration . These data seemed to support the hypothesis that the orotic acid induced Patty liver is mediated by enhanced lipid peroxidation, reported by 1Cinsella(3 .4) . This interpretation has been suggested by the comparison of the findings of the orotic acid experiments with the pathophysiological behaviour of rat liver after CC14 poisoning, which stimulates
liver lipid peroxidation( . 15-18) " However, only these results,
out of those obtained in our experiments, might be considered as support for the before mentioned hypothesis . In fact, the presence of either adenine or orotic acid does not affect the spontaneous production of malonyldialdehyde in vitro when liver homogenate (pH either 5 .6 or 7 .4) contains either of these compounds at concentrations ranging between 10-5 and 10-2 M . In addition, it is yell known that in the fatty liver induced by CC14 or white phosphorus an early damage of the subcellular particles, namely the shifting of the double bonds in the
unsaturated Fatty acids, has been detected after poisoning( 1 9r 20 ), In our experiments, this line of work Failed to confirm the hypothesis that lipoperoxidation of the cytoplasmic particles is involved in the pathogenesis of orotic acid fatty liver . The data reported in Figure 2 clearly shoes that no peroxidatioe alterations take place in either mitochondria or microsomes within 8 days of dietary treatment . Furthermore, the peroxidatioe attack on the unsaturated lipids might be followed by detecting the exhaustion oP the substrate oP the reaction . For instance, after CC14 intoxication,
Vol . 10, No. 2
Lipoperaaidation in Rat Liver
109
a lover content oP arachidonic acid in the liver microso~mes has been qualitatively and quantitatively Pound(~ .9) . On the contrary, our experiments demonstrate .that orotic acid Feeding does not affect significantly either the per cent distribution of the unsaturated fatty acid in membrane phospholipids or the hepatic content oP mitochondrial and microsomal polar lipids . These observations seem to rule out any direct damage by lipid peroxidation on the subcellular particles which are involved in the triglyceride secretion and are inconsistent with the hypothesis that lipoperoxidation is dtirectly responsible Por the orotic acid induced fatty liver. The enhanced malonyldialdehyde production by liver homogenate possibly depends on a lowered stores of dietary or endogenous antioxidants occurring at the onset of fat infiltration . The protective ePFect of the administration of DPPD in vivo could reflect the reversal of this phenomenon . Treatment with this lipid soluble free radical scavenger has been reported to increase the antioxidant concentration in the liver oP both normal aad poisoned rats( 2~) . The explanation of the protective effect of DPPD on hepatic triglyceride accumulation does not yet appear conclusive i.n supporting the peroxidative point of view . This antioxidant, injected daily into the peritoneal cavity from 2 up to 5 days, progressively lowers the efficiency of the enzymes of endoplasmic reticulum (hexobarbital sleeping time)( 22 ) . These results suggest that, in addition to its antioxidant properties, DPPD may also prevent fatty liver by depressing the enzymatic activities involved in the metabolism of CC14 and make less cogent the indirect "ex-adiuvantibus" criterion used by several investigators in supporting the peroxidative view of the pathogenesis of some types of toxic Patty liver.
11 0
Lipoperaaddation im Rat Liver
Yol. 10, No . 2
SUMMARY The non-enzymatic production of malonyldialdehyde by liver homogenate is enhanced during the earliest stages of dietary administration of orotic acid . These alterations are prevented by injecting either DPPD or adenine . However, the ultraviolet absorption spectrum of total lipids and the concentration of plyunsaturated Patty acids of phospholipids of the mitochondria) and microsomal fractions were unchanged during the steatogenic treatment . These findings are not consistent with the hypothesis which envisions a direct attack by lipoperoxi.dation, induced by the metabolism of orotfc acid, on the unsaturated lipids of the cell membranes .
Acknowledgements : This work has been supported by "Consiglio Naziorale delle Ricerche", Koma .
RSFSRSNCSS 1 . H.G . Vindmueller, J . Hioi . Chew . 2~, 530 (1964) . 2 . P .3 . Roheim, 3 . Svitzer, A. Girard and H .A . Sder, Hiochem . Hiophys . Res . Coaunun. ~, 416 (1965 ) . 3 . J" 8. tinselia, Hiochim . Biopiers . Acta ~, 205 (1967) . 4 . J" g. tinsella, Canad. J . Biochem. x,1206 (1967) . 5 . M.V . Tonfell and G . Ugazio, Life 3ci . 9, 1 (1970) . 6 . 3 . Lui, S .M . 3abesin and X .J . Isse3bacher, Fed: Proc . _ 29, 756 (1970) . 7 . R .O . Recknagel and A .X . Ghoshal, Lab . Invest : 1~, 132 (1966) . 8 . R.O . Recknagel and A.X . ßhoshal, Sxp. Mol . Pathol . 5, 413 s 9 . M . Comporti, S . Hutdino and G . Ugazio, in preparation
Vol . 10, No. 2
Lipoperaoddaüon in Rat Liver
111
10 . J " G " Bieri and A .A . Anderson, Arch . Biochem . 90, 105 (1964) . 11 . J" Folch, M . Lees and G .H . Sloane-Stanley, J . Biol . Chem . 226, 497 (1957) . 12 . S .P . Chiang, C .F . Gessert and O .H . Lovry, Current list of the medical literature (Air University, Schoôl of Aviation Medicine, USAF) Research Report no . 56-113 (1957) . 13 . E . Van Handel and D .B . Zilversmit, J. Lab . Clin . Med . 15 2 (1957) . 14 . A .M . Chalvardjian, Hiochem . J . 90, 518 (1964) . 15 . M . Comporti, Boll . Soc . ital . Hiol . seer . 41, 222 (1965) " 16 . M. Comporti and C . Saccocci, Boll . Soc . ital . Hiol . spec . 41, 1066 (1965) . 17 . M. Comporti, C . Saccocci and IK .U . Dianzani, Snzymologia =, 185 (1965) . 18 . A .X . Ghoshal and R .O . Recknagel, Life ici . 4, 1521
(1965) "
19 . S .S . Rao and R .O . Recknagel, Bxp . Moles . Pathol . 9, 271 (1968) . 20 . A .X . Ghoshal, E .A . Porta and W .S . Hartroft, Am . J . Pathol . 54, 275 (1969) . 21 . N .R . Di Luzio and A .D . Hartman, Exp . Moles . Pathol . 11, 38 (1969) . 22 . 2~I .V . Torrielli, L . Gabriel and G . .Ugazio, in preparation.
Pergamon Press
BEHAVIOUR OF LIPOFEROXIDATION IN RAT LIVER DURING OROTIC ACID TREATN.EIv^P N~ .V . Torrielli, N:. U. Dianzani and G . Ugazio Istituto di Patologia Generale dell'Università di Torino 10125 Torino - Italy (Received 28 September 1970 ; in final form 7 December 1970) Experimental evidence indicates that both protein synthesis( ) and assembly of very low density lipoproteins( 2 ) are impaired in the livers of rats fed on a diet containing orotic acid . This treatment has been reported to cause an increase in the liver's content of lipid soluble compounds reacting with thiobarbituric acid, referred to by Kinsella( 3 ) as malonyldialdehyde . The orotic acid induced fatty liver has been prevented by a,-tocopherol( 4 ) . These observations were offered as evidence for the involvement of lipoperoxidation in the pathogenesis of the observed liver fat accumulation( 3 ' 4) . Recent work in our laboratory has demonstrated that DPPD (N,N'-biphenyl-p-phenylendiamine), a lipid soluble free radical scavenger, prevents fatty liver in rats when the diet contains orotic acid( 5 ) . However , although a severe derangement of the morphologic organization ofendoplasmic reticulum occurs very early after orotic acid administration( 6 ), to date, no evidence other than that of Kinsella( 3 ' 4 ) is available indicating that peroxidative alteration of membrane lipids is involved .
Experiments reporteà here took into consideration lines of
work previously used in order to evaluate in vivo lipid peroxidation of liver cell particles(~' 8 ' 9 ) . Both the ultraviolet absorption spectrum of total lipids and the arachidonic acid concentration in phospholipids were determined in mitochondria and microsomes of rat liver during dietary administration of orotic acid . In
addition, the spontaneous lipoperoxidation of liver homogenates 99
j,ipoperaaidation in Rat Liver
100
Vol . 10, No .
2
vas studied in rats under steatogenic treatment either in the presence or in the absence of DPPD or adenine injection . MATERIALS AND METHODS In vitro non-enzymatic lipid peroxidation oP liver hom
mates .
Male rats, 90-100 g initial body weight, of the Wistar strain, were randomly allotted into Four groups . Control animals (group A) were offered a diet containing casein (20X), corn starch (70X),
cellulose (3X), corn oil (4X), minerals (2X) and vitamins (1X) . This diet vas devoid of any non-natural antioxidant (Piccioni, Brescia, Italy) . Experimental rats (groups B, C and D) received the same diet provided with 1 .5X orotic acid (B .D .H ., Poole, England) which replaced an equal amount of corn starch . Fifty mg DPPD (Schuchardt, Munich, Germany) per 100 g of body weight or 20 adenine sulphate (H .D .H ., Poole, England) per 100 g of body weight, prepared as previously reported(5), were daily injected into the peritoneal cavity of the rats belonging to the groups C and D, respectively . Animals from each group were sacrificed after 2, 4 and 8 days of treatment . Liver homogenates (6X v/v), prepared at 4°C in a Potter-Elvehjem apparatus with 0 .05 M phosphate buffer, ph 5 .6 or 7 .4, containing 0 .15 M LC1, were shaken in a metabolic incubator (Dubnoff, 37 °C) For up to 240 minutes . Malonyldialdehyde production vas determined on the trichloroacetic acid-soluble Fraction oP the homogenate after incubation and treatment with thiobarbituric acid( 10 ), Followed by XOH(17), IIltraviolet absorpti on spectrwn of lipids from mitochondria and microsomes . Male rats, 200-220 g initial body weight, oP the Wistar strain, were used in this series of experiments . The animals, divided into two groups, were fed on the before mentioned diet either devoid of or provided with orotic acid (1 .5X), respectively . They were starved for 16 hours and then sacrificed at 2, 4 or 8 days oP dietary treatment . Liver homogenates (10X v/v), prepared at 4°C in
Vol. 10, No . 2
Lipoperaaidation in Rat Liver
101
a Potter-Flvehjem apparatus, with 0 .25 M sucrose containing 3 mM EDTA (sodium salt ; Fisher Chem . Co ., Fair Lawn, N .J .), vas spun at 1,000 x g For 10 minutes in a Servall centrifuge . The decanted supernate was made up to the initial volume and then centrifuged at 10,000 x g for 25 minutes . The microsomal fraction vas harvested by centrifuging the second supernate at 78,500 x g for 80 minutes (Spinco preparative ultracentrifuge, rotor no . 30) .
Both the
10,000 x ~ and the 78,500 x ~ sediments were extracted according to the method of Folch, Lees and Sloane-Stanley( 11 ) . The washed lipids were dried down under an oxygen-free nitrogen stream, dissolved in cyclohexane (spectrophotometric grade: C . Erba, Milano, Italy) and scanned in the ultraviolet region with a Beckman spectrophotometer (model DH) .
The absorption spectrum vas
normalized on the basis of the lipid concentration (1 mg per ml) which was assayed with the potassium dichromate-sulfuric acid method of Chiang, Gessert and Lovry( 12 ) . Lipid Fractions of the cytoplasmic particles . The lipids extracted from both mitochondria and microsomes oP rat liver at the eighth day of orotic acid treatment were further fractionated . Triglycerides were separated by means of thin layer chromatography and estimated with the chromotropic acid method of Van Handel and Zilversmit( 13 ) . Neutral and polar lipid Fractions were eluted with chloroform and methanol, respectively, from silicic acid columns (~iallinckrodt Chem . Works, ST Louis, Mo) and then photometrically determined( 12 ) . Fatty acid pattern in mitochondrial and microsomal phospholipids . An aliquot of the lipids extracted from the cytoplasmic particles was fractionated on silica gel thin-layer plates . Phospholipids were scraped off and submitted to hydrolytic methyl ation( 14 ) . Fatty acid methyl esters were then separated with a gas liquid chromatography apparatus (Fractovap, model GV, C . Erba,
102
Lipoperoaidation in Rat Liver
Vol. 10, No . 2
hilano, Italy), according to a procedure as reported by Comporti, Bardino and Ugazio( 9 ) . Data are expressed as per-cent distribution of the fatty acid fractions . RSSULTS Treatment with orotic acid in vivo induces an increase in the spontaneous production of malonyldialdehyde by rat liver homogenato when incubation occurs in an acidic medium (pH 5 .6) (Figures 1, 1a, 1b) . This phenomenon is particularly evident 2 days after
p H 5.6
p H 7.4
FIG . 1 - Rate of in vitro malonyldialdehyde production by liver homogenate from rats fed on a diet containing 1 .5X orotic acid for 2 days .
Ordinate : optical density at 543 nm of the compounds reacting with thiobarbituric acid, produced by 45 mg-equiv liver, per ml of final volume .
Abscissa : incubation time, in hours .
Treatments : C : diet devoid of orotic acid ; OA : diet containing orotic acid ; OA-DPPD : diet containing orotic acid fed to animals daily treated with 50 mg DPPD per 100 g b .wt, intraperitoneally ; OA-Ad : diet containing orotic acid, fed to animals daily injected with 20 mg adenine sulphate per 100 g b .wt, intraperitoneally . Plotted points represent the values obtained with duplicate estimations on samples of 4 pooled livers .
Vol: 10, No . 2
Lipoperaa~idation in Rat Liver
pH
s.e
103
pH 7.4
FIG . 1a - Rate of in vitro malonyldialdehyde production by liver homogenate from rats Fed on a diet containing 1 .5X orotic acid For 4 days . For other details see Figure 1 .
C
OA
_
I
~~ 0 .1 0 0
OA+OrPO
OAtAd
FIG . 1b - Rate of in vitro malonyldialdehyde production by liver homogenate from rats fed on a diet containing 1 .5X orotic acid for 8 days . For other details see Figure 1 .
104
Lipoperoaidation in Rat Liver
Yol . 10, No . 2
orotic acid feeding and is prevented by the concomitant injection of either DPPD or adenine both of arhich are kno~m to inhibit the orotic acid induced triglyceride accumulation in the liver( 5 ) . Figures 2, 2a and 2b shov that no peroxidative alteration
~.s ~.o 45 ti. ,,., .._,\~ 0 N
FIG . 2 - Ultraviolet absorption spectrum of hepatic mitochondria) (A) and microsomal (B) lipids after 2 days of dietary treatment ~rith orotic acid (1 .5X) " Ordinate : optical density Abscissa : wavelenght (nm) .
(fi0 .1X) . 1 cm
Plotted points are means of either 4 treated (~~) or 4 control (~---0) rats .
occurs in the mitochondria) and microsomal lipids during the experimental period, as far as the shifting of the double bonds in the unsaturated lipids is concerned.
Vol . 10, No . Z
Lipoperoaddation in Rat Liver
105
r~ L5
A
LO
FIG . 2a - Ultraviolet absorption spectrum of hepatic mitochondrial (A) and microsomal (H) lipids after 4 days of dietary treatment with orotic acid (1 .5?~) " For other details see fig . 2 .
0 N L8
ns 0
_ . ., . : . t20
2"0
2~0
ä0
700 nm
L6
A
1A
0 .5
0 d,_______- .- .
.
.ti
For other details see fig . 2 .
~s
B
La
Q8
0
2L0 ' 220 '
FIG . 2b - Ultraviolet absorption spectrum of hepatic mitochondrial (A) and microsomal (B) lipids after 8 days of dietary treatment with orotic acid (1 .5i) "
280
280
300 n m
108
Lipoperaaidation in Rat Liver
vol . 10, No . 73
In addition, no significant change in the fatty acid pattern of the phospholipids of the cytoplasmic particles occurs within 8 days of orotic acid treatment ( Figures 3 and 4 ) .
100 C 1&0 C 16=1
c .Q
60
in
i0
v c v â
C 16~0 C 18~1 C 16i2 C 20~i
0
C
OA
2 days
C
OA
4 days
C
OA
8days
FIG . 3 - Fatty acid pattern in hepatic mitochondrial phospholipids of rats fed For 2, 4 and 8 days on a diet devoid of (C) or supplemented (OA) with orotic acid (1 .5x) . 100 60
- C 16~0
_ C 16t1 C tB0 60
'v u
i0 20
v
â
C18~1 C16~2
Ill il~~
C20~~
0 C OA 2 days
C OA 4days
C OA 8 days
FIG . 4 - Fatty acid pattern in hepatic microsomal phospholipids of rats fed For 2, 4 and 8 days on a diet devoid of (:) or supplemented (OA) with orotic acid (1 .570 .
Vol. 10, No . 2
Fatty acids : C16 :0 C16 :1 C18 :0 C18 :1 C18 :2 C20 :4
Lipoperaaddation in Rat Liver
paLnitic acid palmitoleic acid stearic acid oleic acid linoleic acid arachidonic acid
Values are the means From groups of 4 animals .
The results reported in Figure 5 demonstrate that the alterations in lipid content of mitxhondria and microsomes are accounted Por by an increase in triglycerides, while the polar lipids are not affected after 8 days oP-treatment .
e aays
h"CROSOMAL FRACTION
FIß .
MITOCFpNORIAL FRACTION
5
Hepatic concentration in mitochondrial and microsomal lipids of rats treated with orotic acid For 8 days . Treatments : C : diet devoid of orotic acid OA : diet containing orotic acid . Values represent the means t standard error (vertical bar) of 4 esperiments .
10 8
Lipoperaaddation in Rat Liver
Vol. 10, No . 2
DISCIISSION The present findings confirm our previous results shoving that spontaneous lipid peroxidation of liver homogenate in vitro is enhanced by dietary treatment with orotic acid(5) .
The increase
in malonyldialdehyde production occurs earlier than the detectable onset of triglyceride accumulation (2 days) . The administration of either DPPD or adenine prevents both the changes in lipid peroxidation and fat infiltration . These data seemed to support the hypothesis that the orotic acid induced Patty liver is mediated by enhanced lipid peroxidation, reported by 1Cinsella(3 .4) . This interpretation has been suggested by the comparison of the findings of the orotic acid experiments with the pathophysiological behaviour of rat liver after CC14 poisoning, which stimulates
liver lipid peroxidation( . 15-18) " However, only these results,
out of those obtained in our experiments, might be considered as support for the before mentioned hypothesis . In fact, the presence of either adenine or orotic acid does not affect the spontaneous production of malonyldialdehyde in vitro when liver homogenate (pH either 5 .6 or 7 .4) contains either of these compounds at concentrations ranging between 10-5 and 10-2 M . In addition, it is yell known that in the fatty liver induced by CC14 or white phosphorus an early damage of the subcellular particles, namely the shifting of the double bonds in the
unsaturated Fatty acids, has been detected after poisoning( 1 9r 20 ), In our experiments, this line of work Failed to confirm the hypothesis that lipoperoxidation of the cytoplasmic particles is involved in the pathogenesis of orotic acid fatty liver . The data reported in Figure 2 clearly shoes that no peroxidatioe alterations take place in either mitochondria or microsomes within 8 days of dietary treatment . Furthermore, the peroxidatioe attack on the unsaturated lipids might be followed by detecting the exhaustion oP the substrate oP the reaction . For instance, after CC14 intoxication,
Vol . 10, No. 2
Lipoperaaidation in Rat Liver
109
a lover content oP arachidonic acid in the liver microso~mes has been qualitatively and quantitatively Pound(~ .9) . On the contrary, our experiments demonstrate .that orotic acid Feeding does not affect significantly either the per cent distribution of the unsaturated fatty acid in membrane phospholipids or the hepatic content oP mitochondrial and microsomal polar lipids . These observations seem to rule out any direct damage by lipid peroxidation on the subcellular particles which are involved in the triglyceride secretion and are inconsistent with the hypothesis that lipoperoxidation is dtirectly responsible Por the orotic acid induced fatty liver. The enhanced malonyldialdehyde production by liver homogenate possibly depends on a lowered stores of dietary or endogenous antioxidants occurring at the onset of fat infiltration . The protective ePFect of the administration of DPPD in vivo could reflect the reversal of this phenomenon . Treatment with this lipid soluble free radical scavenger has been reported to increase the antioxidant concentration in the liver oP both normal aad poisoned rats( 2~) . The explanation of the protective effect of DPPD on hepatic triglyceride accumulation does not yet appear conclusive i.n supporting the peroxidative point of view . This antioxidant, injected daily into the peritoneal cavity from 2 up to 5 days, progressively lowers the efficiency of the enzymes of endoplasmic reticulum (hexobarbital sleeping time)( 22 ) . These results suggest that, in addition to its antioxidant properties, DPPD may also prevent fatty liver by depressing the enzymatic activities involved in the metabolism of CC14 and make less cogent the indirect "ex-adiuvantibus" criterion used by several investigators in supporting the peroxidative view of the pathogenesis of some types of toxic Patty liver.
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Lipoperaaddation im Rat Liver
Yol. 10, No . 2
SUMMARY The non-enzymatic production of malonyldialdehyde by liver homogenate is enhanced during the earliest stages of dietary administration of orotic acid . These alterations are prevented by injecting either DPPD or adenine . However, the ultraviolet absorption spectrum of total lipids and the concentration of plyunsaturated Patty acids of phospholipids of the mitochondria) and microsomal fractions were unchanged during the steatogenic treatment . These findings are not consistent with the hypothesis which envisions a direct attack by lipoperoxi.dation, induced by the metabolism of orotfc acid, on the unsaturated lipids of the cell membranes .
Acknowledgements : This work has been supported by "Consiglio Naziorale delle Ricerche", Koma .
RSFSRSNCSS 1 . H.G . Vindmueller, J . Hioi . Chew . 2~, 530 (1964) . 2 . P .3 . Roheim, 3 . Svitzer, A. Girard and H .A . Sder, Hiochem . Hiophys . Res . Coaunun. ~, 416 (1965 ) . 3 . J" 8. tinselia, Hiochim . Biopiers . Acta ~, 205 (1967) . 4 . J" g. tinsella, Canad. J . Biochem. x,1206 (1967) . 5 . M.V . Tonfell and G . Ugazio, Life 3ci . 9, 1 (1970) . 6 . 3 . Lui, S .M . 3abesin and X .J . Isse3bacher, Fed: Proc . _ 29, 756 (1970) . 7 . R .O . Recknagel and A .X . Ghoshal, Lab . Invest : 1~, 132 (1966) . 8 . R.O . Recknagel and A.X . ßhoshal, Sxp. Mol . Pathol . 5, 413 s 9 . M . Comporti, S . Hutdino and G . Ugazio, in preparation
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10 . J " G " Bieri and A .A . Anderson, Arch . Biochem . 90, 105 (1964) . 11 . J" Folch, M . Lees and G .H . Sloane-Stanley, J . Biol . Chem . 226, 497 (1957) . 12 . S .P . Chiang, C .F . Gessert and O .H . Lovry, Current list of the medical literature (Air University, Schoôl of Aviation Medicine, USAF) Research Report no . 56-113 (1957) . 13 . E . Van Handel and D .B . Zilversmit, J. Lab . Clin . Med . 15 2 (1957) . 14 . A .M . Chalvardjian, Hiochem . J . 90, 518 (1964) . 15 . M . Comporti, Boll . Soc . ital . Hiol . seer . 41, 222 (1965) " 16 . M. Comporti and C . Saccocci, Boll . Soc . ital . Hiol . spec . 41, 1066 (1965) . 17 . M. Comporti, C . Saccocci and IK .U . Dianzani, Snzymologia =, 185 (1965) . 18 . A .X . Ghoshal and R .O . Recknagel, Life ici . 4, 1521
(1965) "
19 . S .S . Rao and R .O . Recknagel, Bxp . Moles . Pathol . 9, 271 (1968) . 20 . A .X . Ghoshal, E .A . Porta and W .S . Hartroft, Am . J . Pathol . 54, 275 (1969) . 21 . N .R . Di Luzio and A .D . Hartman, Exp . Moles . Pathol . 11, 38 (1969) . 22 . 2~I .V . Torrielli, L . Gabriel and G . .Ugazio, in preparation.