SNORT
EFFECT
OF
IN
COMMUNICATIONS
4-PENTENOIC
PLASMA
Tadashi
FUJITA
AND
Japan. J. Pharmacol. 25, 73 (1975)
ACID LIVER
and Masahide
OF
ON
LIPIDS
RATS
YASUDA
Department of FTvgicnic Chemistry, Osaka College of Pharmacy, A-latsrrhara, Osaka, Japan Accepted October 4, 1974
It has been reported that 4-pentenoic acid (4-PA) inhibited fatty acid oxidation in in vitro systems, but causes little inhibition of oxidative phosphorylation in mitochondria (1, 2, 3). Kleinman et al. (4) have suggested that in vivo perfusion of dog kidneys with 4-PA produces a natriuresis, kaliuresis, and bicarbonate diuresis independent of changes in renal hemodynamics and which appear to be due to inhibition of the metabolic energy sources for active transport. To consider the biological action of 4-PA, we have examined the effects of 4-PA on free fatty acids (FFA) and triglycerides (TG) in plasma and liver of rats, and also attempted to determine its effect on the peroxidation of polyunsaturated fatty acid. Wistar strain male rats, weighing 150 to 250g, were used as test subjects. 4-PA in saline solution or saline alone for control was injected i.p.. 4-PA was given once a day at a dose of 50 mg/kg for 3 days. Blood samples were taken from all rats after which the animals were exsanguinated. The liver was removed 24 hr after the final treatment. Plasma and liver homogenate (prepared in ice-cold 0.25 M sucrose, 1: 9, w/v) were used as samples to be assayed for FFA and TG. Mitochondrial fraction was prepared by the method of Hogeboom (5). Protein was determined by the method of Lowry et al. (6).
SHORT
COMMUNICATIONS Japan. J. Pharmacol.
25, 74 (1975)
Mitochondrial respiration was measured according to the procedure described by Hagi hara (7), lipid peroxidation of mitochondria was estimated by the thiobarbituric acid (TBA) reaction, as described in a previous paper (8), and fluorescent products derived from mitochondria were measured by the technique of Bidlack et al. (9). Concentration of TG in plasma and liver was assayed colorimetrically by the procedure of Naito et al. (10), and FFA by the method of Itaya et al. (11). The fatty acid composition of plasma and liver was determined by gas-liquid chromatography (12). Following i.p. administration of 4-PA in a dose of 50 mg/kg/day for 3 consecutive days, concentration of TG in liver decreased from 6.99 0.44 mg/g tissue of the control experiment to 3.51 0.35 mg/g tissue, and also decreased in plasma by approx. 32'%, as compared to that of control animals. Concentration of FFA in plasma and liver of con trol animals was 0.488='0.035 mFq/ l, 15.54-L-0.62 /ig/g tissue, respectively. By treat ment with 4-PA for 3 consecutive days, content of FFA in the liver was decreased by ap prox. 45",1,and by 60",' in plasma. The major saturated fatty acids in both plasma and liver of normal animals were ('16:0 and Cis :o, and the major unsaturated fatty acids were C,9:1, C18:2,C20:4and C22,6 In the individual fatty acids of the plasma,a consistentlyhigher percentage of C18:1and CI8:2was found while the percentage of C18:owas found to be lower than in the liver. However,there was no significant difference in the changes of fatty acid composition in both plasma and liver from animals given 4-PA for 3 days (Table 1). TABLE: 1. Fatty nistration
acid composition in plasma of 4-pentenoic acid')
and liver
after
intraperitoneal
admi
a) i.p. administration of 4-pentenoic acid (50 mg/kg/day, x 3) All values are the mean±S.E. of percentage of total fatty acids in 5 experiments. Only the major acids are listed.
Lipid peroxidation
has been noted as a metabolic
but the mechanism
in rim remains
in vivo is inhibited
by the administration
endogenous
polyunsaturated
18), we have determined mitochondria
of 4-PA,
fatty acids.
the fluorescent
According products
after daily i.p. administration
effect on the lipid peroxidation the endogenous
to be elucidated
respiration
(13, 14).
related
to several diseases,
If the fatty acid oxidation
lipid peroxides to Tappel
and TBA-reactive
of 4-PA for 3 days.
in mitochondria.
of mitochondria
disorder
On the other
must
be derived
and his co-workers substances However,
(15
in rat liver
4-PA
hand, oxygen
from
had no
utilized
in
and in the state 3 was 15.39 and 102 ,tM, re
SHORT
C'0,1 I M UNJC'A TIO NS
Japan.
J. Pharmacol.
25, 75 (1975)
FIG. 1. A comparison of the extent of oxygen consumption in rat liver mitochondria after i.p. administration of 4-pentenoic acid (50 mg/kg/day, x 3). Rates of oxygen consumption were measured with a oxygen electrode (Yanagi moto) in 2 ml closed chamber at 25°C. The reaction medium contained 8 mg of mitochondrial protein in a buffer containing 0.25 M sucrose, 0.01 M KCI, 2 mM MgCI2, 0.2 mM EDTA, 0.01 M Tris-chloride and 0.01 M potassium phosphate, pH 7.2. The additions were 0.2 M succinate and 0.042 M ADP. ; Control rate, - ; Effect of 4-pentenoic acid
spectively
(Fig.
1).
treatment
with 4-PA
The former
was inhibited
about
i.p.) for 3 days.
is known of the regulatory
steps of plasma is considered,
in mitochondria
in lipid metabolism
Acknowledgements: Tamura
for technical
TG transport.
Further
are grateful
to Mr.
Con
after daily administration
is difficult
to explain
since little
When the result of oxygen con
the effect of 4-PA may be a decrease
of the liver.
The authors
significantly
This development
sumption
activity
30", , by
of lipid in plasma and liver is still unknown.
tent of TG and FFA in plasma and liver decreased
tional
about
for 3 days.
The effect of 4-PA on mobilization
of 4-PA (50 mg/kg,
50")" and the latter
studies
of the func
are in progress.
K. Matsumoto
and
Miss Y.
assistance.
REFERENCES 1) BRENDFL,K., CORREDOR,C.F. AND BRESSLER,R.: Blocheln. biophys. Res. Commun. 34, 340 (1969); 2) FUKAMT,M.H. ANDWILLIAMSON, J.R.: J. biol. Chem. 246, 1206 (1971); 3) SENIOR, A.E., ROBSON,B. AND SI4ERRATT,H.S.A.: Biochem. J. 110, 511 (1968); 4) KLEINMAN,J.G., MANDELBAUM, .1. AND LEVIN, M.L.: Am. J. Physiol. 224, 95 (1973); 5) HOGEBOOM,G.H.: Method in F_n:vmrmlogv,Fdited by COIOwWK, S.Y. AND KAPI.AN,N.(7., Vol. 1, p.16, Academic Press, NcNv York and London (1955); (i) LoWRV, O.H., RostBRODU!!, N.H., FARtz, A.F,. ANh RANDALt, R..1.:.1. hint. Chem. 193, 265 (1951); 7) I IAU1nARA,B.: Protein, Nucleic acid, En
SHORT
COMMUNICATIONS
Japan. J. Pharmacol. 25, 76 (1975)
zyme 10, 1689 (1965) (in Japanese); 8) FUJITA,T.: Yakugaku Zasshi 93, 756 (1973) (in Japanese); 9) BIDLACK,W.R. ANDTAPPEL,A.L.: Lipids 8, 177 (1973); 10) NAITO,C., USUI, M., KOHAYA KAWA,K., OKANIWA,H. AND1CHIDA,T.: Igaku noayumi 57, 551 (1966) (in Japanese); 11) ITAYA, K. AND UI, M.: J. Lipid Res. 6, 16 (1965); 12) FUJITA, T. AND YASUDA,M.: Japan. J. Phar macol. 23, 899 (1973); 13) FUJITA, T.: Yakugaku Zasshi 93, 765 (1973) (in Japanese); 14) FUJITA, T.: Yakugaku Zasshi 94, 215 (1974) (in Japanese); 15) CHIo, K.S. AND TAPPEL,A.L.: Biochemistry 8, 2821 (1969); 16) DILLARD,C.J. AND TAPPEL, A.L.: Lipids 6, 715 (1971); 17) MALSI-IET,V.G. AND TAPPEL, A.L.: Lipids 8, 194 (1973); 18) BIDLACK, W.R. AND TAPPEL, A.L.: Lipids 8, 203 (1973)
EFFECT
OF
IN
COMMUNICATIONS
4-PENTENOIC
PLASMA
Tadashi
FUJITA
AND
Japan. J. Pharmacol. 25, 73 (1975)
ACID LIVER
and Masahide
OF
ON
LIPIDS
RATS
YASUDA
Department of FTvgicnic Chemistry, Osaka College of Pharmacy, A-latsrrhara, Osaka, Japan Accepted October 4, 1974
It has been reported that 4-pentenoic acid (4-PA) inhibited fatty acid oxidation in in vitro systems, but causes little inhibition of oxidative phosphorylation in mitochondria (1, 2, 3). Kleinman et al. (4) have suggested that in vivo perfusion of dog kidneys with 4-PA produces a natriuresis, kaliuresis, and bicarbonate diuresis independent of changes in renal hemodynamics and which appear to be due to inhibition of the metabolic energy sources for active transport. To consider the biological action of 4-PA, we have examined the effects of 4-PA on free fatty acids (FFA) and triglycerides (TG) in plasma and liver of rats, and also attempted to determine its effect on the peroxidation of polyunsaturated fatty acid. Wistar strain male rats, weighing 150 to 250g, were used as test subjects. 4-PA in saline solution or saline alone for control was injected i.p.. 4-PA was given once a day at a dose of 50 mg/kg for 3 days. Blood samples were taken from all rats after which the animals were exsanguinated. The liver was removed 24 hr after the final treatment. Plasma and liver homogenate (prepared in ice-cold 0.25 M sucrose, 1: 9, w/v) were used as samples to be assayed for FFA and TG. Mitochondrial fraction was prepared by the method of Hogeboom (5). Protein was determined by the method of Lowry et al. (6).
SHORT
COMMUNICATIONS Japan. J. Pharmacol.
25, 74 (1975)
Mitochondrial respiration was measured according to the procedure described by Hagi hara (7), lipid peroxidation of mitochondria was estimated by the thiobarbituric acid (TBA) reaction, as described in a previous paper (8), and fluorescent products derived from mitochondria were measured by the technique of Bidlack et al. (9). Concentration of TG in plasma and liver was assayed colorimetrically by the procedure of Naito et al. (10), and FFA by the method of Itaya et al. (11). The fatty acid composition of plasma and liver was determined by gas-liquid chromatography (12). Following i.p. administration of 4-PA in a dose of 50 mg/kg/day for 3 consecutive days, concentration of TG in liver decreased from 6.99 0.44 mg/g tissue of the control experiment to 3.51 0.35 mg/g tissue, and also decreased in plasma by approx. 32'%, as compared to that of control animals. Concentration of FFA in plasma and liver of con trol animals was 0.488='0.035 mFq/ l, 15.54-L-0.62 /ig/g tissue, respectively. By treat ment with 4-PA for 3 consecutive days, content of FFA in the liver was decreased by ap prox. 45",1,and by 60",' in plasma. The major saturated fatty acids in both plasma and liver of normal animals were ('16:0 and Cis :o, and the major unsaturated fatty acids were C,9:1, C18:2,C20:4and C22,6 In the individual fatty acids of the plasma,a consistentlyhigher percentage of C18:1and CI8:2was found while the percentage of C18:owas found to be lower than in the liver. However,there was no significant difference in the changes of fatty acid composition in both plasma and liver from animals given 4-PA for 3 days (Table 1). TABLE: 1. Fatty nistration
acid composition in plasma of 4-pentenoic acid')
and liver
after
intraperitoneal
admi
a) i.p. administration of 4-pentenoic acid (50 mg/kg/day, x 3) All values are the mean±S.E. of percentage of total fatty acids in 5 experiments. Only the major acids are listed.
Lipid peroxidation
has been noted as a metabolic
but the mechanism
in rim remains
in vivo is inhibited
by the administration
endogenous
polyunsaturated
18), we have determined mitochondria
of 4-PA,
fatty acids.
the fluorescent
According products
after daily i.p. administration
effect on the lipid peroxidation the endogenous
to be elucidated
respiration
(13, 14).
related
to several diseases,
If the fatty acid oxidation
lipid peroxides to Tappel
and TBA-reactive
of 4-PA for 3 days.
in mitochondria.
of mitochondria
disorder
On the other
must
be derived
and his co-workers substances However,
(15
in rat liver
4-PA
hand, oxygen
from
had no
utilized
in
and in the state 3 was 15.39 and 102 ,tM, re
SHORT
C'0,1 I M UNJC'A TIO NS
Japan.
J. Pharmacol.
25, 75 (1975)
FIG. 1. A comparison of the extent of oxygen consumption in rat liver mitochondria after i.p. administration of 4-pentenoic acid (50 mg/kg/day, x 3). Rates of oxygen consumption were measured with a oxygen electrode (Yanagi moto) in 2 ml closed chamber at 25°C. The reaction medium contained 8 mg of mitochondrial protein in a buffer containing 0.25 M sucrose, 0.01 M KCI, 2 mM MgCI2, 0.2 mM EDTA, 0.01 M Tris-chloride and 0.01 M potassium phosphate, pH 7.2. The additions were 0.2 M succinate and 0.042 M ADP. ; Control rate, - ; Effect of 4-pentenoic acid
spectively
(Fig.
1).
treatment
with 4-PA
The former
was inhibited
about
i.p.) for 3 days.
is known of the regulatory
steps of plasma is considered,
in mitochondria
in lipid metabolism
Acknowledgements: Tamura
for technical
TG transport.
Further
are grateful
to Mr.
Con
after daily administration
is difficult
to explain
since little
When the result of oxygen con
the effect of 4-PA may be a decrease
of the liver.
The authors
significantly
This development
sumption
activity
30", , by
of lipid in plasma and liver is still unknown.
tent of TG and FFA in plasma and liver decreased
tional
about
for 3 days.
The effect of 4-PA on mobilization
of 4-PA (50 mg/kg,
50")" and the latter
studies
of the func
are in progress.
K. Matsumoto
and
Miss Y.
assistance.
REFERENCES 1) BRENDFL,K., CORREDOR,C.F. AND BRESSLER,R.: Blocheln. biophys. Res. Commun. 34, 340 (1969); 2) FUKAMT,M.H. ANDWILLIAMSON, J.R.: J. biol. Chem. 246, 1206 (1971); 3) SENIOR, A.E., ROBSON,B. AND SI4ERRATT,H.S.A.: Biochem. J. 110, 511 (1968); 4) KLEINMAN,J.G., MANDELBAUM, .1. AND LEVIN, M.L.: Am. J. Physiol. 224, 95 (1973); 5) HOGEBOOM,G.H.: Method in F_n:vmrmlogv,Fdited by COIOwWK, S.Y. AND KAPI.AN,N.(7., Vol. 1, p.16, Academic Press, NcNv York and London (1955); (i) LoWRV, O.H., RostBRODU!!, N.H., FARtz, A.F,. ANh RANDALt, R..1.:.1. hint. Chem. 193, 265 (1951); 7) I IAU1nARA,B.: Protein, Nucleic acid, En
SHORT
COMMUNICATIONS
Japan. J. Pharmacol. 25, 76 (1975)
zyme 10, 1689 (1965) (in Japanese); 8) FUJITA,T.: Yakugaku Zasshi 93, 756 (1973) (in Japanese); 9) BIDLACK,W.R. ANDTAPPEL,A.L.: Lipids 8, 177 (1973); 10) NAITO,C., USUI, M., KOHAYA KAWA,K., OKANIWA,H. AND1CHIDA,T.: Igaku noayumi 57, 551 (1966) (in Japanese); 11) ITAYA, K. AND UI, M.: J. Lipid Res. 6, 16 (1965); 12) FUJITA, T. AND YASUDA,M.: Japan. J. Phar macol. 23, 899 (1973); 13) FUJITA, T.: Yakugaku Zasshi 93, 765 (1973) (in Japanese); 14) FUJITA, T.: Yakugaku Zasshi 94, 215 (1974) (in Japanese); 15) CHIo, K.S. AND TAPPEL,A.L.: Biochemistry 8, 2821 (1969); 16) DILLARD,C.J. AND TAPPEL, A.L.: Lipids 6, 715 (1971); 17) MALSI-IET,V.G. AND TAPPEL, A.L.: Lipids 8, 194 (1973); 18) BIDLACK, W.R. AND TAPPEL, A.L.: Lipids 8, 203 (1973)
