- Email: [email protected]
Effect of Orotic Acid on the Distribution of Phosphatidylcholine [32P]
Japan. J. Pharmacol. 25, 345 (1975)
SHORT COMMUNICATIONS
EFFECT
OF
OROTIC
ACID
ON
THE
OF
PHOSPHATIDYLCHOLINE
DISTRIBUTION [12p]
Isao NEGISHI and Yoshio AIZAWA Department of Pharmacology, Tokyo College of Pharmacy, Shinjuku-ku, Tokyo, Japan Accepted February 10, 1975
Triglyceride accumulation in the liver can be brought about by a disease state in man, and by the administration of a large variety of agents (1, 2). A block in the release of hepatic beta-lipoprotein into blood stream appears to be responsible for the development of a number of experimentally induced fatty liver conditions (3). Our previous results demonstrated that a decreased release of beta-lipoprotein from liver slices in rats fed orotic acid was restored by the addition of phosphatidylcholine. It was further found that phospholipid has a curative effect on fatty liver induced by orotic acid or ethionine. These findings suggest that phospholipid is an important factor in the normal release of beta-lipoprotein. Accordingly, it is of interest to examine the fate of ingested phosphatidylcholine. The present experiments were done to examine the distribution of phosphatidylcholine [32P]in rats fed orotic acid. Female Wistar strain rats weighing 180 to 210 g were first fed a semisyntheticdiet (4) for 2 days. Control rats were given a semisyntheticdiet alone. Groups of animals then were put on a dietary supplement of I ;o orotic acid for 7 days after which 0.373 or 0.824 /iCi of phosphatidylcholine [32P]to which was added 14 mg of non-radioactive phosphatidyl choline as carrier in 0.5 nil of saline solution (1.91 or 4.23 /tCi/Kg body weight) was in jected intraperitoneally. At the indicated time after administration of the phospholipid, the rats were sacrificed by heart puncture under ether anesthesia. Tissues were removed, homogenized with ice-cold 5 trichloroacetic acid, and centrifuged. The precipitate was washed twice with ice-cold trichloroacetic acid. Lipids were then extracted once with 80% ethanol, once with 100% ethanol, twice with chloroform-ethanol (1: 1, by vol.), and once with ether. Trichloroaceticacid was added to the serum, and centrifuged. Serum lipids were then extracted with chloroform-methanol (2: 1, by vol.). Radioactivity of acid-soluble and lipid fraction was determined in a liquid scintillation spectrometer using dioxan and toluene-phosphor solution, respectively. Phosphatidylcholine [32P]was prepared as follows: Liver slices were incubated with 1.25 mCi orthophosphate [32P]per ml of Krebs-Ringer bicarbonate buffer at 37' in 02 CO2 (95: 5, by vol.) for 5 hr. Lipids were extracted as described above. Phosphatidyl choline was separated by paper chromatography according to the method of Marinetti et al. (5). The specificradioactivity of the prepared phosphatidylcholine[32P]was 18.41eCi/
SHORT TABLE 1.
Effect
CO111 Al UNICA
of orotic
acid
TIO.VS
Japan.
on the distribution
J. Pharinacol.
of phosphatidylcholine
25, 346 (1975, ['°P]
Value in the serum as dpm/ml serum. Each value represents mean standard error of 4 rats. * Mean significant difference from control (P<0 .05). ** Mean significant difference from control (P<0 .01). Rats were sacrificed 6 hr after administration of 0.373 1lCi of phosphatidylcholine [32P]to which was added 14 nagof non-radioactive phosphatidylcholine. Acid-soluble and lipid fractions were determined as described in Methods.
mg P.
The purity was checked by rechromatography.
found to be in the phosphatidylcholine
Over 90")/' of the radioactivity was
fraction.
As shown in Table 1, at 6 hr after administration of the phospholipid, the radioactivity in the lipid fraction of serum and liver decreased, but not that in the acid-soluble fraction. On the contrary, the incorporation
of phosphatidylcholine
[32P] into the lipid fraction of
adipose tissue was significantly increased by orotic acid.
However, orotic acid had no
effect on the incorporation of phosphatidylcholine
[32P] into both fractions of the spleen,
although a large amount of radioactivity was found.
The radioactivity in both fractions
of most other tissues was also unchanged by orotic acid, but that in acid-soluble fraction of the skeletal muscle was decreased. The radioactivity in lipid fraction of serum, liver, and adipose tissue at various periods after administration of phosphatidylcholine
[32P]is shown in Fig. 1.
In the serum lipid
fraction, the radioactivity in both control and rats fed orotic acid was highest at 3 hr and declined to 32 and 14° of the maximum value, respectively, at 24 hr.
At 3 hr, the incor
poration of phosphatidylcholine ['2P] into this fraction in rats fed orotic acid was more than that in the control, but the radioactivity in the rats fed orotic acid was less at 6 hr. At 12 and 24 hr, there was no difference in this fraction between control and rats fed oro tic acid.
In the liver lipid fraction, the highest radioactivity in both groups was found
at 6 hr after administration of phospholipid and then declined gradually. At 3 hr, the incorporation of phosphatidylcholine [32P] into this fraction in rats was also more than that in the control, but thereafter less radioactivity was observed as compared with the con trol.
In adipose tissues of rats fed orotic acid, the highest radioactivity was found 3 hr
after giving the phospholipid and then this level remained almost unchanged tip to 24 hr. In the control, the incorporation
of phosphatidylcholine
[32P] increased gradually with
SHORT
COJ%111IUNICATIONS
Japan. J. Pharnzacol. 25, 347 (1975)
FIG. 1. Effect of orotic acid on the incorporation of phosphatildylcholine [22P]into lipid fraction in serum, liver, and adipose tissue. Rats were given 0.824 pCi of phosphatidylcholine [32P] to which was added 14 mg of non-radioactive phosphatidylcholine. Each point represents the mean of 3 rats and the vertical lines represent standard error of the mean. •--• Control, 0-0 Orotic acid
time and the maximum
level was found
at 12 hr after administration
In an early stage, within 6 hr, the radioactivity higher
in this fraction
of the phospholipid.
of rats fed orotic acid was
than that in the control.
The results
of the present
study show that
the incorporation
of phosphatidylcholine
[32P] into lipid fraction but not into acid-soluble fraction in serum, liver, and adipose tissue was changed by ingestion of orotic acid. Changes in serum fraction by orotic acid are attributed
to the variation
of serum
lipoprotein
concentration,
since the absorption
of
phosphatidylcholine appeared to be unaffected by orotic acid. Accordingly, high radio activity in rats fed orotic acid at 3 hr after administration of phospholipid suggests that the releasa of lipoprotein lipid.
This suggestion
from the liver is facilitated
is further
tion of the liver and adipose administration
by the administration
by the fact that the radioactivity
tissue was also higher than that in the control
of the phospholipid.
thesis was proposed
supported
This effect disappeared
by Yagi et al. (6-8).
They suggested
within 6 hr.
of phospho in lipid frac at 3 hr after
A similar hypo
that phosphatidylinositol
plays
SHORT an important
COALUUNICATIONS
role in the release of beta-lipoprotein
Japan. J. Pharmacol. 25, 348 (1975) in fatty liver induced
by a low-protein
diet. Thus, perimental
the present
work demonstrates
that phospholipid
has a curative
fatty liver and that the effect is due to the release of lipoprotein
effect on ex from the liver.
REFERENCES 1) RoviLLER, C.H.: The Lii°cr, Edited by RoULLER,C.H., Vol. 2, p. 394, Academic Press, New York and London (1964); 2) SHAPIRO, B.: Lipids and Lipidoscs, Edited by SCILETTLER, G., p. 52, Springer-Verlag, Berlin, Heidelberg, and New York (1967); 3) LANIBORDI,B.: Fe(hi. Proc., 24, 1200 (1965); 4) HANDSCIIMMACHER, R.E., CREASEY,W.A., JAFFIi,J.J., PASTERNAK, C.A., AND HANKIN, L.: Proc. natty. Aca(I. Sci. 46, 128 (1960); 5) MARINETTI,G.V., ERBLAND, .1., AND KOCHEN, J.: Fe(In. Proc., 16, 837 (1957); 6) KOTAKi, A., SAKURAT,T., OKUMURA, M., AND YAGI, K.: Vitamins, 39, 168 (1969); 7) YAM, K., AND KOTAKI, A.: Ann. N.Y. Acad. Sci., 165, 710 (1969); 8) HASAN,S.H., NAKAGAWA,Y., NISHIGAKI,I., AND YAGI, K.: J. Vita minol., 17, 159 (1971)
SHORT COMMUNICATIONS
EFFECT
OF
OROTIC
ACID
ON
THE
OF
PHOSPHATIDYLCHOLINE
DISTRIBUTION [12p]
Isao NEGISHI and Yoshio AIZAWA Department of Pharmacology, Tokyo College of Pharmacy, Shinjuku-ku, Tokyo, Japan Accepted February 10, 1975
Triglyceride accumulation in the liver can be brought about by a disease state in man, and by the administration of a large variety of agents (1, 2). A block in the release of hepatic beta-lipoprotein into blood stream appears to be responsible for the development of a number of experimentally induced fatty liver conditions (3). Our previous results demonstrated that a decreased release of beta-lipoprotein from liver slices in rats fed orotic acid was restored by the addition of phosphatidylcholine. It was further found that phospholipid has a curative effect on fatty liver induced by orotic acid or ethionine. These findings suggest that phospholipid is an important factor in the normal release of beta-lipoprotein. Accordingly, it is of interest to examine the fate of ingested phosphatidylcholine. The present experiments were done to examine the distribution of phosphatidylcholine [32P]in rats fed orotic acid. Female Wistar strain rats weighing 180 to 210 g were first fed a semisyntheticdiet (4) for 2 days. Control rats were given a semisyntheticdiet alone. Groups of animals then were put on a dietary supplement of I ;o orotic acid for 7 days after which 0.373 or 0.824 /iCi of phosphatidylcholine [32P]to which was added 14 mg of non-radioactive phosphatidyl choline as carrier in 0.5 nil of saline solution (1.91 or 4.23 /tCi/Kg body weight) was in jected intraperitoneally. At the indicated time after administration of the phospholipid, the rats were sacrificed by heart puncture under ether anesthesia. Tissues were removed, homogenized with ice-cold 5 trichloroacetic acid, and centrifuged. The precipitate was washed twice with ice-cold trichloroacetic acid. Lipids were then extracted once with 80% ethanol, once with 100% ethanol, twice with chloroform-ethanol (1: 1, by vol.), and once with ether. Trichloroaceticacid was added to the serum, and centrifuged. Serum lipids were then extracted with chloroform-methanol (2: 1, by vol.). Radioactivity of acid-soluble and lipid fraction was determined in a liquid scintillation spectrometer using dioxan and toluene-phosphor solution, respectively. Phosphatidylcholine [32P]was prepared as follows: Liver slices were incubated with 1.25 mCi orthophosphate [32P]per ml of Krebs-Ringer bicarbonate buffer at 37' in 02 CO2 (95: 5, by vol.) for 5 hr. Lipids were extracted as described above. Phosphatidyl choline was separated by paper chromatography according to the method of Marinetti et al. (5). The specificradioactivity of the prepared phosphatidylcholine[32P]was 18.41eCi/
SHORT TABLE 1.
Effect
CO111 Al UNICA
of orotic
acid
TIO.VS
Japan.
on the distribution
J. Pharinacol.
of phosphatidylcholine
25, 346 (1975, ['°P]
Value in the serum as dpm/ml serum. Each value represents mean standard error of 4 rats. * Mean significant difference from control (P<0 .05). ** Mean significant difference from control (P<0 .01). Rats were sacrificed 6 hr after administration of 0.373 1lCi of phosphatidylcholine [32P]to which was added 14 nagof non-radioactive phosphatidylcholine. Acid-soluble and lipid fractions were determined as described in Methods.
mg P.
The purity was checked by rechromatography.
found to be in the phosphatidylcholine
Over 90")/' of the radioactivity was
fraction.
As shown in Table 1, at 6 hr after administration of the phospholipid, the radioactivity in the lipid fraction of serum and liver decreased, but not that in the acid-soluble fraction. On the contrary, the incorporation
of phosphatidylcholine
[32P] into the lipid fraction of
adipose tissue was significantly increased by orotic acid.
However, orotic acid had no
effect on the incorporation of phosphatidylcholine
[32P] into both fractions of the spleen,
although a large amount of radioactivity was found.
The radioactivity in both fractions
of most other tissues was also unchanged by orotic acid, but that in acid-soluble fraction of the skeletal muscle was decreased. The radioactivity in lipid fraction of serum, liver, and adipose tissue at various periods after administration of phosphatidylcholine
[32P]is shown in Fig. 1.
In the serum lipid
fraction, the radioactivity in both control and rats fed orotic acid was highest at 3 hr and declined to 32 and 14° of the maximum value, respectively, at 24 hr.
At 3 hr, the incor
poration of phosphatidylcholine ['2P] into this fraction in rats fed orotic acid was more than that in the control, but the radioactivity in the rats fed orotic acid was less at 6 hr. At 12 and 24 hr, there was no difference in this fraction between control and rats fed oro tic acid.
In the liver lipid fraction, the highest radioactivity in both groups was found
at 6 hr after administration of phospholipid and then declined gradually. At 3 hr, the incorporation of phosphatidylcholine [32P] into this fraction in rats was also more than that in the control, but thereafter less radioactivity was observed as compared with the con trol.
In adipose tissues of rats fed orotic acid, the highest radioactivity was found 3 hr
after giving the phospholipid and then this level remained almost unchanged tip to 24 hr. In the control, the incorporation
of phosphatidylcholine
[32P] increased gradually with
SHORT
COJ%111IUNICATIONS
Japan. J. Pharnzacol. 25, 347 (1975)
FIG. 1. Effect of orotic acid on the incorporation of phosphatildylcholine [22P]into lipid fraction in serum, liver, and adipose tissue. Rats were given 0.824 pCi of phosphatidylcholine [32P] to which was added 14 mg of non-radioactive phosphatidylcholine. Each point represents the mean of 3 rats and the vertical lines represent standard error of the mean. •--• Control, 0-0 Orotic acid
time and the maximum
level was found
at 12 hr after administration
In an early stage, within 6 hr, the radioactivity higher
in this fraction
of the phospholipid.
of rats fed orotic acid was
than that in the control.
The results
of the present
study show that
the incorporation
of phosphatidylcholine
[32P] into lipid fraction but not into acid-soluble fraction in serum, liver, and adipose tissue was changed by ingestion of orotic acid. Changes in serum fraction by orotic acid are attributed
to the variation
of serum
lipoprotein
concentration,
since the absorption
of
phosphatidylcholine appeared to be unaffected by orotic acid. Accordingly, high radio activity in rats fed orotic acid at 3 hr after administration of phospholipid suggests that the releasa of lipoprotein lipid.
This suggestion
from the liver is facilitated
is further
tion of the liver and adipose administration
by the administration
by the fact that the radioactivity
tissue was also higher than that in the control
of the phospholipid.
thesis was proposed
supported
This effect disappeared
by Yagi et al. (6-8).
They suggested
within 6 hr.
of phospho in lipid frac at 3 hr after
A similar hypo
that phosphatidylinositol
plays
SHORT an important
COALUUNICATIONS
role in the release of beta-lipoprotein
Japan. J. Pharmacol. 25, 348 (1975) in fatty liver induced
by a low-protein
diet. Thus, perimental
the present
work demonstrates
that phospholipid
has a curative
fatty liver and that the effect is due to the release of lipoprotein
effect on ex from the liver.
REFERENCES 1) RoviLLER, C.H.: The Lii°cr, Edited by RoULLER,C.H., Vol. 2, p. 394, Academic Press, New York and London (1964); 2) SHAPIRO, B.: Lipids and Lipidoscs, Edited by SCILETTLER, G., p. 52, Springer-Verlag, Berlin, Heidelberg, and New York (1967); 3) LANIBORDI,B.: Fe(hi. Proc., 24, 1200 (1965); 4) HANDSCIIMMACHER, R.E., CREASEY,W.A., JAFFIi,J.J., PASTERNAK, C.A., AND HANKIN, L.: Proc. natty. Aca(I. Sci. 46, 128 (1960); 5) MARINETTI,G.V., ERBLAND, .1., AND KOCHEN, J.: Fe(In. Proc., 16, 837 (1957); 6) KOTAKi, A., SAKURAT,T., OKUMURA, M., AND YAGI, K.: Vitamins, 39, 168 (1969); 7) YAM, K., AND KOTAKI, A.: Ann. N.Y. Acad. Sci., 165, 710 (1969); 8) HASAN,S.H., NAKAGAWA,Y., NISHIGAKI,I., AND YAGI, K.: J. Vita minol., 17, 159 (1971)