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Biosynthesis of fatty acids from [1-14C]acetate in the perfused rat heart
576
SHORT COMMUNICATIONS
BBA 53238
Biosynthesis of fatty acids from [l -l%]acetate
in the perfused rat heart
Fatty acid biosynthesis in the heart has been ascribed chiefly to the mitochondria192 in contrast to organs such as the liver, adipose tissue and mammary gland in which the fatty acid synthesizing enzymes are mainly in the cell cytoplasm. Synthesis in the mitochondria is generally by the process of elongation contrasted to the de tioz’o synthesis which usually occurs in cell cytoplasm. WHEREAT, HULL AND ORISHIMO~ reported that rabbit heart mitochondria synthesis and, when incubated with [I-14C]acetate,
were very active in fatty acid yielded shorter (12-16 carbons)
fatty acids synthesized de nova and longer-chain fatty acids formed by the elongation of preformed acyl units with the added labeled acetate. HOWARD~ concluded that de nova synthesis occurred in the outer membrane of the mitochondrion, while others2+*6 have suggested that little or no de novo synthesis takes place. The question arises as to the types of fatty acids synthesized and the mode of synthesis
occurring
in the intact
heart
in vivo. In rat heart
perfused
with
[I-‘%]
acetate, we have found the major portion of the incorporated 14C in saturated and monoenoic acids of 14-, r6- and r8-carbon chain lengths. Palmitic acid was synthesized by a de nova pathway, while stearic acid was formed by a pathway involving elongation. Hearts taken from adult female Sprague-Dawley rats weighing between 250 and 300 g and maintained on Purina rat chow were perfused in an apparatus similar to that described by MORGAN et al.’ for coronary perfusion in a recirculating system. The gas mixture used was O,-CO, (95 :5, v/v), and the perfusion medium was KrebsHenseleit bicarbonate buffer (pH 7.4) maintained at 37.5”. The heart was removed from the animals
under nembutal
anesthesia,
transferred
to the perfusion
and washed for 3 min with medium before starting on the recirculation 25 ml of perfusion medium containing the labeled substrate. Both
chamber
system with the washing
medium and the perfusing medium contained glucose at a concentration of 5 mM. The labeled fatty acid precursor was 5 ,L of [r-laC]acetate at a concentration of 5 mM. Perfusion was carried on for 2 h. If the heart beat, which generally was 200220 per min, dropped below 160, the preparation was discarded. Fatty acids were isolated by extraction after hydrolysis with ethanolic KOH (containing 0.1 ml of 0.5% solution of hydroquinone as antioxidant) and subsequent acidification. Radioactivity of total fatty acids was determined in a liquid scintillation spectrometer. Radioactivity of individual fatty acids was determined by gas-liquid radiochromatography of the methyl esters using a heated continuous-flow proportional detector (Nuclear Chicago Model 4998). Diethylene glycol succinate polyester (12%) on 110/120 mesh Anakrom ABS was used for column packing. For isolation of pure palmitic and stearic acids for degradation, a preparative column packed with diethylene glycol succinate polyester (200/L) on Chromosorb W (100/140) mesh was used, and the samples were collected on siliconized glass wool packed in long tubes which were heated at the end connected to the exit port of the mass detector. Collected fractions were subjected to final purification by column chromatography on silicic acid-AgNO, (25% AgNO,) using the elution schedule of DE VRIES~. For chemical degradations, the method of DAUBEN et aL9 was used. Total lipids extracted Biochim.
Riophys.
Ada,
187 (1969) 576-578
577
SHORT COMMUNICATIONS
with Folch mixture (chloroform-methanol) were fractionated into lipid classes by thin-layer chromatography using silicic acid. The fractions were eluted from the was obtained from New plates by the method of BIEZENSKI lo. Sodium [I-Xlacetate England Nuclear Corporation, column packings for gas chromatography from Analabs, and standards for gas chromatography and silicic acid for chromatography from Applied Science Laboratories. Incorporation of [r-14C]acetate into fatty acids determined in perfused hearts of IO rats averaged 0.24 & 0.05~/~ (S.E.) of the added substrate. Results of gas-liquid radiochromatographic analyses of these ten samples are given in Table I in the form of average values & SE. Very little 14C activity was present in fatty acids of chain length shorter than 12 carbons. The major portion of the incorporated radioactivity TABLE I DISTRIBUTION OF 14C FROM ACIDS
SYNTHESIZED
Fatty acid
6 Cl0:o
c 12.0, c 14.0.
14:1
cl,:, ,
16:l
18.0,
Is:1
c
Go:,
12:1
c,o:, >c,o: 4
BY
[I-X]ACETATE
PERFUSED
IN FATTY
HEART
o/oof recovered ‘% (average ofIO samples
0.7 * 0.4 2.8 f 0.9 (includes 9.1 f 1.3 (includes
f
S.E.)
trace of approx. 48.1 5 4.3 (includes approx. 26.6 + 3.0 (includes approx. 2.2 f 0.6 3.4 f 1.8 f
C,,: J 0.50/6 14: I) 2.0% 16: I) g.o”% 18: I)
1.2 0.g
was
in the 14-, 16- and r8-carbon fatty acids (saturated and monoenoic). Small amounts of radioactivity were present in fractions identified by retention time as 20 : 2 and 20 : 4, in other compounds of higher degree of unsaturation than 4 double bonds (possibly 20 : 5) and also of 22-carbon chain length. Confirmation of the latter was obtained by hydrogenation of an aliquot of one of these samples and determination of the 14C activity of the resulting fatty acids by gas-liquid radiochromatography. The distribution of the radioactivity in the chain was determined by decarboxylating pure palmitic and stearic acids isolated from pooled samples. Results of these analyses are given in Table II. The average ratio (specific activity carboxyl carbon to specific activity average carbon in the entire chain) resulting from analyses of two different pooled samples of palmitic acid was 2.0, which is the theoretical value expected if this acid had been synthesized de nono from [I-Xlacetate. On the other hand, a ratio greater than two, as obtained for stearic acid, indicates that some degree of elongation occurred in the synthesis of this acid. The 14C activity of fatty acids of various lipid fractions separated from a total lipid extract by thin-layer chromatography was distributed in the following manner : phospholipids 78%, triglycerides 17%, free fatty acids 4% and cholesterol esters 0.5%. Our results indicate that the perfused rat heart used [Wlacetate for synthesis of palmitic acid by a de novo pathway and of stearic acid at least partially by elongation. Results similar to ours were obtained by WHEREAT et aL3 using rabbit heart mitochondria. There is some disagreement among investigators on whether there is de novo B&him.
Biophys. Acta, 187 (1969) 576-578
SHORT
578 TABLE
II
SPECIFICACTIVITYOFAVERAGEFATTYACIDCARBONANDOFCARBOXYLCARBON AND STEARIC ACIDS ISOLATED FROM POOLED HEART FATTY ACIDS
Fatty acids
16:o
18:o _
COMMUNICATIONS
Specijic activity (counts/min per mg C)
Sample Sample Sample Sample
synthesis
I 2 I 2
OFPUREPALMITIC
Ratio B/A
Average fatty acid carbon (A)
Carboxyl carbon (B)
30.0 12.1
65.0 21.2
2.2 I.8
4.6 0.93
42.7 6.5
9.3 7.0
in mitochondria
2~4~11-14 It has also been suggested
by
CHRIST
AND
Hii~s-
that the mitochondrion is the major intracellular site of fatty acid synthesis in the heart. If this is indeed so and if there is little or no de novo synthesis in mitoMANN’~
chondria, then results of our experiments in the intact perfused heart do not agree with those obtained by others with isolated particles. Although the results of studies reported in this paper do not define in what part of the cell palmitic and stearic acids were synthesized, the data clearly indicate that de nova synthesis and elongation of fatty acids are both used by the perfused rat heart and presumably, intact
rat heart in v&o. This investigation
was supported
by U.S.
Public
Health
therefore,
Service
by the
Grants
AM
06483 and GM 00300. We are grateful to Dr. H. C. Meng and to Dr. M. Ghosal, Department of Physiology, Department Nashville,
for help with the perfusion.
of Biochemistry, Term.
37203
Vanderbilt
(U.S.A.)
University,
JHARNA
GHOSAL
TOM WHITWORTH JOHN G. CONIGLIO
I W. C. HOLSMANN, Biochim. Biophys. Acta, 58 (1962) 417. 2 J. V. DAHLEN AND J. W. PORTER, Arch. Biochem. Biophys., 127 (1968) 207. F. E. HULL, M. W. ORISHIMO AND J. L. RABINOWITZ, J. Bid. 3 A. F. WHEREAT,
Chem., 242
(1967) 4013. C. F. HOWARD,
Jr., Biochim. Biophys. Acta, 164 (1968) 448. E. J. V. J. CHRIST, Biochim. Biophys. Acta, 152 (1968) 50. E. QUAGLIARIELLO,~. LANDRISCINA AND P. CORATELLI, Biochim.Biophys. Acta, 164(1968) 12. H. E. MORGAN,M. J.HENDERSON,D.M.REGEN AND C. R. PARK, J.BioZ.Chem., 236(rg61)
253.
8 B. 9 W. IO J. E. II 12 W. I3 E. 74 W. I5 E.
DE VRIES, J. Am. Oil Chemists’ Sot., 40 (1963) 184. G. DAUBEN, E. HOERGER AND J. W. PETERSEN, J. Am. Chem. Sot., 75 (1953) 2347. J. BIEZENSKI, Federation Proc., 23 (1964) 503. M. WIT-PEETERS, Biochim. Biophys. Acta, 176 (1969) 453. R. HARLAN AND S. J. WAKIL, J. Biol. Chem., 238 (1963) 3216. J. BARRON, Biochim. Biophys. Acta, 116 (1966) 425. C. H~LSMANN AND D. S. Dow, Biochim. Biophys. Acta, 84 (1964) 486. J. CHRIST AND W. C. HOLSMANN, Biochim. Biophys. Ada, 60 (1962) 72.
Received
July
rpth, 1969
Biochim. Biophys. Acta, 187 (1969) 576-578
SHORT COMMUNICATIONS
BBA 53238
Biosynthesis of fatty acids from [l -l%]acetate
in the perfused rat heart
Fatty acid biosynthesis in the heart has been ascribed chiefly to the mitochondria192 in contrast to organs such as the liver, adipose tissue and mammary gland in which the fatty acid synthesizing enzymes are mainly in the cell cytoplasm. Synthesis in the mitochondria is generally by the process of elongation contrasted to the de tioz’o synthesis which usually occurs in cell cytoplasm. WHEREAT, HULL AND ORISHIMO~ reported that rabbit heart mitochondria synthesis and, when incubated with [I-14C]acetate,
were very active in fatty acid yielded shorter (12-16 carbons)
fatty acids synthesized de nova and longer-chain fatty acids formed by the elongation of preformed acyl units with the added labeled acetate. HOWARD~ concluded that de nova synthesis occurred in the outer membrane of the mitochondrion, while others2+*6 have suggested that little or no de novo synthesis takes place. The question arises as to the types of fatty acids synthesized and the mode of synthesis
occurring
in the intact
heart
in vivo. In rat heart
perfused
with
[I-‘%]
acetate, we have found the major portion of the incorporated 14C in saturated and monoenoic acids of 14-, r6- and r8-carbon chain lengths. Palmitic acid was synthesized by a de nova pathway, while stearic acid was formed by a pathway involving elongation. Hearts taken from adult female Sprague-Dawley rats weighing between 250 and 300 g and maintained on Purina rat chow were perfused in an apparatus similar to that described by MORGAN et al.’ for coronary perfusion in a recirculating system. The gas mixture used was O,-CO, (95 :5, v/v), and the perfusion medium was KrebsHenseleit bicarbonate buffer (pH 7.4) maintained at 37.5”. The heart was removed from the animals
under nembutal
anesthesia,
transferred
to the perfusion
and washed for 3 min with medium before starting on the recirculation 25 ml of perfusion medium containing the labeled substrate. Both
chamber
system with the washing
medium and the perfusing medium contained glucose at a concentration of 5 mM. The labeled fatty acid precursor was 5 ,L of [r-laC]acetate at a concentration of 5 mM. Perfusion was carried on for 2 h. If the heart beat, which generally was 200220 per min, dropped below 160, the preparation was discarded. Fatty acids were isolated by extraction after hydrolysis with ethanolic KOH (containing 0.1 ml of 0.5% solution of hydroquinone as antioxidant) and subsequent acidification. Radioactivity of total fatty acids was determined in a liquid scintillation spectrometer. Radioactivity of individual fatty acids was determined by gas-liquid radiochromatography of the methyl esters using a heated continuous-flow proportional detector (Nuclear Chicago Model 4998). Diethylene glycol succinate polyester (12%) on 110/120 mesh Anakrom ABS was used for column packing. For isolation of pure palmitic and stearic acids for degradation, a preparative column packed with diethylene glycol succinate polyester (200/L) on Chromosorb W (100/140) mesh was used, and the samples were collected on siliconized glass wool packed in long tubes which were heated at the end connected to the exit port of the mass detector. Collected fractions were subjected to final purification by column chromatography on silicic acid-AgNO, (25% AgNO,) using the elution schedule of DE VRIES~. For chemical degradations, the method of DAUBEN et aL9 was used. Total lipids extracted Biochim.
Riophys.
Ada,
187 (1969) 576-578
577
SHORT COMMUNICATIONS
with Folch mixture (chloroform-methanol) were fractionated into lipid classes by thin-layer chromatography using silicic acid. The fractions were eluted from the was obtained from New plates by the method of BIEZENSKI lo. Sodium [I-Xlacetate England Nuclear Corporation, column packings for gas chromatography from Analabs, and standards for gas chromatography and silicic acid for chromatography from Applied Science Laboratories. Incorporation of [r-14C]acetate into fatty acids determined in perfused hearts of IO rats averaged 0.24 & 0.05~/~ (S.E.) of the added substrate. Results of gas-liquid radiochromatographic analyses of these ten samples are given in Table I in the form of average values & SE. Very little 14C activity was present in fatty acids of chain length shorter than 12 carbons. The major portion of the incorporated radioactivity TABLE I DISTRIBUTION OF 14C FROM ACIDS
SYNTHESIZED
Fatty acid
6 Cl0:o
c 12.0, c 14.0.
14:1
cl,:, ,
16:l
18.0,
Is:1
c
Go:,
12:1
c,o:, >c,o: 4
BY
[I-X]ACETATE
PERFUSED
IN FATTY
HEART
o/oof recovered ‘% (average ofIO samples
0.7 * 0.4 2.8 f 0.9 (includes 9.1 f 1.3 (includes
f
S.E.)
trace of approx. 48.1 5 4.3 (includes approx. 26.6 + 3.0 (includes approx. 2.2 f 0.6 3.4 f 1.8 f
C,,: J 0.50/6 14: I) 2.0% 16: I) g.o”% 18: I)
1.2 0.g
was
in the 14-, 16- and r8-carbon fatty acids (saturated and monoenoic). Small amounts of radioactivity were present in fractions identified by retention time as 20 : 2 and 20 : 4, in other compounds of higher degree of unsaturation than 4 double bonds (possibly 20 : 5) and also of 22-carbon chain length. Confirmation of the latter was obtained by hydrogenation of an aliquot of one of these samples and determination of the 14C activity of the resulting fatty acids by gas-liquid radiochromatography. The distribution of the radioactivity in the chain was determined by decarboxylating pure palmitic and stearic acids isolated from pooled samples. Results of these analyses are given in Table II. The average ratio (specific activity carboxyl carbon to specific activity average carbon in the entire chain) resulting from analyses of two different pooled samples of palmitic acid was 2.0, which is the theoretical value expected if this acid had been synthesized de nono from [I-Xlacetate. On the other hand, a ratio greater than two, as obtained for stearic acid, indicates that some degree of elongation occurred in the synthesis of this acid. The 14C activity of fatty acids of various lipid fractions separated from a total lipid extract by thin-layer chromatography was distributed in the following manner : phospholipids 78%, triglycerides 17%, free fatty acids 4% and cholesterol esters 0.5%. Our results indicate that the perfused rat heart used [Wlacetate for synthesis of palmitic acid by a de novo pathway and of stearic acid at least partially by elongation. Results similar to ours were obtained by WHEREAT et aL3 using rabbit heart mitochondria. There is some disagreement among investigators on whether there is de novo B&him.
Biophys. Acta, 187 (1969) 576-578
SHORT
578 TABLE
II
SPECIFICACTIVITYOFAVERAGEFATTYACIDCARBONANDOFCARBOXYLCARBON AND STEARIC ACIDS ISOLATED FROM POOLED HEART FATTY ACIDS
Fatty acids
16:o
18:o _
COMMUNICATIONS
Specijic activity (counts/min per mg C)
Sample Sample Sample Sample
synthesis
I 2 I 2
OFPUREPALMITIC
Ratio B/A
Average fatty acid carbon (A)
Carboxyl carbon (B)
30.0 12.1
65.0 21.2
2.2 I.8
4.6 0.93
42.7 6.5
9.3 7.0
in mitochondria
2~4~11-14 It has also been suggested
by
CHRIST
AND
Hii~s-
that the mitochondrion is the major intracellular site of fatty acid synthesis in the heart. If this is indeed so and if there is little or no de novo synthesis in mitoMANN’~
chondria, then results of our experiments in the intact perfused heart do not agree with those obtained by others with isolated particles. Although the results of studies reported in this paper do not define in what part of the cell palmitic and stearic acids were synthesized, the data clearly indicate that de nova synthesis and elongation of fatty acids are both used by the perfused rat heart and presumably, intact
rat heart in v&o. This investigation
was supported
by U.S.
Public
Health
therefore,
Service
by the
Grants
AM
06483 and GM 00300. We are grateful to Dr. H. C. Meng and to Dr. M. Ghosal, Department of Physiology, Department Nashville,
for help with the perfusion.
of Biochemistry, Term.
37203
Vanderbilt
(U.S.A.)
University,
JHARNA
GHOSAL
TOM WHITWORTH JOHN G. CONIGLIO
I W. C. HOLSMANN, Biochim. Biophys. Acta, 58 (1962) 417. 2 J. V. DAHLEN AND J. W. PORTER, Arch. Biochem. Biophys., 127 (1968) 207. F. E. HULL, M. W. ORISHIMO AND J. L. RABINOWITZ, J. Bid. 3 A. F. WHEREAT,
Chem., 242
(1967) 4013. C. F. HOWARD,
Jr., Biochim. Biophys. Acta, 164 (1968) 448. E. J. V. J. CHRIST, Biochim. Biophys. Acta, 152 (1968) 50. E. QUAGLIARIELLO,~. LANDRISCINA AND P. CORATELLI, Biochim.Biophys. Acta, 164(1968) 12. H. E. MORGAN,M. J.HENDERSON,D.M.REGEN AND C. R. PARK, J.BioZ.Chem., 236(rg61)
253.
8 B. 9 W. IO J. E. II 12 W. I3 E. 74 W. I5 E.
DE VRIES, J. Am. Oil Chemists’ Sot., 40 (1963) 184. G. DAUBEN, E. HOERGER AND J. W. PETERSEN, J. Am. Chem. Sot., 75 (1953) 2347. J. BIEZENSKI, Federation Proc., 23 (1964) 503. M. WIT-PEETERS, Biochim. Biophys. Acta, 176 (1969) 453. R. HARLAN AND S. J. WAKIL, J. Biol. Chem., 238 (1963) 3216. J. BARRON, Biochim. Biophys. Acta, 116 (1966) 425. C. H~LSMANN AND D. S. Dow, Biochim. Biophys. Acta, 84 (1964) 486. J. CHRIST AND W. C. HOLSMANN, Biochim. Biophys. Ada, 60 (1962) 72.
Received
July
rpth, 1969
Biochim. Biophys. Acta, 187 (1969) 576-578