22
Biochimica et Biophysics Acta, 431 (1976) 22-32 @ Elsevier Scientific Publishing Company, Amsterdam
- Printed
in The Netherlands
BBA 56760
METABOLISM RAT HEART S.C. VASDEV
PERFUSED
and K.J. KAKO *
of Physiology,
Department (Received
OF ERUCIC ACID IN THE ISOLATED
September
Faculty
30th,
of Medicine,
University
of Ottawa,
Ottawa
(Canada)
1975)
Summary In the present work the uptake and utilization of [‘“Cl erucic acid by the perfused rat heart has been investigated and compared with those of [‘“Cl palmitic acid. Both fatty acids were found to be taken up by the heart at the same rate. On the other hand, the incorporation of erucic acid into tissue lipid during 30 min perfusion were significantly high and CO, production low as compared with palmitic acid. Incorporation of erucic acid into diacylglycerol, triacylglycerol and cholesterol ester was considerably higher than that of palmitic acid. During a 30-min period, a large amount of [‘“Cl erucic acid was accumulated in tissue fatty acid fraction. Similarly, relatively high labelling was found in the fatty acid and diacylglycerol fraction during the initial 300 s of perfusion with erucic acid. When [‘“Cl erucic acid and unlabelled palmitic acid was used, the radioactivity was very high in the fatty acid fraction of the heart lipid in comparison with the experiment when [ 14C] palmitate and unlabelled erucic acid was used. Therefore, erucic acid is poorly oxidized by the heart and is preferentially incorporated into heart lipids. There was relatively high incorporation of [ 14C] erucic acid into diacylglycerol and addition of unlabelled palmitic acid tended to decrease it, probably converting more diacylglycerol to triacylglycerol. When [ 14C] palmitic acid and erucic acid were used together, incorporation to triacylglycerol was high and diacylglycerol low. These results, therefore suggest that palmitic acid is a more suitable acyl donor than erucic acid for the C-3 position of triacylglycerol, especially when the diacylglycerol contains erucoyl moieties. Introduction Erucic acid (cis-13-docosenoic acid) forms a large portion of the total fatty acids of some strains of rapeseed. Dietary intake of rapeseed oil containing erucic acid produces in various species of animals myocardial lesions which include deposition of fat globules, myocytolytic changes, myocarditis and focal * To
whom
reprint
requests
should
be addressed.
23
areas of necrosis [l--6] . Both biochemical and ultrastructural changes in heart mitochondria have also been described [6,7]. Feeding of this oil in rats causes an accumulation of lipids in the heart muscle which are mainly in the form of triacylglycerol [ 71. The composition of triacylglycerol showed a high proportion of erucic acid, reflecting the dietary fatty acids [8] . The pathogenecity of rapeseed oil has been attributed to its component erucic acid [9] . It has been shown that erucic acid can be oxidized in the animal in vivo [lo] . On the other hand experiments in vitro have shown that it is a poor substrate for mitochondrial enzymes responsible for fatty acid oxidation [11,12]. The mechanism of cardiac lipid accumulation that occurs in rats fed diet containing high erucic acid is not clear. Therefore, we have studied the cellular metabolism of erucic acid in comparison to that of palmitic acid in the perfused rat heart. The results demonstrate distinct features of the erucic acid incorporation into complex lipids in the myocardium. Materials and Methods Perfusion medium. The perfusion medium consisted of Krebs-Henseleit bicarbonate buffer [ 131 (pH 7.4), 5 mM glucose and bovine serum albumin (3%, w/v). The perfusate was gassed with O,/COZ (95 : 5, v/v) before starting the experiment and during perfusion. The fatty acid was bound to the albumin by adding the warm sodium salt of fatty acid dropwise to the albumin solution during continuous agitation with a magnetic stirrer. The perfusion fluid was filtered before use. The final fatty acid concentration in the perfusion medium was made to 0.5 mM giving a final molar ratio of fatty acid to albumin as 1 : 1. The bovine serum albumin, fraction V, was obtained from Sigma, which was essentially free from fatty acid (less than 0.005%). This was used without further purification. [ 14-14C] Erucic acid (spec. act. 47.2 Ci/mol) was obtained from Schwarz/Mann (New York). [1-14C] Palmitic acid (spec. act. 53 Ci/mol) was obtained from New England Nuclear Cor., Montreal. Both fatty acids were checked for purity by thin-layer chromatography and were found to be 98% pure. Both erucic acid and palmitic acid were obtained from Sigma Chemical Co. and were approx. 99% pure. The perfusion apparatus and technique. The perfusion apparatus used was a modification of recirculation system described by Morgan et al. [ 141. The pump was a polystaltic type obtained from Buchler Instruments (U.S.A.). All tubing was made of white silicon rubber which was rinsed in distilled water and ethanol extensively before use. Male albino rats (175-210 g) of a SpragueDawley strain were purchased from Canadian Breeding Farm and Laboratory Ltd., Montreal. The animals were fed ad libitum a Purina laboratory chow diet and had free access to water. All the animals were fasted overnight before use. The animals were sacrificed by decapitation and heart removed immediately along with aorta from the thorax and immersed in ice-cold 0.9% NaCl. A fine pair of small forceps and scissors was used for all this procedure. After the heart beat stopped, a 19-gauge cannula was inserted into the aorta and tied in place with silk ligature. The left ventricle was punctured to prevent the accumulation of fluid. The heart was then perfused with 50 ml of Krebs-Henseleit
24
bicarbonate buffer, without fatty acid and albumin so as to flush the coronary vasculature. The preperfusion buffer was discarded. All the preparations which were not found satisfactory were not used. The preparation was considered satisfactory when the heart beat was regular, ranging from 180 to 220 contractions per min and the coronary flow was constant and ranged from 5 to 7 ml per min. The perfusion pressure was maintained at 60-80 mm Hg with the help of peristaltic pump. After the preperfusion, 15 ml of perfusing medium containing bovine serum albumin and 0.5 mM fatty acid (approx. 1 /..&i) was added and the heart transferred to the proper recirculating perfusion system. The perfusion was carried out at 37°C for 30 min unless otherwise stated in some experiments. During this period, the perfusing fluid dripping on the sintered glass was constantly exposed to a mixture of 95% 0, and 5% COz. At the end of perfusion, the heart was removed quickly and incised to open the chambers. All the chambers were washed three times with chilled buffer so as to remove the labelled precursor. The ventricles were blotted dry and two pieces of the muscle were cut, one for dry weight and the other for lipid profile. The tissue for lipid extraction was frozen in liquid nitrogen and kept in a freezer at -60°C until processed. The perfusion fluid was collected and made up to 25 ml with buffer which was used to rinse the perfusion apparatus. Kinetics of labelling of fatty acid during perfusion of a short duration. To study the time course of labelling of fatty acids, the perfusing fluid containing either [ 14-‘4C] erucic acid or [ 1-‘4C] palmitic acid was injected after preperfusion into modified apparatus. The heart was removed after 30, 60, 90, 120, 180, and 300 s of perfusion. After the perfusion, the heart tissue and perfusate were processed as described earlier. Analytical procedure: Isolation and analysis of lipids. A portion of heart was homogenized in a glass homogenizer with Teflon pestle in a solvent mixture (20 ml per g of heart) containing chloroform/methanol (2 : 1, v/v). The chloroform/methanol extract was removed and the residue extracted three times with 5 ml of solvent mixture, and the extracts combined. The lipids were further processed and purified by the method of Folch et al. [ 151. Neutral lipids were separated by thin-layer chromatography on silica gel G plates (precoated plates from Analabs, Inc., North Haven, Conn.) by developing in a solvent mixture containing N-hexane, diethylether, methanol and acetic acid (90 : 20 : 2 : 3, v/v) [ 161. Phospholipids in the chloroform/methanol extracts were separated by thin-layer chromatography using silica gel G plates and developing in a solvent system containing chloroform, methanol and water (65 : 25 : 4, v/v). It was not possible to quantitatively separate some fraction such as sphingomyelin fraction (containing sphingomyelin, lysophosphatidylethanolamine and phosphatidic acid) and lysophosphatidylcholine fraction (containing lysophosphatidylcholine, phosphatidylserine, phosphatidylinositol and lysophosphatidic acid). Each lipid fraction was identified by using authentic lipid standards, i.e. cholesterol oleate, cholesterol, palmitic acid, erucic acid, For palmitoylglycerol, 1,2-dipalmitoylglycerol and tripalmitoylglycerol. phospholipids, various standards were run simultaneously on thin-layer plates. Lipid standards were obtained from Serdary Research Laboratory, London, Ontario. Radioactivity determination. Each lipid fraction from thin-layer plates was
25
scraped and transferred into a scintillation vial. To this 10 ml of Econofluor (New England Nuclear, Boston) was added, and radioactivity dete~ined in Beckman, Model LS-150 liquid scintillation spectrometer. Efficiency was approx. 90%. Measurement of fatty acid uptake and CO, production. The uptake of [ l-14C] palmitic acid and [ 14-14C] erucic acid was derived from the radioactivity disappearing from medium during perfusion. At the beginning and end of perfusion, the radioactivity of the perfusion fluid was determined by taking l-ml aliquots in quad~plicates, adding 10 ml of Aquasol (New England Nuclear, Boston, Mass.) and counting them by a scintillation counter. For measurements of 14C02 content, an aliquot (1 ml) of the perfusate was acidified (pH 2.0) by an addition of 0.1 ml 11 M HCI and then counted after addition of Aquasol. The difference between radioactivities before and after acidification gave the value for 14C02 in the perfusate [ 171, The uptake of fatty acid by the heart was calculated from the initial radioactivity in the perfusing fluid before starting the experiment and the radioactivity found in the above acidified sample. The validity of the method of estimatind 14C02 production was checked by different experiments. (1) Labelled NaH14C03 was added to the perfusion medium. The perfusate was recirculated for 30 min without attaching heart to the cannula. The i4C02 released from the acidified perfusion fluid was absorbed in a NaOH solution and was measured by a liquid scintillation counter. The recovery of 14C02 was found to be almost 100%. (2) In another experiment NaH14C0, was taken in a counting vial in 1 ml buffer containing bovine serum albumin. To this 0.1 ml HCl was added to acidify (pH 2.0) and “C0, allowed to escape for 2 h. There were no counts in the vial after acidification. The same procedure was carried out by using [l-‘4C] palmitic acid and [l-‘“Cl erucic acid. No loss in radioactivity occurred by acidification of fatty acids. (3) Lastly, the heart was perfused with labelled fatty acid for 30 min in a recirculatory system, and the system was made alkaline at the end of perfusion. The radioactivities in the alkaline perfusate, in the CO2 absorber and in the alkaline heart digest were released measured. These experiments confirmed that 14C02 is qu~titatively from an acidified perfusate, so that remaining radioactivity in the perfusate is only due to the fatty acid or its intermediates. All the data were expressed as the mean I S.E. and the significance of difference was calculated with Student’s t-test. Results Myocardia~ up take of [l -“CT]palmitic acid and [I 4-14Cj erucic acid The amount of palmitate uptake by the perfused heart examined at 5, 10, 20 and 30 min of perfusion indicated that uptake increased linearly during this period. Hearts perfused for 30 min were shown to take up both labelled palmitic acid and erucic acid at the same rate from the perfusing fluid (Table I). The incorporation of erucic acid into tissue lipid was significantly higher than palmitic acid. Conversely, the production of 14C02 by the heart was lower from erucic acid as compared to palmitic acid. In the experiments where both palmitie acid and erucic acid were used together in equimolar concentration in the perfusate (0.25 mM each), the results were slightly different. The uptake was higher
I
UPTAKE
Uptake (percent of available fatty acid) Tissue lipid (nmol/heart) Tissue lipid (percent of uptake) 14CO2 production (nmol/heart) 14~02 produktion (percent of uptake)
Uptake (nmol/heart)
For
OF
LABELLED
ERUCIC
ACID
AND
LABELLED
PALMITIC
ACID
IN
PERFUSED
RAT
HEARTS
t
67.9
3.7 **
f
2.7 *
+ 161.60 *
f
n=6
1436.14
32.1
17.85 **
2.50
+ 155.29
661.68 Z
2’7.5
2098.83
Expt. A [ 14~1 Palmitic acid
51.2
4.3 **
72.32 **
2.4
L
4.7 *
_+ 94.05 *
2
f
i
+ 167.04
II = 7
1068.64
48.7
1021.01
28.2
2089.64
Expt. B [ 1 4 C] Erucic acid
+
59.0
+
t.
c
n=3
786.04
41.0
2.0
i
84.97 *
2.0 ***
52.93
5.2 ***
***
acid +
i 194.93
539.20 +
35.3
1324.93
Expt. C [’ 4Cl Palmitic erucic acid
D
0.9 ***
+
6.3 ***
+ 28.44
i
36.2
2.3
rz= 3
t
+
283.25 t 62.51 +
63.8
488.95
20.6
772.87 + 35.18 ***
[ ’ 4 I Erucic acid + palmitic acid
l3xpt.
.___-__
the experimental detail, see the text. The final concentration of fatty acids was 0.5 mii’I. The molar ratio of fatty acid to albumin was 1.0, the the perfusion time 30 min. All the values are mean 2 S.E. Values of Expt. A and B and Expt. C and D are compared. Significance of difference is indicated as * (P < 0.05), ** (P < 0.01). *** (P < 0.02) and t (P < 0.002). -____
MYOCARDIAL
TABLE
II
OF INCORPORATION
OF PALMITIC
ACID
AND
ERUCIC
ACID
INTO
NEUTRAL
LIPID
FRACTIONS
OF TIILC I’ERFUSED
RAT
HEART
Cholesterol ester Triacylglycerol Fatty aicds Diacylgkcerol PhosphoIipids
2.60 451.96 49.49 26.56 198.48
n=6
r 0.42 f 21.31 f 2.81 f 1.05 f 15.08 *
0.4 62.1 6.8 3.5 27.3
46.10 566.05 251.99 127.66 245.53 n=7
f 2.94 + 50.37 + 25.79 + 9.21 + 21.53 *
nmo1/g
nmol/g %
Expt. B [ 14CI Erucic acid
Expt. A [14CJ PaImitic acid
3.7 45.7 20.4 10.3 19.8
% 1.02 376.71 17.83 28.44 114.93
nmolfg
n=3
rt 0.01 ?: 22.61 t 2.63 + 2.36 t 6.43
Expt. C [14C1 Palmitic erucic acid acid +
0.12 69.9 3.3 5.3 21.3
%
37.23 232.97 144.99 39.75 76.31
nmol/g ~._
n=3
+ 8.63 f 20.22 t 16.02 i 8.52 f. 2.13
Expt. D [ 14C 1 Erucic acid + palmitic acid
7.0 43.9 27.2 7.5 14.4
%
Hearts were perfused and lipids extracted and separated by thin-layer chromatography as described in text. Values are expressed as nmol per g wet weight (mean + SE.) and percent of total incorporation into tissue lipid. Differences in value between Expt. A and B and those in Expt. C and D are significant (P < 0.05) except *.
COMPARISON
TABLE
28
where [ l-i4C] palmitic acid was used along with unlabelled erucic acid (Expt. C) in comparison with [ 14-*4C] erucic acid and unlabelled palmitic acid (Expt. D). The data was expressed as amounts of labelled fatty acid transformed; therefore the uptake of [‘“Cl palmitic acid in Expt. C was higher than that when only palmitic acid or only erucic acid was used in the perfusion medium (1325 > 209812 in nmoljg). The inco~oration into tissue lipid in percentage of the uptake was higher and the production of 14C0, was smaller in Expt. D than from [ 14-14C] erucic in Expt. C. A very low production of 14C02 production acid, when palmitic acid is also present suggests that palmitic acid is preferentially oxidized by the heart as compared to erucic acid. The data demonstrates that erucic acid is preferentially incorporated into heart lipids and palmitic acid is preferentially oxidized to CO;? by the heart. Fatty acid incorporation in to neutral lipids and phospholipids Fractionation of heart lipids showed significantly greater incorporation of erucic acid than that of palmitic acid into fatty acids, diacylglycerol and cholesterol ester both when calculated as absolute amounts and as percentage incorporation. The rate of incorporation of erucic acid was greater into both triacylglycerol and phospholipids than those of palmitate, but the values represented smaller fractions of total incorporation into tissue lipids than those observed with palmitic acid (Table II). In the hearts perfused with labelled erucic acid and unlabelled palmitic acid (Expt. D), the incorporation of erucic acid was significantly higher in fatty acids, diacylglycerol and cholesterol ester whereas it was lower in triacylglycerol and phospholipids as compared to experiments with labelled palmitic acid and unlabelled erucic acid (Expt. C) (Table II). There was decreased labelling in the sphingomyelin-phosphatidic acid fraction and increased labelling in the lysophosphatidylcholine fraction in phospholipids of rat hearts perfused with erucic acid (Table III).
TABLE III COMPARISON OF INCORPORATION OF [l- 14C] PALMITIC INTO PHOSPHOLIPIDS OF THE PERFUSED RAT HEART
ACID AND [ 14-14C1 ERUCIC
ACID
Sphingomyelin fraction contains sphinogomyelin, Iysophosphatidylethanolamine and phosphatidic acid; lysophosphatidylcholine fraction Wmtains lysophosphatidylcholine, phosphatidylserine. phosphatidylinositol and lysophosphatidic acid. All values are represented as mean t S.E. For further information see Tables i and II.
Phosphatidylethanolamine Phosphatidylcholine Sphingomyelin fraction Lysophosphatidylcholine fraction
Expt. A Palmitic acid (nmol/g wet weight)
Expt. B Erucic acid (nmol/g wet weight)
P
20.22 rt 1.53 24.07 + 1.82 134.83 f 10.21
33.90 + 3.21 34.01 f 2.98 106.16 f 9.31