Journal of MolecularandCellularCardiology( 1977) 9, 617-631
Incorporation o f Fatty Acids into Rat Heart Lipids. In Vivo and In Vitro S t u d i e s S. C. VASDEV AND K . J . K A K O
Department of Physiology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada (Received 21 May 1976, accepted in revisedform 7 September 1976) S. C, VASD~VAm) K.J. KaKo. Incorporation of Fatty Acids into Rat Heart Lipids. In Vivo and In VitroStudies. 3ournalof MolevularandCellularCardiology(1977) 9, 617-631. Incorporation of different [14C] labelled long chain fatty acids into complex lipids of myocardium was studied using three different preparations, i.e., isolated perfused heart, heart slices and in vivo animals. Neutral and phospholipids were fractionated by thin layer chromatography. The results showed that labelling of triacylglycerol was highest when labelled oleic acid was used as precursor. On the other hand phospholipid labeUing was maximum with linoleic acid or linolenic acid as the labelled substrate in the medium. Both linoleic and linolenic acid were extracted and oxidized by the perfused heart at a higher rate than other fatty acids. Erucic and stearic acids were accumulated in the myocardium probably because they were oxidized at a slow rate. With labelled erucate as the precursor, the formation of labelled diacylglycerol was relatively large. Of the individual phospholipid fractions studied, labelling of phosphatidylcholine was high with linoleic acid, but phosphatidylethanolamine was labelled equally well, irrespective of fatty acid precursors. Percentage incorporation of erucic and linoleic acid into phosphatidic acid was high probably because the latter molecule was utilized relatively slowly for further synthetic pathway. Labelling into lysophosphatidylcholine was relatively more than that into phosphatidylcholine in the experiment with stearic acid, suggesting that this fatty acid is less suitable than other fatty acids for position 2 of phosphatidylcholine molecules. These results show that the pattern of incorporation of individual fatty acids in lipids of heart muscle varies in each of the three different experimental procedures.
K~Y WORDS:Palmitic acid; Oleic acid; Stearic acid; Linoleic acid; Linolenic acid; Erucic acid; Heart perfusion; Heart slices; Phosphatidylcholine; Phosphatidylethanolamine; Phosphatidic acid; Phosphafidylserine; Lysophosphatidylcholine; Cholesterol ester; Triacylglycerol; Diacylglycerol; Acylcarnitine.
1. Introduction T h e c o n t r i b u t i o n of long c h a i n fatty acids as a p r i n c i p a l fuel of respiration of c a r d i a c muscle has b e e n clearly d e m o n s t r a t e d i n the h u m a n as well as i n the isolated h e a r t p r e p a r a t i o n [4]. F u r t h e r m o r e , the relative p e r c e n t a g e of p l a s m a oleic acid decreased a n d that of p a l m i t i c acid increased d u r i n g passage of the blood t h r o u g h the c o r o n a r y vessel i n the h u m a n , suggesting t h a t several fatty acids comm o n l y available to the h e a r t as p l a s m a F F A are n o t e q u a l l y utilized b y the m y o c a r d i u m [15, 21, 28]. Studies with isolated perfused rat hearts using [14C] fatty acids i n d i c a t e d t h a t the p a t t e r n of u t i l i z a t i o n of fatty acid b y the m y o c a r d i u m m a y
618
S . C . V A S D E V A N D K.
j.
KAKO
change depending upon the quantity and type of fatty acids [II, 18, 24]. The rate of fatty acid uptake by rat hearts was proportional to its concentration and molar ratio of fatty acid to albumin in the perfusing fluid [I0, 13, 23]. Since a part of the fatty acids taken up from the circulation is converted to tissue lipids [4, I0, 16, 18], it is of interest to know patterns of incorporation of individual long chain fatty acids into tissue lipids in the myocardium. In the present study, we examined the metabolism of long chain fatty acids which form the main constituents of the dietary fat. In addition we studied the metabolism of erucic acid (cis-13-docosenoic acid) which forms a large portion of the total fatty acids of some strains of rapeseed oil, since dietary intake of this oil produces myocardial lesions in various species of animals [3]. An effort was also made to compare the incorporation of fatty acids in three different experiments, i.e. in the in vivo heart, perfused heart and heart slice.
2. M a t e r i a l s a n d M e t h o d s
Male albino rats (150-210 g) of a Sprague-Dawley 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 animals were fasted overnight before use. Krebs-Ringer bicarbonate or phosphate buffer (pH 7.4) was prepared according to Umbreit et al. [25]. To this buffer, glucose (5 raM) and bovine serum albumin (3%, w]v) were added. 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 final fatty acid concentration in the 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, which was essentially free from fatty acids (less than 0.005~o), was obtained from Sigma. [1-14C] palmitic acid (spec. act. 52 mCi/mmol), [1J4C] oleic acid (spec. act. 54 rnCi/mmol), [1-14C] stearic acid (spec. act. 54 mCi/mmol), [ I J 4 C ] linoleic acid (spee. act. 50 mCi/mmol) were purchased from New England Nuclear Corp. Boston, and [1J4C] Iinolenic-acid (spec. act. 42.3 mCi/mmol) and [14-14C] erucic acid (spec. act. 47 mCi/mmoI) were from Amersham/Searle Corp. Chicago and Schwartz] Mann, New York, respectively. All the fatty acids were checked for purity by thin layer chromatography and were found to be approximately 98% pure. All the nonlabelled fatty acids were obtained from Sigma Chemical Co. and approximately 99 % pure.
Perfusion studies The perfuslon apparatus and technique were described in detail elsewhere [27]. T h e animals were killed by decapitation and then hearts excised and immersed in ice-cold Krebs-Henseleit bicarbonate buffer which had been filtered and aerated with 95% 02 and 5% CO~. Visible fat and connective tissue were removed, the
F A T T Y ACIDS I N H E A R T L I P I D S
619
aorta cannulated and the heart was pre-perfused with 50 ml of buffer without fatty acid and albumin. Following the pre-perfusion, the heart was transferred to the proper recirculadng perfusion system containing 15 ml of perfusion fluid. Different fatty acids were used for different experiments. The perfusion was carried out at 37~ for 30 mln. During this period the perfusate dripping on the sintered glass was constantly exposed to a O~.-COz mixture. At the end of perfusion, the ventricles were blotted dry and two pieces cut, one for determination of dry weight and the other for lipid profile.
Extraction and analyses of lipids A portion of heart was homogenized in a glass homogenizer with a Teflon pestle in a solvent mixture (20 vol) containing chloroform-methanol (2:1, v/v). The lipids were further processed and purified by the method of Folch et al. [12]. Neutral liplds were separated by thin layer chromatography on silica gel G plates (precoated plates from Analab Inc. North Haven) by developing in a solvent system containing aV-hexane-diethyl ether-methanol-acetic acid (90:20:2:3, v/v/v]v) [22]. Phospholipids in the chIoroform-methanol extracts were separated by two dimensional thin layer chromatography [2] using solvent system 1 containing chloroform-methanol-cone. NH4OH (65:25:4, v]v/v) and system 2 containing chloroform-methanol-acetic acid (65:25:8, v/v/v). Chromatographic plates were activated immediately prior to application at 100~ for 1 h. Each lipid fraction was identified by using authentic lipid standards, which were purchased from Serdary Research Lab. London, Ontario.
Radioactivity determination Each lipid fraction from thin layer plates was scraped and transferred into a scintillation vial. To this 10 ml of Econofluor (New England Nuclear Corp) was added, and radioactivity determined by a Beckman, LS-150, liquid scintillation spectrometer. Efficiency was approximately 90%. The uptake of [14C] fatty acid was derived from the radioactivity disappeared from the medium during perfusion, as described previously [27]. For measurements of 14CO~ content, an aliquot of the perfusate was acidified (pH 2.0) and counted in Aquasol. The difference between radioactivity before and after acidification gave the value for 14CO2 in the perfusate. The COs released from the perfusion chamber was collected in an NaOH solution and the radioactivity in the latter solution measured. This 14CO~ value, together with the ~4CO~ in the perfusate, formed the total 14CO~ production by the heart in 30 min. The validity of this method was tested and described earlier
[27]. Heart slice experiment Hearts were taken from overnight fasted rats. Slices were prepared using glass slides and a sharp razor blade [25]. Slices weighing 300-400 mg (3-4 pieces) were
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s.c. VASDEVAND K. J. r~AKO
incubated in each flask for 30 min at 37 ~ in 4.0 ml of Krebs-Ringer phosphate buffer (pH 7.4) containing 5 ham glucose and 0.5 mM [14C] fatty acid bound to 3% bovine albumin. The flasks were shaken vigorously in a Dubnoff incubator. At the end of incubation, the slices were rinsed three times with chilled buffer and then frozen in liquid nitrogen until processed. The fipids were extracted, purified, separated and radioactivities determined, as described above.
In vivo incorporation studies Male rats which were fasted overnight were anesthetized by a subcutaneous injection of 0.25 ml/100 g body weight of 2% chloralose in 20% urethane. [14C] fatty acid bound to albumin was injected into the jugular vein. The injected volume (0.5 ml) contained 5 ~Ci (0.11 ~mol) fatty acid and 15 mg albumin. The animals were sacrificed after 75, 150 or 300 s by decapitation, and the heart and liver were rapidly frozen in liquid nitrogen. The tissue was processed for lipid analyses.
Identification of fatty acylcarnitine Very high radioactivities were found in the sphingomyelin fraction of heart lipids in the perfusion experiment. This fraction was further analyzed and it was found that the labelling almost exclusively occurred in fatty acylcarnitine esters. In the chromatographic system routinely used in our study (see above), standard palmitoylcarnitine (supplied from Otsuka Pharmaceutical Co. Japan) gave the same RF value as sphingomyelin. We have eluted the substance from the region of thin layer chromatography corresponding to sphingomyelin with chloroform-methanolwater (30:50:2, v/v/v). The substance was (i) re-chromatographed with a solvent system containing chloroform-methanol-acetic acid-water (50:25:8:4, v]v[v[v), which separated acylcarnitine and sphingomyelin; and (ii) hydrolyzed by the method of Dawson et al. [7]. Upon hydrolysis, approximately 90% of the radioactivity of the sphingomyelin-acylcarnitine fraction appeared in the fatty acid fraction; standard sphingomyelin was not hydrolyzed under these conditions. We have also found that two dimensional thin layer chromatography using chloroform-methanol-ammonia-water (50:35:3:3, v/v/v]v) and chIoroform-methanolglacial acetic acid-water (50:25:8:4, by vol) [29] separated fatty acylcarnitine. The plates were first exposed to iodine vapour, which stained both acylcarnitine and phospholipids, and to the molybdenum blue reagent of Dittmer and Lester [8], which stained only phospholipids. Incorporation of fatty acid into acylcarnitine in the perfused heart preparation was further verified by perfusing the heart in the presence of DL-carnitine (methyl-[14C]) hydrochloride (ICN Chemical & Radioisotope Division, Waltham, Mass) and non-labelled fatty acid. Two dimensional
FATTY
A C I D S IN H E A R T
LIPIDS
621
thin layer chromatographic analyses showed that all the radioactivity was located in the sphingomyelin-acylcarnitine spot.
3. R e s u l t s
Studies with perfused hearts Uptake of [14C]fatty acid Albumin-bound [14C] linoleic acid was taken up at the highest rate during 30 rain perfusion in this preparation. The rates of uptake of linolenate, palmitate and oleate were lower than that of linoleate (Table 1). Incorporation of erucic acid into tissue lipids was highest and its oxidation lowest among all fatty acids examined. Linoleic and linolenic acids were oxidized at the highest rate as compared to all other fatty acids.
Incorporation of [14C]fatty acid into tissue lipids Linoleic acid incorporation was highest in phospholipid fraction both in the absolute quantity and as the percentage of total lipids (Table 2). Although the incorporation of linolenate in the total tissue lipids was considerably below that of other fatty acids, comparably a large fraction of the total tissue radioactivity was found in phospholipid fraction. The incorporation into acylcarnitine was greatest with labelled palmitic acid (Table 2). Palmitoylcarnitine accumulated probably because palmitate was activated and transferred to carnitine at a faster rate, or because palmitoylcarnitine was oxidized at a slower rate as compared to other fatty acids. Comparison ofdistributlon of various fatty acids revealed that labelling of diacylglycerol was the highest in the experiment with erucic acid. Significant differences were also observed in the incorporation pattern of fatty acids into phospholipid classes (Table 2). The formation of radioactive phosphatidylcholine was highest with linoleic acid, followed by palmitic acid, as the labelled substrate in the perfusate. The radioactivity in the phosphaddylserine fraction was highest with oleic acid, whereas phosphatidic acid labelling was greatest with erucic acid and linolenic acid.
Incorporation of [14C] fatty acid into tissue lipids in heart slices The distribution of labelled fatty acid in tissue lipids is shown in Table 3. The highest incorporation into tissue lipid was found with erucic acid, followed by stearic, palmitic and oleic acids. At least 70-90% of the total radioactivity was
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TABLE 3. Comparison of incorporation of [14C] fatty acid into tissue lipids in heart slices Lipid fraction Cholesterol ester Triacylglycerol Diacylglycerol Fatty acid Phospholipids Total Iipids
Palmitic acid
Oleic acid
Stearic acid
Erucic acid
2.9 -4- 0.2 7.8 -4- 0.7 6.8 ztz 0.5 108.0 4- 1.1 15.0 q- 0.9 I40.6 4- 1.9
0.69 q- 0.1 6.1 -4- 0.3 3.5 -4- 0.2 100.3 -4- 5.1 6.9 q- 0.1 117.6 4- 5.1
1.4 + 0.1 5.9 • 0.4 4.3 4- 0.2 193.2 4- 8.6 11.0 -t- 0.4 217.9 -4- 10.3
94.5 4- 4.3 28.7 =k 2.2 51.5 i 8.9 382.3zt: 29.0 20.5 ~- 1.5 577.4 ~ 27.0
All values are presented as nmol fatty acid incorporated per g of the wet weight (mean -]- S.E.M.). Number of experiments were 4 in each group. recovered in the fatty acid fraction. T h e radioactivity in the fatty acid fraction was highest in the experiment with erucic acid, followed by that with stearic acid. I t is possible that these two fatty acids penetrate the cell m e m b r a n e at a faster rate, and therefore, would accumulate in the tissue. O n the other hand, the oxidation of these acids by the heart slices m a y be slower as compared to other fatty acids. However, in the present study the oxidation of fatty acid was not measured in this experiment. While the amount oferucic acid incorporated into total lipids in the perfused hearts was only slightly greater than the amount of palmitic acid, approximately four times more erucic acid was incorporated in the tissue slices than was palmitic acid. T h e cause for this difference was not further studied. Molar ratios of newly formed triacylglycerol, diacylglycerol and phospholipids in heart slices were approximately 1:1:2 with palmitate as a precursor, 2:1:2 with oleate, 1.5:1:3 with stearate, and 1.5:2.5:1 with erucate. These ratios clearly indicated that oleic acid was incorporated relatively more into triacylglycerol than into phospholipids, as compared to palmitic and stearic acid. Furthermore, erucic acid greatly incorporated into diacyl- and triacylglycerol. A m o n g phospholipid classes, approximately 60% of the IabeUed palmitate was recovered in phosphatidylcholine but m u c h less ~o of the total activity was accounted for by the incorporation of other fatty acids into phosphatidylcholine (Table 4). T h e radioactivity in phosphatidic acid formed the major fraction in the experiment with erucic acid. Relatively high percentages of the total activities were found in the lysophosphatidylcholine fraction with stearic and oleic acid as the precursor (Table 4).
Incorporation of [14C] fatty acid into heart lipids in vivo T h e incorporation of injected [14C] fatty acid into different lipid fractions of rat hearts is shown in Table 5. T h e data are presented in terms of percentages of the injected radioactivity. It should reflect reasonably well the specific activities of each lipid fraction, because, in a relatively short period, absolute amounts of esterified fatty acids in tissue must have remained constant. As early as 150 s after injection of radioactive fatty acid, labelling in the triacyl-
FATTY ACIDS IN HEART LIPIDS
625
TABLE 4. Comparison of incorporation [14C] fatty acid into phospholipids in heart slices Palmitic acid nmol of fatty acid incorporated into total tissue phospholipids per g wet weight 15.02 4- 0.99
Oleic acid
Stearic acid
Erucic acid
6.95 4- 0.06
11.02 4-. 0.37
20.47 4- 1.50
Phospholipids class
Radioactivity as percentages of total labelled phospholipids
Phosphatidic acid Phosphatidylserine Lysophosphatidylcholine Phosphatidylcholine Phosphatidylethanolamine
2.7 zk 0.I 16.0 4- 1.2
14.5 + 1.2 14.7 4- 0.7
8.6 -4- 0.6 19.5 4- 1.0
45.5 -4- 1.0 2.9 4- 0.5
14.3 4- 0.9 59.3 zk 0.8
23.4 4- 2.4 37.6 + 1.8
33.2 4- 0.8 26.2 + 1.3
9.6 4- 2.1 36.0 4- 1.6
7.8 ~ 0.5
9.8 -4- 0.4
12.5 4- 0.5
6.1 4- 0.7
Values are expressed as mean • S.~.M.The number of experiments were 4 in each group. glycerol i n the h e a r t b e c a m e highest a m o n g all the lipid fractions, regardless of the k i n d of fatty acids. Between 75 s a n d 300 s after injection, the r a d i o a c t i v i t y i n the phospholipid fraction (in terms of % of the total activity) decreased; this was p a r t i c u l a r l y e v i d e n t with oleic acid, i n w h i c h case the label i n triacylglycerol increased a n d that i n diacylglycerol decreased with time. Erucic acid b e h a v e d v e r y differently from other fatty acids; the r a d i o a c t i v i t y i n triacylglycerol was relatively low a n d that i n the tissue fatty acid was relatively high, suggesting t h a t this acid was poorly oxidized a n d was poorly transformed to triacylglycerol. TABLE 5. In vivo incorporation of [14C] fatty acid into heart lipids Palmitic acid Time after injection Amount of total radioactive tissue lipids* Fraction Cholesterol ester Tr{acylglycerol Diacylglycerol Fatty acid Acy!carnitine Phospholipids
Oleic acid
Erucic acid
150 s
300 s
75 s
150 s
300 s
150 s
300 s
0.887
1.109
0.148
0.393
0.434
0.463
0.418
Percentage distribution of the radioactivity recovered in tissue lipid . 64.3 15.7 5.0 0.6 14.4
. 68.4 8.4 10.4 1.2 11.5
.
. 39.3 10.8 17.7 1.8 30.4
. 65.2 8.9 9.2 3.2 13.5
80.9 6.0 5.3 1.2 13.6
1.8 35.7 9.8 30.3 5.0 17.6
1.2 52.1 9.7 17,5 4,0 15.5
Number of experiments were 2 in each group except with erucic acid where there were four experiments. Values are means of the observations. *Amounts of total radioactive lipids were expressed as percentages of the injected radioactivity (5 ptCi). The values were normalized as per g wet weight.
626
s . c . VASDEVAND K. J. XAKO
I n the experiment with palmitate, the label in the lysophosphatidylcholine fraction decreased and that in the phosphatidylcholine increased with increasing time (Table 6). I n the experiment with oleic acid, the radioactivity in phosphatidic acid increased with time but this increase was not reflected in the corresponding increase in phosphatidylcholine or other phospholipids. T h e phospholipid labelling from erucic acid was different again in that it incorporated greatly into phosphatidylserine and lysophosphatidylcholine fraction. Phosphatidylcholine fraction was comparably less labelled. T h e pattern changed little with the increasing time after injection, suggesting low reaction rates of some synthetic steps with erucic acid. TABLE 6. In vivo incorporation of [t4C] fatty acid into heart phospholipid fractions Palmitic acid Time after injection Amounts of total radioactive phospholipids*
Oleic acid
Erucic acid
150 s
300 s
75 s
150 s
300 s
150 s
300 s
0.127
0.127
0.044
0.053
0.059
0.081
0.065
Fraction Percentage distribution of the radioactivity recovered in tissue phospholipids Phosphatidic acid 3.9 3.9 16.4 30.3 28.9 7.3 8.4 Phosphatidylserine 26.4 26.6 15.3 22.4 23.6 32.7 29.4 Lysophosphatidylcholine 34.2 18.9 11.4 16.9 11.4 28.3 26.4 Phosphatidylcholine 27.6 39.0 48.8 21.6 21.3 17.8 17.3 Phosphatidylethanolamine 7.8 11.6 8.0 8.8 14.8 14.0 18.5 Number of experiments were 2 in each group, except with erucic acid at 300 s where there were 4 experiments. *Amounts of total radioactive phospholipids were expressed as percentages of the injected radioactivity (5 ~tCi).The values were normalized as per g wet weight of tissue. Formation of radioactive acylcarnitine was very small in the in vivo experiment (0.0135-0.0169% of the injected radioactivity). Similarly only trace amounts of radioactivity were found in acylcarnitine in the tissue slice experiment. The higher labelling of acylcarnitine in the perfused heart than in the in vivo experiment could be due to higher specific activity of the intracellular free fatty acid pool in the recirculating perfused heart than in the in situ heart, which only received a transit exposure to a small bolus of fatty acid. T h e low level of acylcarnitine in the slice experiment could be due to a slow rate of oxidative metabolism in this preparation. 4. D i s c u s s i o n
I n the present study the distribution of various labelled fatty acids into tissue lipids was examined by using three different heart preparations. We found that the metabolic rates of individual fatty acids differ both quantitatively and qualitatively in heart muscle.
FATTY ACIDS IN HEART
LIPIDS
627
Labelled oleic acid was incorporated preferentially into triacylglycerol and comparably less into phospholipids both in the heart perfusion experiment and in the heart slice experiment. Similarly in the in vivo experiment, labelling of triacylglycerol with oleic acid was increased rapidly within 75 s following injection, while the incorporation of oleic acid into the phosphatidic acid fraction was comparably higher and its incorporation into phosphatidylcholine fraction lower than that of palmitic acid. Therefore, when oleic is a labelled precursor, phosphatidylcholine was synthesized at a reIatively low rate and triacylglycerol at a relatively high rate from phosphatidate. The rate of incorporation of linoleic acid into heart lipids in the perfused rat heart was higher than palmitic acid, oleic acid and linolenic acid. The incorporat i o n into individual fractions indicated that linoleic acid was incorporated into phospholipids to a greater extent than other fatty acids. The labelling of phosphatidylcholine was the highest with this fatty acid. Relatively large incorporation of linoleic acid into phospholipid fraction has also been found by others [20, 24], and is in part due to the acyl exchange reactions. The rate of incorporation of linolenic acid into heart lipids during perfusion was lowest among five fatty acids studied. However, the percentage incorporation of linolenic acid into phospholipids was high (20%). O f the individual phospholipid fractions, the incorporation of linolenic acid into phosphatidic acid was comparably high and into phosphatidylcholine comparably low, suggesting either that phosphatidic acid containing linolenic acid was utilized at a slow rate for the synthesis of phosphatidylcholine or that direct acylation of linolenic acid was a slow process. As discussed below, it is also possible that linolenic acid was converted to other species of fatty acid in the cell. Phosphatidylethanolamine was labelled almost equally well regardless of fatty acid donors used. In a separate study we found that both palmitoyl-CoA and linoleoyl-CoA are able to serve equally well as the acyl donor for the synthesis of lysophosphatidic acid and phosphatidic acid by cardiac mitochondria and microsomes [17, 30]. In other words, the acyltransferases of isolated cardiac subcellular fractions do not possess substantiaI fatty acid specificity. Therefore, asymmetric fatty acid distribution found in tissue lipids is largely due to the result of selective utilization of phosphatidate and diacylglycerol for further synthetic process. Previous reports indicated that turnover rates of subspecies of phospholipids having different fatty acid composition are not equal [1, 26]. The present work suggests that the high triacylglycerol formation from oleic acid, rapid rate of phospholipid formation from linoleic and linolenic acid and low rate of triacylglycerol formation from linolenic acid may be regulated by diacylglycerol transfer reaction; subspecies of diacylglycerol containing a large amount of oleic acid may be directed to a great extent to the formation of triacylglycerol, whereas subspecies of phosphatidate or diacylglycerol containing a large amount oflinoleic acid may be directed more for the formation of phosphatidylcholine.
628
S . G . VASDEV AND K. J. KAKO
Elovson observed that stearic acid, which was injected into the portal vein of rats, was rapidly converted into C-18, C-20 and C-22 fatty acids of various degrees of unsaturation in the in situ liver [9]. Similar studies have not yet, to the authors' knowledge, been carried out with heart muscle. However, it is possible that, in our experiment, part of the labelled fatty acid may have been converted to other fatty acids and then incorporated into complex lipids. I f the magnitude of such interconversion was of significant degree in heart tissue, calculation of the rate of incorporation based on the specific activity of the labelled fatty acid that was administered, as was done in this study, would have resulted in some overestimation. However, the identification of individual fatty acids in each of the lipid fractions in rat heart is near the limit of the resolution of the analytical techniques available, and thus was not attempted at this time. Under the experimental conditions adopted, the rate of uptake of linoleic and linolenic acid by the perfused heart was high. These results disagree with those of Stein and Stein [24] who showed no difference in the uptake of different fatty acids in the rat heart perfusion with a low (0.14 mm) concentration of fatty acid and serum albumin. Evans [11] reported that oleic acid was taken up by the perfused rat heart in preference to saturated fatty acid of equal chain length and to linoleic acid. He suggested that the cellular transport of individual fatty acids is determined by the difference in their solubility in aqueous media, strength of binding to albumin, affinity for tissue binding sites and fate of intracellular utilization [ 1/]. T h e incorporation of labelled erucic acid into tissue lipids both in the perfused heart and tissue slice experiments was greater than that of other fatty acids. The high radioactivity was found in the tissue fatty acid fraction in all three experiments and may in part be due to the relatively low rate of oxidation of erucic acid [5, 6, 27]. T h e incorporation of this acid into triacylglycerol was relatively small in both perfusion and in vivo experiments, despite the fact that its incorporation into phosphatidate and into diacylglycerol was not low, suggesting the existence of substrate specificity of diacylglycerol acyltransferase, as postulated previously [27]. In the tissue slice experiment with labelled stearic acid, a relatively large amount of the label accumulated in the tissue fatty acid fraction; this is probably an indication of a low rate of oxidation of this acid. Labelled lysophosphatidylcholine accumulated and formation of phosphafidylcholine was slow, suggesting that stearic acid was a less suitable substrate than other fatty acids for position 2 of phosphatidylcholine. T h e incorporation of different fatty acids into complex lipids in the tissue slice experiment was considerably slow as compared to perfused heart. However, labelling of the free fatty acid fraction was very large. It is possible that in this preparation the activation of fatty acid might have been below normal due to the low rate of energy production, although glucose was present in the incubation medium. Alternatively, the diffusion of oxygen might have been limited. Nevertheless, measurements with tissue slices were performed, because numerous studies
FATTY ACIDS IN HEART LIPIDS
629
have previously been carried out by using this preparation by a number of investigators, and therefore, a comparative metabolic study with different heart preparations was felt necessary. Recently, it has been shown that slices cut by the method similar to that used in our experiment are suitable for in vitro studies of myocardial function; heart slices were able to regulate cell volume and to establish high intracellular concentration of potassium [14]. Changes in the fatty acid incorporation into lipids in the in vivo experiment are kinetically complicated, since although the heart receives a bolus of fatty acid precursor, the latter is subsequently diluted by non-labelled fatty acids. However, a comparison can be made between the behaviour of different fatty acids and between different lipid fractions. In general, the metabolic pattern in the perfused heart and that in the in vivo experiment resembled closely. Results with tissue slices differed a little from the above two preparations, probably due, among other reasons, to the negligible work conditions. It has been shown recently that oxygen consumption and fatty acid oxidation increased linearly with the product of peak systolic pressure and heart rate [19]. The non-oxidative fate of fatty acids in the myocardium under increased work load of the heart is as yet to be investigated.
Acknowledgements This work was supported by grants from the Medical Research Council of Canada and the Ontario Heart Foundation. T h e authors gratefully acknowledge the assistance of Ms R. Tourangeau.
REFERENCES 1. AxzssoN,B., ELOVSON,J. & AnWDSON,G. Initial incorporation into rat liver glycerolipids of intraportaUy injected [sH] glycerol. Biochimica et biophysiva acta 210, 15-27 (1970). 2. AMEs, G. F. Lipids of Salmonella typhimurium and Escherichia roli. Structure and metabolism. Journal of Bacteriology 95, 833-843 (1968). 3. B E ~ - R o ~ R s , J. L. & NEnA, E. A. Cardiac fatty acids and histDpathology of rats, pigs, monkeys and gerbils fed rapeseed oil. Comparative Biochemistry and Physiology 41B, 793-800 (1972). 4. BING,R. J. Cardiac metabolism. PhysiologicalReviews 45, 171-213 (1965). 5. CHENG,C. & PANDr, S. V. Erueic aeid metabolism in rat heart preparations. Lipid 10, 335-339 (1975). 6. CHI~.ISTOPHER$EN, B. O. & BREMER,J. Erucic acid--An inhibitor of fatty acid oxidation in the heart. Biochimica et biophysicaacta 280, 506-514 (1972). 7. DAWSON,R. M. C., HEMINGTON,N. & DAVEm'ORT,J. B. Improvements in the method of determining individual phospholipids in a complex mixture by successive chemical hydrolyses. BiochemicalJournal84, 497-501 (1962).
630 8. 9. 10. 11. 12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
25. 26. 27. 28.
s . c . VASDEVAND K. J. KAKO DITTMER,J. C. & LES~R, R. L. A simple, specific spray for the detection of phospholipids on thin layer chromatography. Journal of Lipid Research 5, 126-127 (1964). ELOVSON,J. Immediate fate of albumin-bound [1-14(]] stearic acid following intraportal injection into carbohydrate re-fed rats. Biochimiea et biophysica acta 106, 480--494 (1965). EVANS,J. R., OPIE, L. H. & SmPP, J. C. Metabolism of palmitic acid in perfused rat heart. AmericanjToumal of Physiology 205, 766-770 (1963). EVANS,J. R. Cellular transport of long chain fatty acids. Canadian Journal of Biochemistry 42,955-969 (1964). FOLCH,J., LEES, M. & STANLEY,C. H. S. A simple method for isolation and purification of total lipids from animal tissues. ~7ournalof Biological Chemistry 226, 497-509 (1957). GORDON,R. S. Unesterified fatty acid in human blood plasma. II. The transl~rt function ofunesterified fatty acid. Journal of Clinkal Investigation 36, 810-815 (1957). GROeHOWSm, E. C., GANOTE, C. E., HmL, M. L. & JEm~INOS, R. B. Experimental myocardial ischemic injury. I. A comparison of Stadie-Riggs and free-hand slicing techniques on tissue ultrastructure, water and electrolytes during in vitro incubation. .yournal of Molecular and Cellular Cardiology 8, 173-187 (I 976). HARMS, P., CHLOUVERA~IS,J. GLOSTER,J. &JONES,J. H. Arterio-venous differences in the composition of plasma free fatty acids in various regions of the body. Clinical Science 22, 113-118 (1962). KAKO, K. J., Lxt~, M. S. & THOR~rroN, M . J . Changes in fatty acid composition of myocardial triglyceride following a single administration of ethanol to rabbits, aT0urnal of Molecular and Cellular Cardiology 5, 473-489 (1973). LIv, M. S. & KAKO, K.J. Characteristics of mitochondrial and microsomal monoaeyland diacylglycerol 3-phosphate biosynthesis in rabbit heart. Bioclwmical Journal 138, 11-21 (t974). NEELEY,J. R. & MORGAN,H. E. Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Annual Review of Physiology 36, 413-459 (1974). NF.Er~v, J. R., W m a ~ R , K. M. & MosmzuKb S. Effects of mechanical activity and hormones on myocardial glucose and fatty acid utilization. Circulation Research, Suppl. 38, 1-22-1-30 (1976). NESTEL,P.J. & Sa'EINB~RG,D. Fate ofpalmitate and oflinoleate perfused through the isolated rat liver at high concentrations, oTournalof Lipid Research 4, 461-469 (1963). ROTHHN,M. E. & BING, R . J . Extraction and release of individual free fatty acids by the heart and fat depots. Journal of Clinical Investigation 40, 1380-1386 (I 961). SEm)AR~VlCH,B. & CARROL,K. K. In vivo incorporation of labelled acetate into liver and serum lipids of rats on different dietary regimens. Canadian dTournal of Biochemistry 50, 557-562 (1972). SHTACH~R,G. & SHAFRm, E. Uptake and distribution of fatty acids in rat diaphragm and heart muscles in vitro. Archives of Biochemistry and Biophysics 100, 205-213 (1963). STEIN, O. & STEm, Y. Metabolism of fatty acids in the isolated perfused rat heart. Biochemica et biophysica acta 70, 517-530 (1963). UMBREIT,W. W., BURRIS,R. H., STAUFFER,J. F. Manometric Techniques and Tissue metabolism. 2nd Ed. pp. 110-120. Burgess, Minneapolis (1951). VAN DEN BOSCH,H., VAN GOLDE, L. M. G. & VAN DEENEN, L. L. M. Dynamics of phosphoglycerides. Reviews of Physiology 66, 13-145 (1972). VASDEV,S. C. & KAKO, K. J. Metabolism oferucic acid in the isolated perfused rat heart. Biochimica et biophysica acta 431, 22-32 (1976). WILLERERANDS,A. F. Myocardial extraction of individual non-esterified fatty acids,
F A T T Y ACIDS I N H E A R T LIPIDS
631
esterified fatty acids and acetoacetate in the fasting human. Clinlca chimica acta 10, 435--446 (1964). 29. WIanmLS, B. & BR.ESSLSR,R. Two-dlmensional thin layer chromatographic isolation of fatty acylcarnifines. Journal of Lipid Research 6, 313-314 ( 1965 ). 30. ZARoR-BEmU~NS, G. & KAKO, K . J . Positional and fatty acid specificity of monoacyland diacyl-glycerol-3-phosphate formations by rabbit heart microsomes. Biochimica et biophysica acta 441, I-13 (1976).
Incorporation o f Fatty Acids into Rat Heart Lipids. In Vivo and In Vitro S t u d i e s S. C. VASDEV AND K . J . K A K O
Department of Physiology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada (Received 21 May 1976, accepted in revisedform 7 September 1976) S. C, VASD~VAm) K.J. KaKo. Incorporation of Fatty Acids into Rat Heart Lipids. In Vivo and In VitroStudies. 3ournalof MolevularandCellularCardiology(1977) 9, 617-631. Incorporation of different [14C] labelled long chain fatty acids into complex lipids of myocardium was studied using three different preparations, i.e., isolated perfused heart, heart slices and in vivo animals. Neutral and phospholipids were fractionated by thin layer chromatography. The results showed that labelling of triacylglycerol was highest when labelled oleic acid was used as precursor. On the other hand phospholipid labeUing was maximum with linoleic acid or linolenic acid as the labelled substrate in the medium. Both linoleic and linolenic acid were extracted and oxidized by the perfused heart at a higher rate than other fatty acids. Erucic and stearic acids were accumulated in the myocardium probably because they were oxidized at a slow rate. With labelled erucate as the precursor, the formation of labelled diacylglycerol was relatively large. Of the individual phospholipid fractions studied, labelling of phosphatidylcholine was high with linoleic acid, but phosphatidylethanolamine was labelled equally well, irrespective of fatty acid precursors. Percentage incorporation of erucic and linoleic acid into phosphatidic acid was high probably because the latter molecule was utilized relatively slowly for further synthetic pathway. Labelling into lysophosphatidylcholine was relatively more than that into phosphatidylcholine in the experiment with stearic acid, suggesting that this fatty acid is less suitable than other fatty acids for position 2 of phosphatidylcholine molecules. These results show that the pattern of incorporation of individual fatty acids in lipids of heart muscle varies in each of the three different experimental procedures.
K~Y WORDS:Palmitic acid; Oleic acid; Stearic acid; Linoleic acid; Linolenic acid; Erucic acid; Heart perfusion; Heart slices; Phosphatidylcholine; Phosphatidylethanolamine; Phosphatidic acid; Phosphafidylserine; Lysophosphatidylcholine; Cholesterol ester; Triacylglycerol; Diacylglycerol; Acylcarnitine.
1. Introduction T h e c o n t r i b u t i o n of long c h a i n fatty acids as a p r i n c i p a l fuel of respiration of c a r d i a c muscle has b e e n clearly d e m o n s t r a t e d i n the h u m a n as well as i n the isolated h e a r t p r e p a r a t i o n [4]. F u r t h e r m o r e , the relative p e r c e n t a g e of p l a s m a oleic acid decreased a n d that of p a l m i t i c acid increased d u r i n g passage of the blood t h r o u g h the c o r o n a r y vessel i n the h u m a n , suggesting t h a t several fatty acids comm o n l y available to the h e a r t as p l a s m a F F A are n o t e q u a l l y utilized b y the m y o c a r d i u m [15, 21, 28]. Studies with isolated perfused rat hearts using [14C] fatty acids i n d i c a t e d t h a t the p a t t e r n of u t i l i z a t i o n of fatty acid b y the m y o c a r d i u m m a y
618
S . C . V A S D E V A N D K.
j.
KAKO
change depending upon the quantity and type of fatty acids [II, 18, 24]. The rate of fatty acid uptake by rat hearts was proportional to its concentration and molar ratio of fatty acid to albumin in the perfusing fluid [I0, 13, 23]. Since a part of the fatty acids taken up from the circulation is converted to tissue lipids [4, I0, 16, 18], it is of interest to know patterns of incorporation of individual long chain fatty acids into tissue lipids in the myocardium. In the present study, we examined the metabolism of long chain fatty acids which form the main constituents of the dietary fat. In addition we studied the metabolism of erucic acid (cis-13-docosenoic acid) which forms a large portion of the total fatty acids of some strains of rapeseed oil, since dietary intake of this oil produces myocardial lesions in various species of animals [3]. An effort was also made to compare the incorporation of fatty acids in three different experiments, i.e. in the in vivo heart, perfused heart and heart slice.
2. M a t e r i a l s a n d M e t h o d s
Male albino rats (150-210 g) of a Sprague-Dawley 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 animals were fasted overnight before use. Krebs-Ringer bicarbonate or phosphate buffer (pH 7.4) was prepared according to Umbreit et al. [25]. To this buffer, glucose (5 raM) and bovine serum albumin (3%, w]v) were added. 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 final fatty acid concentration in the 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, which was essentially free from fatty acids (less than 0.005~o), was obtained from Sigma. [1-14C] palmitic acid (spec. act. 52 mCi/mmol), [1J4C] oleic acid (spec. act. 54 rnCi/mmol), [1-14C] stearic acid (spec. act. 54 mCi/mmol), [ I J 4 C ] linoleic acid (spee. act. 50 mCi/mmol) were purchased from New England Nuclear Corp. Boston, and [1J4C] Iinolenic-acid (spec. act. 42.3 mCi/mmol) and [14-14C] erucic acid (spec. act. 47 mCi/mmoI) were from Amersham/Searle Corp. Chicago and Schwartz] Mann, New York, respectively. All the fatty acids were checked for purity by thin layer chromatography and were found to be approximately 98% pure. All the nonlabelled fatty acids were obtained from Sigma Chemical Co. and approximately 99 % pure.
Perfusion studies The perfuslon apparatus and technique were described in detail elsewhere [27]. T h e animals were killed by decapitation and then hearts excised and immersed in ice-cold Krebs-Henseleit bicarbonate buffer which had been filtered and aerated with 95% 02 and 5% CO~. Visible fat and connective tissue were removed, the
F A T T Y ACIDS I N H E A R T L I P I D S
619
aorta cannulated and the heart was pre-perfused with 50 ml of buffer without fatty acid and albumin. Following the pre-perfusion, the heart was transferred to the proper recirculadng perfusion system containing 15 ml of perfusion fluid. Different fatty acids were used for different experiments. The perfusion was carried out at 37~ for 30 mln. During this period the perfusate dripping on the sintered glass was constantly exposed to a O~.-COz mixture. At the end of perfusion, the ventricles were blotted dry and two pieces cut, one for determination of dry weight and the other for lipid profile.
Extraction and analyses of lipids A portion of heart was homogenized in a glass homogenizer with a Teflon pestle in a solvent mixture (20 vol) containing chloroform-methanol (2:1, v/v). The lipids were further processed and purified by the method of Folch et al. [12]. Neutral liplds were separated by thin layer chromatography on silica gel G plates (precoated plates from Analab Inc. North Haven) by developing in a solvent system containing aV-hexane-diethyl ether-methanol-acetic acid (90:20:2:3, v/v/v]v) [22]. Phospholipids in the chIoroform-methanol extracts were separated by two dimensional thin layer chromatography [2] using solvent system 1 containing chloroform-methanol-cone. NH4OH (65:25:4, v]v/v) and system 2 containing chloroform-methanol-acetic acid (65:25:8, v/v/v). Chromatographic plates were activated immediately prior to application at 100~ for 1 h. Each lipid fraction was identified by using authentic lipid standards, which were purchased from Serdary Research Lab. London, Ontario.
Radioactivity determination Each lipid fraction from thin layer plates was scraped and transferred into a scintillation vial. To this 10 ml of Econofluor (New England Nuclear Corp) was added, and radioactivity determined by a Beckman, LS-150, liquid scintillation spectrometer. Efficiency was approximately 90%. The uptake of [14C] fatty acid was derived from the radioactivity disappeared from the medium during perfusion, as described previously [27]. For measurements of 14CO~ content, an aliquot of the perfusate was acidified (pH 2.0) and counted in Aquasol. The difference between radioactivity before and after acidification gave the value for 14CO2 in the perfusate. The COs released from the perfusion chamber was collected in an NaOH solution and the radioactivity in the latter solution measured. This 14CO~ value, together with the ~4CO~ in the perfusate, formed the total 14CO~ production by the heart in 30 min. The validity of this method was tested and described earlier
[27]. Heart slice experiment Hearts were taken from overnight fasted rats. Slices were prepared using glass slides and a sharp razor blade [25]. Slices weighing 300-400 mg (3-4 pieces) were
620
s.c. VASDEVAND K. J. r~AKO
incubated in each flask for 30 min at 37 ~ in 4.0 ml of Krebs-Ringer phosphate buffer (pH 7.4) containing 5 ham glucose and 0.5 mM [14C] fatty acid bound to 3% bovine albumin. The flasks were shaken vigorously in a Dubnoff incubator. At the end of incubation, the slices were rinsed three times with chilled buffer and then frozen in liquid nitrogen until processed. The fipids were extracted, purified, separated and radioactivities determined, as described above.
In vivo incorporation studies Male rats which were fasted overnight were anesthetized by a subcutaneous injection of 0.25 ml/100 g body weight of 2% chloralose in 20% urethane. [14C] fatty acid bound to albumin was injected into the jugular vein. The injected volume (0.5 ml) contained 5 ~Ci (0.11 ~mol) fatty acid and 15 mg albumin. The animals were sacrificed after 75, 150 or 300 s by decapitation, and the heart and liver were rapidly frozen in liquid nitrogen. The tissue was processed for lipid analyses.
Identification of fatty acylcarnitine Very high radioactivities were found in the sphingomyelin fraction of heart lipids in the perfusion experiment. This fraction was further analyzed and it was found that the labelling almost exclusively occurred in fatty acylcarnitine esters. In the chromatographic system routinely used in our study (see above), standard palmitoylcarnitine (supplied from Otsuka Pharmaceutical Co. Japan) gave the same RF value as sphingomyelin. We have eluted the substance from the region of thin layer chromatography corresponding to sphingomyelin with chloroform-methanolwater (30:50:2, v/v/v). The substance was (i) re-chromatographed with a solvent system containing chloroform-methanol-acetic acid-water (50:25:8:4, v]v[v[v), which separated acylcarnitine and sphingomyelin; and (ii) hydrolyzed by the method of Dawson et al. [7]. Upon hydrolysis, approximately 90% of the radioactivity of the sphingomyelin-acylcarnitine fraction appeared in the fatty acid fraction; standard sphingomyelin was not hydrolyzed under these conditions. We have also found that two dimensional thin layer chromatography using chloroform-methanol-ammonia-water (50:35:3:3, v/v/v]v) and chIoroform-methanolglacial acetic acid-water (50:25:8:4, by vol) [29] separated fatty acylcarnitine. The plates were first exposed to iodine vapour, which stained both acylcarnitine and phospholipids, and to the molybdenum blue reagent of Dittmer and Lester [8], which stained only phospholipids. Incorporation of fatty acid into acylcarnitine in the perfused heart preparation was further verified by perfusing the heart in the presence of DL-carnitine (methyl-[14C]) hydrochloride (ICN Chemical & Radioisotope Division, Waltham, Mass) and non-labelled fatty acid. Two dimensional
FATTY
A C I D S IN H E A R T
LIPIDS
621
thin layer chromatographic analyses showed that all the radioactivity was located in the sphingomyelin-acylcarnitine spot.
3. R e s u l t s
Studies with perfused hearts Uptake of [14C]fatty acid Albumin-bound [14C] linoleic acid was taken up at the highest rate during 30 rain perfusion in this preparation. The rates of uptake of linolenate, palmitate and oleate were lower than that of linoleate (Table 1). Incorporation of erucic acid into tissue lipids was highest and its oxidation lowest among all fatty acids examined. Linoleic and linolenic acids were oxidized at the highest rate as compared to all other fatty acids.
Incorporation of [14C]fatty acid into tissue lipids Linoleic acid incorporation was highest in phospholipid fraction both in the absolute quantity and as the percentage of total lipids (Table 2). Although the incorporation of linolenate in the total tissue lipids was considerably below that of other fatty acids, comparably a large fraction of the total tissue radioactivity was found in phospholipid fraction. The incorporation into acylcarnitine was greatest with labelled palmitic acid (Table 2). Palmitoylcarnitine accumulated probably because palmitate was activated and transferred to carnitine at a faster rate, or because palmitoylcarnitine was oxidized at a slower rate as compared to other fatty acids. Comparison ofdistributlon of various fatty acids revealed that labelling of diacylglycerol was the highest in the experiment with erucic acid. Significant differences were also observed in the incorporation pattern of fatty acids into phospholipid classes (Table 2). The formation of radioactive phosphatidylcholine was highest with linoleic acid, followed by palmitic acid, as the labelled substrate in the perfusate. The radioactivity in the phosphaddylserine fraction was highest with oleic acid, whereas phosphatidic acid labelling was greatest with erucic acid and linolenic acid.
Incorporation of [14C] fatty acid into tissue lipids in heart slices The distribution of labelled fatty acid in tissue lipids is shown in Table 3. The highest incorporation into tissue lipid was found with erucic acid, followed by stearic, palmitic and oleic acids. At least 70-90% of the total radioactivity was
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TABLE 3. Comparison of incorporation of [14C] fatty acid into tissue lipids in heart slices Lipid fraction Cholesterol ester Triacylglycerol Diacylglycerol Fatty acid Phospholipids Total Iipids
Palmitic acid
Oleic acid
Stearic acid
Erucic acid
2.9 -4- 0.2 7.8 -4- 0.7 6.8 ztz 0.5 108.0 4- 1.1 15.0 q- 0.9 I40.6 4- 1.9
0.69 q- 0.1 6.1 -4- 0.3 3.5 -4- 0.2 100.3 -4- 5.1 6.9 q- 0.1 117.6 4- 5.1
1.4 + 0.1 5.9 • 0.4 4.3 4- 0.2 193.2 4- 8.6 11.0 -t- 0.4 217.9 -4- 10.3
94.5 4- 4.3 28.7 =k 2.2 51.5 i 8.9 382.3zt: 29.0 20.5 ~- 1.5 577.4 ~ 27.0
All values are presented as nmol fatty acid incorporated per g of the wet weight (mean -]- S.E.M.). Number of experiments were 4 in each group. recovered in the fatty acid fraction. T h e radioactivity in the fatty acid fraction was highest in the experiment with erucic acid, followed by that with stearic acid. I t is possible that these two fatty acids penetrate the cell m e m b r a n e at a faster rate, and therefore, would accumulate in the tissue. O n the other hand, the oxidation of these acids by the heart slices m a y be slower as compared to other fatty acids. However, in the present study the oxidation of fatty acid was not measured in this experiment. While the amount oferucic acid incorporated into total lipids in the perfused hearts was only slightly greater than the amount of palmitic acid, approximately four times more erucic acid was incorporated in the tissue slices than was palmitic acid. T h e cause for this difference was not further studied. Molar ratios of newly formed triacylglycerol, diacylglycerol and phospholipids in heart slices were approximately 1:1:2 with palmitate as a precursor, 2:1:2 with oleate, 1.5:1:3 with stearate, and 1.5:2.5:1 with erucate. These ratios clearly indicated that oleic acid was incorporated relatively more into triacylglycerol than into phospholipids, as compared to palmitic and stearic acid. Furthermore, erucic acid greatly incorporated into diacyl- and triacylglycerol. A m o n g phospholipid classes, approximately 60% of the IabeUed palmitate was recovered in phosphatidylcholine but m u c h less ~o of the total activity was accounted for by the incorporation of other fatty acids into phosphatidylcholine (Table 4). T h e radioactivity in phosphatidic acid formed the major fraction in the experiment with erucic acid. Relatively high percentages of the total activities were found in the lysophosphatidylcholine fraction with stearic and oleic acid as the precursor (Table 4).
Incorporation of [14C] fatty acid into heart lipids in vivo T h e incorporation of injected [14C] fatty acid into different lipid fractions of rat hearts is shown in Table 5. T h e data are presented in terms of percentages of the injected radioactivity. It should reflect reasonably well the specific activities of each lipid fraction, because, in a relatively short period, absolute amounts of esterified fatty acids in tissue must have remained constant. As early as 150 s after injection of radioactive fatty acid, labelling in the triacyl-
FATTY ACIDS IN HEART LIPIDS
625
TABLE 4. Comparison of incorporation [14C] fatty acid into phospholipids in heart slices Palmitic acid nmol of fatty acid incorporated into total tissue phospholipids per g wet weight 15.02 4- 0.99
Oleic acid
Stearic acid
Erucic acid
6.95 4- 0.06
11.02 4-. 0.37
20.47 4- 1.50
Phospholipids class
Radioactivity as percentages of total labelled phospholipids
Phosphatidic acid Phosphatidylserine Lysophosphatidylcholine Phosphatidylcholine Phosphatidylethanolamine
2.7 zk 0.I 16.0 4- 1.2
14.5 + 1.2 14.7 4- 0.7
8.6 -4- 0.6 19.5 4- 1.0
45.5 -4- 1.0 2.9 4- 0.5
14.3 4- 0.9 59.3 zk 0.8
23.4 4- 2.4 37.6 + 1.8
33.2 4- 0.8 26.2 + 1.3
9.6 4- 2.1 36.0 4- 1.6
7.8 ~ 0.5
9.8 -4- 0.4
12.5 4- 0.5
6.1 4- 0.7
Values are expressed as mean • S.~.M.The number of experiments were 4 in each group. glycerol i n the h e a r t b e c a m e highest a m o n g all the lipid fractions, regardless of the k i n d of fatty acids. Between 75 s a n d 300 s after injection, the r a d i o a c t i v i t y i n the phospholipid fraction (in terms of % of the total activity) decreased; this was p a r t i c u l a r l y e v i d e n t with oleic acid, i n w h i c h case the label i n triacylglycerol increased a n d that i n diacylglycerol decreased with time. Erucic acid b e h a v e d v e r y differently from other fatty acids; the r a d i o a c t i v i t y i n triacylglycerol was relatively low a n d that i n the tissue fatty acid was relatively high, suggesting t h a t this acid was poorly oxidized a n d was poorly transformed to triacylglycerol. TABLE 5. In vivo incorporation of [14C] fatty acid into heart lipids Palmitic acid Time after injection Amount of total radioactive tissue lipids* Fraction Cholesterol ester Tr{acylglycerol Diacylglycerol Fatty acid Acy!carnitine Phospholipids
Oleic acid
Erucic acid
150 s
300 s
75 s
150 s
300 s
150 s
300 s
0.887
1.109
0.148
0.393
0.434
0.463
0.418
Percentage distribution of the radioactivity recovered in tissue lipid . 64.3 15.7 5.0 0.6 14.4
. 68.4 8.4 10.4 1.2 11.5
.
. 39.3 10.8 17.7 1.8 30.4
. 65.2 8.9 9.2 3.2 13.5
80.9 6.0 5.3 1.2 13.6
1.8 35.7 9.8 30.3 5.0 17.6
1.2 52.1 9.7 17,5 4,0 15.5
Number of experiments were 2 in each group except with erucic acid where there were four experiments. Values are means of the observations. *Amounts of total radioactive lipids were expressed as percentages of the injected radioactivity (5 ptCi). The values were normalized as per g wet weight.
626
s . c . VASDEVAND K. J. XAKO
I n the experiment with palmitate, the label in the lysophosphatidylcholine fraction decreased and that in the phosphatidylcholine increased with increasing time (Table 6). I n the experiment with oleic acid, the radioactivity in phosphatidic acid increased with time but this increase was not reflected in the corresponding increase in phosphatidylcholine or other phospholipids. T h e phospholipid labelling from erucic acid was different again in that it incorporated greatly into phosphatidylserine and lysophosphatidylcholine fraction. Phosphatidylcholine fraction was comparably less labelled. T h e pattern changed little with the increasing time after injection, suggesting low reaction rates of some synthetic steps with erucic acid. TABLE 6. In vivo incorporation of [t4C] fatty acid into heart phospholipid fractions Palmitic acid Time after injection Amounts of total radioactive phospholipids*
Oleic acid
Erucic acid
150 s
300 s
75 s
150 s
300 s
150 s
300 s
0.127
0.127
0.044
0.053
0.059
0.081
0.065
Fraction Percentage distribution of the radioactivity recovered in tissue phospholipids Phosphatidic acid 3.9 3.9 16.4 30.3 28.9 7.3 8.4 Phosphatidylserine 26.4 26.6 15.3 22.4 23.6 32.7 29.4 Lysophosphatidylcholine 34.2 18.9 11.4 16.9 11.4 28.3 26.4 Phosphatidylcholine 27.6 39.0 48.8 21.6 21.3 17.8 17.3 Phosphatidylethanolamine 7.8 11.6 8.0 8.8 14.8 14.0 18.5 Number of experiments were 2 in each group, except with erucic acid at 300 s where there were 4 experiments. *Amounts of total radioactive phospholipids were expressed as percentages of the injected radioactivity (5 ~tCi).The values were normalized as per g wet weight of tissue. Formation of radioactive acylcarnitine was very small in the in vivo experiment (0.0135-0.0169% of the injected radioactivity). Similarly only trace amounts of radioactivity were found in acylcarnitine in the tissue slice experiment. The higher labelling of acylcarnitine in the perfused heart than in the in vivo experiment could be due to higher specific activity of the intracellular free fatty acid pool in the recirculating perfused heart than in the in situ heart, which only received a transit exposure to a small bolus of fatty acid. T h e low level of acylcarnitine in the slice experiment could be due to a slow rate of oxidative metabolism in this preparation. 4. D i s c u s s i o n
I n the present study the distribution of various labelled fatty acids into tissue lipids was examined by using three different heart preparations. We found that the metabolic rates of individual fatty acids differ both quantitatively and qualitatively in heart muscle.
FATTY ACIDS IN HEART
LIPIDS
627
Labelled oleic acid was incorporated preferentially into triacylglycerol and comparably less into phospholipids both in the heart perfusion experiment and in the heart slice experiment. Similarly in the in vivo experiment, labelling of triacylglycerol with oleic acid was increased rapidly within 75 s following injection, while the incorporation of oleic acid into the phosphatidic acid fraction was comparably higher and its incorporation into phosphatidylcholine fraction lower than that of palmitic acid. Therefore, when oleic is a labelled precursor, phosphatidylcholine was synthesized at a reIatively low rate and triacylglycerol at a relatively high rate from phosphatidate. The rate of incorporation of linoleic acid into heart lipids in the perfused rat heart was higher than palmitic acid, oleic acid and linolenic acid. The incorporat i o n into individual fractions indicated that linoleic acid was incorporated into phospholipids to a greater extent than other fatty acids. The labelling of phosphatidylcholine was the highest with this fatty acid. Relatively large incorporation of linoleic acid into phospholipid fraction has also been found by others [20, 24], and is in part due to the acyl exchange reactions. The rate of incorporation of linolenic acid into heart lipids during perfusion was lowest among five fatty acids studied. However, the percentage incorporation of linolenic acid into phospholipids was high (20%). O f the individual phospholipid fractions, the incorporation of linolenic acid into phosphatidic acid was comparably high and into phosphatidylcholine comparably low, suggesting either that phosphatidic acid containing linolenic acid was utilized at a slow rate for the synthesis of phosphatidylcholine or that direct acylation of linolenic acid was a slow process. As discussed below, it is also possible that linolenic acid was converted to other species of fatty acid in the cell. Phosphatidylethanolamine was labelled almost equally well regardless of fatty acid donors used. In a separate study we found that both palmitoyl-CoA and linoleoyl-CoA are able to serve equally well as the acyl donor for the synthesis of lysophosphatidic acid and phosphatidic acid by cardiac mitochondria and microsomes [17, 30]. In other words, the acyltransferases of isolated cardiac subcellular fractions do not possess substantiaI fatty acid specificity. Therefore, asymmetric fatty acid distribution found in tissue lipids is largely due to the result of selective utilization of phosphatidate and diacylglycerol for further synthetic process. Previous reports indicated that turnover rates of subspecies of phospholipids having different fatty acid composition are not equal [1, 26]. The present work suggests that the high triacylglycerol formation from oleic acid, rapid rate of phospholipid formation from linoleic and linolenic acid and low rate of triacylglycerol formation from linolenic acid may be regulated by diacylglycerol transfer reaction; subspecies of diacylglycerol containing a large amount of oleic acid may be directed to a great extent to the formation of triacylglycerol, whereas subspecies of phosphatidate or diacylglycerol containing a large amount oflinoleic acid may be directed more for the formation of phosphatidylcholine.
628
S . G . VASDEV AND K. J. KAKO
Elovson observed that stearic acid, which was injected into the portal vein of rats, was rapidly converted into C-18, C-20 and C-22 fatty acids of various degrees of unsaturation in the in situ liver [9]. Similar studies have not yet, to the authors' knowledge, been carried out with heart muscle. However, it is possible that, in our experiment, part of the labelled fatty acid may have been converted to other fatty acids and then incorporated into complex lipids. I f the magnitude of such interconversion was of significant degree in heart tissue, calculation of the rate of incorporation based on the specific activity of the labelled fatty acid that was administered, as was done in this study, would have resulted in some overestimation. However, the identification of individual fatty acids in each of the lipid fractions in rat heart is near the limit of the resolution of the analytical techniques available, and thus was not attempted at this time. Under the experimental conditions adopted, the rate of uptake of linoleic and linolenic acid by the perfused heart was high. These results disagree with those of Stein and Stein [24] who showed no difference in the uptake of different fatty acids in the rat heart perfusion with a low (0.14 mm) concentration of fatty acid and serum albumin. Evans [11] reported that oleic acid was taken up by the perfused rat heart in preference to saturated fatty acid of equal chain length and to linoleic acid. He suggested that the cellular transport of individual fatty acids is determined by the difference in their solubility in aqueous media, strength of binding to albumin, affinity for tissue binding sites and fate of intracellular utilization [ 1/]. T h e incorporation of labelled erucic acid into tissue lipids both in the perfused heart and tissue slice experiments was greater than that of other fatty acids. The high radioactivity was found in the tissue fatty acid fraction in all three experiments and may in part be due to the relatively low rate of oxidation of erucic acid [5, 6, 27]. T h e incorporation of this acid into triacylglycerol was relatively small in both perfusion and in vivo experiments, despite the fact that its incorporation into phosphatidate and into diacylglycerol was not low, suggesting the existence of substrate specificity of diacylglycerol acyltransferase, as postulated previously [27]. In the tissue slice experiment with labelled stearic acid, a relatively large amount of the label accumulated in the tissue fatty acid fraction; this is probably an indication of a low rate of oxidation of this acid. Labelled lysophosphatidylcholine accumulated and formation of phosphafidylcholine was slow, suggesting that stearic acid was a less suitable substrate than other fatty acids for position 2 of phosphatidylcholine. T h e incorporation of different fatty acids into complex lipids in the tissue slice experiment was considerably slow as compared to perfused heart. However, labelling of the free fatty acid fraction was very large. It is possible that in this preparation the activation of fatty acid might have been below normal due to the low rate of energy production, although glucose was present in the incubation medium. Alternatively, the diffusion of oxygen might have been limited. Nevertheless, measurements with tissue slices were performed, because numerous studies
FATTY ACIDS IN HEART LIPIDS
629
have previously been carried out by using this preparation by a number of investigators, and therefore, a comparative metabolic study with different heart preparations was felt necessary. Recently, it has been shown that slices cut by the method similar to that used in our experiment are suitable for in vitro studies of myocardial function; heart slices were able to regulate cell volume and to establish high intracellular concentration of potassium [14]. Changes in the fatty acid incorporation into lipids in the in vivo experiment are kinetically complicated, since although the heart receives a bolus of fatty acid precursor, the latter is subsequently diluted by non-labelled fatty acids. However, a comparison can be made between the behaviour of different fatty acids and between different lipid fractions. In general, the metabolic pattern in the perfused heart and that in the in vivo experiment resembled closely. Results with tissue slices differed a little from the above two preparations, probably due, among other reasons, to the negligible work conditions. It has been shown recently that oxygen consumption and fatty acid oxidation increased linearly with the product of peak systolic pressure and heart rate [19]. The non-oxidative fate of fatty acids in the myocardium under increased work load of the heart is as yet to be investigated.
Acknowledgements This work was supported by grants from the Medical Research Council of Canada and the Ontario Heart Foundation. T h e authors gratefully acknowledge the assistance of Ms R. Tourangeau.
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