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Comparative mitochondrial oxidation of fatty acids
BIOCHIMICA ET BIOPHYSICA ACTA
168 BB* 55875
COMPARATIVE
MITOCHONDRIAL
K. J. HUXTABLE*
AND S.4LIH
Department
of Biochemistry,
(Received
December
Duke
OXIDATION
OF FATTY
ACIDS
J. WAKIL** University
Medical
Center, Duvham,
N.C.
27706 (U.S.A.)
just, 1970)
SUMMARY
The comparative oxidation of I-%and U-14C-labeled fatty acids has been studied in beef heart mitochondria. The relative rates of oxidation depended on the conditions employed. Under certain conditions, the rate of oxidation of U-K-labeled acids decreased as unsaturation increased, whereas under the same conditions [I-%]linoleate was oxidized faster than [I-14C]palmitate, as measured by the production of radioactive carbon dioxide. Addition of L-(-)-carnitine and ATP together produced an intense pulse of oxidation. For [I-14C]palmitate, maximum oxidation under such conditions occurred at an energy charge of around 0.65. A search was made for evidence that oxidation of polyunsaturated acids could proceed by “yoxidation” to yield methylmalonic acid via propionic acid. No such evidence could be found, and it is concluded that in beef heart mitochondria, the oxidation of unsaturated fatty acids can be acounted conditions used here.
for solely
on the basis
of ,&oxidation
under
the
INTRODUCTION
While the mitochondrial oxidation of fatty acids has been well-studied, there is a paucity of work which has used uniformly labeled acids. Most work has used carboxyl labeled acids, and it has been assumed that the reactions of these, as followed by the label, are indicative of the complete fatty acid molecule. The /&oxidation pathway for fatty acids has been well established. However, there has been speculation as to the possible existence in mammals of a y-oxidation pathway in the catabolism of long chain unsaturated fatty acidsl-3. Unsaturated acids containing a 9,10 double bond can not undergo total /?-oxidation unless the site of unsaturation shifts. That this, indeed, happens has been demonstrated4+. Because of the possibility of gluconeogenesis from unsaturated acids, the existence of a yoxidation pathway remained an attractive speculation. y-Cleavage of the double * Present address, Department
of Pharmacology, University of Arizona, Tucson, Xriz. 8j72I (U.S.A.). ** Address reprint request to Salih J. Wakil, Duke University Medical Center, Durham, N.C. 27706 (U.S.A.). Biochim.
Biophys.
Acta,
239 (1971) 168-177
~ITOCHONDRIAL
OXIDATION
OF FATTY ACIDS
169
bond in a #I-oxidation pathway intermediate such as 3,6,9-dodecatrienoyl-CoA, derived from linolenic acid by the loss of three C, units, would give propionyl-CoAz. This could enter into gluconeogenesis via methylmalonyl-CoA, succinyl-CoA, and oxaloacetate6. The principle support for a y-oxidation theory has come from the supposedly faster rate of oxidation observed with unsaturated acid+-le. It has been argued that this could be explained by the “self-priming” effect of propionyl-Coil. production on the tricarboxylic acid cycle. By & novo production of tricarboxylic acid cycle intermediates, the flux through the cycle is increased. using rat livers, studied the relative distriHowever, ANTOXY AND LANDAPO, bution of activity in glucose following feeding of [2-%]acetate, [r6-14Cjpalmitate, and [ro-%]oleate. The labeling patterns of the glucose derived from all three were similar enough for the authors to conclude that [ro-14C]oleate underwent exclusive b-oxidation. ANDERSON 21 demonstrated that all the geometric isomers of 9,12octadecadienoic acid, in rat liver mitochondria, were oxidized to only CO, and ketone bodies. The amount of acetoacetate formed was directly proportional to the amount of acid oxidized, and all acids produced the same quantity of CO, under the same conditions. One can conclude that only /$-oxidation is occurring with all isomers. Here, we report some observations on the comparative oxidation of I-‘%- and I?-%-labeled acids by beef heart mitochondria. With U-“*C-labeled acids the rate of oxidation decreased as unsaturation increased as would be expected if B-oxidation was occurring4. No evidence was found for a different oxidation pathway. MATERIALS
AND
METHODS
Heavy beef heart mitochondria were obtained from the Institute for Enzyme Research, University of Wisconsin. They were kept in 0.25 M sucrose at -15" until used. Experiments were done within three months of isolation. A Barber-Coleman Selecta System Series 5000 gas-liquid chromatograp~ equipped with a radium ionization detector was used, with an 8-ft column packed with IS”/ diethylene glycol succinate on Chromosorb W, 60-80 mesh (Analytical Engineering Laboratories, Inc., Hamden, Conn.). For measurement of radioactivity, the eMuent gas was passed through a Nuclear Chicago proportional detector and the radioactivity automatically recorded. Scintillation counting was performed with a Tri-carb Model 314 EX liquid scintillation counter. All counts were corrected for efficiency using an internal standard. [%]Toluene (obtained from New England Nuclear Corp., Boston), measured volumetric~ly, was used to standardize activities of roooo countslmin or above. [14C]Hexadecane (prepared by dilution of a standard hex&cane obtained from the Radiochemical Centre Amersham), measured gravimetrically, was used to standardize activities below IO ooo counts/min. r-14C-labeled linolenic and U-%-labeled linolenic, linoleic, oleic, and palmitic acids were obtained from the Radiochemical Centre, England. I-‘K-labeled palmitic and linoleic acids were obtained from New England Nuclear Corp., Boston, and U14C-labeled linolenic and linoleic acids were additionally obtained from Applied Science Laboratories, Penn. Omnifluor was obtained from New England Nuclear Corp., Boston, carnitine from Nutritional Biochemicals, Cleveland, bovine serum albumin from Sigma, St.
R. J. HWXTABLE,S. J. WAKIL
‘70
Louis, and CoA, NAD+, ATP, ADP, and AMP from P-L Biochemicals, Milwaukee. Radioactive acids were diluted with inactive acids and made up as solutions in ethanol such that 0.01 ml contained the concentration of the acids needed per incubation. The purity of these solutions was checked at intervals by gas-liquid chromatography. The incubation media used were: Medium A: concentrations (mM) ; albumin 0.014,sucrose IOO, EDTA 0.4, KC1 80, sodium phosphate (pH 7.4) 8, M&l, 5, ATP I, NADf 2.5, CoA 0.04, carnitine I, NaHCO, 25. Total volume 2.5 ml. Medium B: as Medium A, with the further addition of maiic and citric acids in concentrations of 2 mM and 0.2 mM, respectively. Protein was determined by the biuret methodm. Where swollen mitochondria were used, these were suspended in water and homogenized by hand with a teflon homogenizer. Incubations were carried out at 37” in erlenmeyer flasks with a short piece of glass tubing welded to the bottom inside. In this tubing a plastic cup was placed. The flask was fitted with a serum cap. Just before the end of the incubation 0.2 ml of hyamine was injected into the cup, and the incubation then stopped by injection of 0.2 ml 3 M H,SO, into the medium. The CO, liberated was absorbed by the hyamine over a r-h period. The cup with the hyamine was then placed in a counting vial, and IO ml of scintillation solution added. The scintillaht used contained: dioxan 2.4 1, anisole 0.4 1, r,z-dimethoxyethane 0.4 1, omnifluor 25.6 g.
At the end of the incubation, 0.05 mg each of succinic and methylm~onic acids were added to the incubation mixture. The pH of this was then adjusted to II, and the solution was left at room temperature for 2 h. The pH was brought to 2 with HCI, and the solution was extracted with pentane until little further activity was removed. It was further extracted with ether (3 times). The ether extracts were reduced to a small volume, and spotted onto a precoated silica gel plate (obtained from Eastman Kodak Co., Rochester, N.Y.), oven-dried immediately before use. The solvent systems used were : benzene-methanol-acetic acid (45 : 8 : 4, by ~01.)~~.with RF values of 0.40 for methylmalonic acid, 0.58 for succinic acid, and 1.00 for fatty acids; benzene-dioxane-acetic acid (90: 25 : 4, by ~01.)~~with RF values of 0.24, 0.41, and 1.00 for methylmalonic, succinic, and fatty acids, respectively; and ethanolwater-25% ammonia (96: 16:16, by v01.)~~with RF values of 0.30, 0.37, and 1.00 for methylmalonic succinic and fatty acids, respectively. After being developed, the plate was oven-dried at 100’ for 0.5 h, and then sprayed with a solution made up of bromophenol blue (50 mg), andcitric acid (zoomg) in water (IOO m&The chromatographic plate was cut into equal sized portions, and each portion shaken with scintillator solution and counted. Isolation and crystallization
ofdicarboxylic
acids
At the end of the incubation, the mixture was divided into two equal halves. To one was added 50 mg succinic acid, and to the other 50 mg of methylmalonic acid. Each solution was worked up as described above. The reisolated succinic acid was recrystallized from acetic acid-chloroform, and methylmalonic acid from ethyl acetate-benzene. All recrystallizations were carried out in Craig tubes31. The activity of the acids (two samples each) were measured after each recrystallization. Biockim. Biapitys. Acta,
239 (1971) 168-177
MITOCIIONDRIAL OXIDATION OF FATTY ACIDS
171
The avidin-treated incubations contained 56 nmoles of avidin. Where biotinpretreated avidin was used, a biotin : avidin mole ratio of 5 : I was employed.
The incubation mixtures were basified to pi3 12, stored for 2 fr, then reacidified to pH. 2. The mixtures were ether extracted (4 times), and the extracts dried and reduced in volume to around 0.5 ml. A solution of diazomethane in ether was added until no more color was discharged. The methylated solutions were kept at 4” overnight before gaschromatographic analysis was carried out. Diazomethane was prepared by the action of base on bis(~-methyl-~-nitroso)terepht~~alamide in ether solution. The diazometh~e was distilled out, and trapped in ice-cold ether. TABLE Co,
I
PRODUCTION
UNDER
VARYING
CONDITIONS
OF INCUBATION
In each experiment, the first column gives the actual activity measured in the CO,, corrected for background and efficiency. The second column gives the nmoles of acid oxidized per mg of protein. All incubations contained 120 nmoles of the acid under study. Conditions used: (I) Whole mitochondtia (g mg) in Medium A, 30 min incubation. (II) Swollen mitochondria (13 mg) in Medium A, 30 min incubation. (III) Swollen mitocbondria (5 mg) in Medium A, 20 min incubation. (IV) Swollen mitochondria (3.5 mg) in Medium B, 30 min incubation.
Fatty acid
Expt. I ~-____ “CO,
Fatty acid
(disint. f oxidized
II-**C]Palmitate
min)
(nmolesjmg p&e+)
37 3oo
0.46
200
?J-W]Palmitate I-**C]Linoleate %-14C]Oleate U-rPC]Linoleate U-%]Linolenate
58 10500 14000 8300 4 7oo
Ex$x. II _-wo,
-._ Fatty acid
(disint. / min)
oxidized fnmoles/mg prorein)
423 9oo
3.6~
7300 IjS200
7-76 3.06 2.14 1‘34
Expt. III IWO, Fatty acid (disint./ oxidized min)
(rtmoleslmg
Expt. IV Fatty acid WO, (disint.j min)
oxidized (nmoleslmg @atein)
28400
0.89
18400 3 200 4 900 3 000 8 600
0.31 0.42 0.29 0.49
p?‘Otein)
1.26
0.40 0.38 0.23 0.13
1.02
II3400
68 300
56000 22400 15000 7 700
6.32 1.86
1.21 0.65
RESULTS
U-14C- and r-lGlabeled fatty acids were oxidized by mito~bo~dria, and the CO, produced trapped and counted. Results obtained under various conditions are shown in Table I. Swollen mitochondria gave much higher activities than whole mitochondria. In Medium A, the rate of oxidation of uniformly labeled fatty acids decreased as the unsaturation increased. However, higher activity was obtained from [I-%]linoleic acid than from [r-l%]palmitic acid. When exogenous Krebs cycle acids were added to the medium (tedium B), the mount of radio-labeled CO, being produced fell, and the relative rates of oxidation of the uniformly labeled acids altered to a near constant level for all the acids, This is presumably a reflection of the competing processes of faster flux through the Krebs cycle, but higher dilution of label into Krebs cycle intermediates. Fig. I shows the comparative rates of oxidation of [I-~%]- and [U-**C]linoleate at different acid concentrations. The curves are complex ones, reflecting the comphxity of the system, but they show that at low concentrations of acids the I-position is oxidized appreciably faster than the molecule, over-all. Since no free intermediary Biochim. Biophys. Acta, 239 (1971) 168-177
R. J. HUXTABLE, S. J. WAKIL
172
+otty Acid Added (n moles]
Fig. 1. Dependence of oxidation on concentration of acid for [I-%]and [U-“%]linoleate. Comparative rates of oxidation of [I-*%]- and [U-*4C]linoleate at different acid concentrations; 2.45 mg protein added per flask. Medium A used with swollen mitochondria.
chain length acids accumulate, the difference between the rate of oxidation of the [I-‘*Cl- and [U1*C]linoleate represents enzyme-bound moities undergoing active oxidation. ~-Oxidation is not an instantaneous process, and starting from the Iposition it takes a finite time to oxidize down to the eqosition. Every time a double bond is reached the process is slowed further due to the need for extra rearrangement steps, as shown by STOFFEL~~~. The noninvolvement of y-oxidation If part of the activity of the CO, produced from unsaturated acids was coming via propiol~ate-n~ethyl malonate-succinate pathway, this part should be biotin dependent. Propionyl-CoA carboxylase is a biotin-containing enzyme, and as such should be completely inhibited by treatment with the protein avidin25. Fatty acids were incubated with swollen mitochondria in the presence of avidin, and biotinpretreated avidin, with the results shown in Table II. Avidin of IO units/mgz6 and TABLE EFFECT
II OF AVIDIN
OK FATTY
ACID
OXIDATION
Medium A used with 120 nmoles of each acid and 3.46 mg mitochondria per flask. Incubation time was 20 min; percent activity is the percentage of activity originally present in the fatty acid which appears in the CO,.
[U-‘*C]Palmitate [U-‘*C]Oleate [U-14C]Linoleate [U-14C]Linolenate
7100 5500 2900 II00
2.01
5000
1.41
1.1.5 0.58
46~=~
0.95
2200 II00
0.45
0.22
0.23
7100 4500 1700
2.02 0.93 0.33
I200
0.24
commercial avidin of 2-5 units/mg were used, and no marked difference was observed between them. Only the oxidation of in-l~C]palmitate was appreciably affected by the presence of avidin. The reason for the effect of avidin on the oxidation of palmitate is not apparent. Nevertheless, the lack of effect of avidin on the oxidation of the unsaturated acids strongly indicates that a propionate-methyl malonate pathway is not involved in the oxidation of the unsaturated acids. Biochim. Biophys. A&a,
239 (x97x) 168-177
MITOCHONDRIAL OXIDATION OF FATTY ACIDS
r73
In order to measure directly the radioactivity in oxidation products apart from carbon dioxide, a gas chromatographic column combined with a proportional counter was used. The acid fractions were isolated from the incubation mixtures, and these fractions were methylated and subjected to gas chromatography. The column was held isothermal at 100’ for 30 min and then raised at 3”/min to 180’. Around 50 mass peaks were obtained, in a highly reproducible manner. The amount of radioactivity placed on the column was determined by liquid scintillation counting. Incubations were performed in Medium B in order to trap radioactivity in the intermediates, and prevent it passing through and appearing as radio-labeled CO, too rapidly. TABLE
III
ME~HYL~ALONIC
AND
SUCCINIC
ACIDS
FORMEU
ON FATTY
ACID
OXIDATION
Incubations performed in Medium B, with IZO nmoles of acid. Second column gives total percent activity found in CO,. Third column gives activity injected onto gas coIumn. Columns four and six give the actual measured activity on the column associated with methylmalonate and succinate, respectively. Acid
[U-i°C]Palmitate [U-‘*C]Oleate [U-W]Linoleate [U-1*C]Linolenate [I-i*C]Palmitate [I-%]Linoleate
Radioactivity in CO,
Disint. lmin placed on column
Radioactivity ~fethylmalonate Disint. /rnilz
-..---_Succinate -.. .-.._-_Total 0/0* Disint./min
0.9
29 100 76200 27 700 34000 109 900 105 700
2250 37.50
11.9 7.8
1.0
0.6 I.8 2.6 3.0
found
0
0
o 0
0 0
0
0
in
6 ooo 16700 8 700 10410 17200 xbooo
.Total
%*
18.9 28.5 16.5 48.5 12.5 27.5
* The total percentage activities associated with methyimalonate and succinate in the incubation. respectively, were caicuiated assuming that the exogenous methyhnalonate and succinate added (zoo yg per incubation) represent ail the acid present. The amount present in the column sample was measured from the mass peak.
The results of such incubations are shown in Table III. Large percentages of activity were found in succinic acid. Labeled methylmalonic acid, however, was only detected on incubation of palmitate and oleate. The activity in succinic acid is higher than, but parallels that found in CO,. The malic and citric acids present in the incubation mixture greatly dilute the pool of Krebs cycle intermediates available. Labeled acetyl-CoA can rapidly enter the pool, but can cycle only once, as the labeled carbons are diluted by the large amount of malate present. TABLE EFFECT
IV OF FLUOROCITRATE
ON FATTY
ACID
Medium B, with 1 wmole fluorocitrate Fatty acid
Activity
OXIDATION
per Aask added where indicated;
20 min incubation.
in CO, (disint. jmin) - Fluorocitrate
+ Fluovocitrate [U-W]Palmitate [U-*4C]Oleate [U-i*C]Linoleate [U-iK]Linolenate ;I-**ClLinolenate _ i
As a further
440 280 770 570 480
indication
8600 26400 18700 29 boo _~________
that
oxidation
of the polyunsaturated Biochim.
Biophys.
Acta,
acids was not 239 (x971) 168-177
R. J. HUXTABLE,
I74 proceeding
via methylmalonic
acid, incubations
were carried
S. J. WAKIL
out in the presence
of
sodium fluorocitrate. This prevented the production of any labeled CO,, as shown in Table IV. Fluorocitric acid would prevent any succinic acid being formed via ,8oxidation and the Krebs cycle. However, fluorocitric acid should not interfere with the generation of succinic acid via propionic acid, if such a pathway is in operation. On examination of the products of fluorocitrate incubation by gas chromatography, no labeled methylmalonic or succinic acid could be detected with any sample. experiments strongly militate against the existence of a y-oxidation pathway system under study.
These in the
An attempt was made to measure radioactivity associated with methylmalonic and succinic acids isolated from fatty acid oxidation. The dicarboxylic acids were diluted out following incubations,
as described
in the experimental
section.
The acid
extracts were subjected to thin-layer chromatography, and following development, the plates were cut into equal sized strips and counted by liquid scintillation. Activity was found to trail down the plate when unsaturated fatty acids had been incubated. No peak of activity was ever detected in a methyl malonate fraction, but activity was usually found associated with succinate. We find this to be an unreliable method for measuring activities in dicarboxylic acids in the presence of radio fatty acid. A recent claim’ to have detected radioactive methyl malonate following incubation of labeled unsaturated fatty acids with various preparations from rat tissue rests on a similar chromatographic The dicarboxylic
technique, and on these grounds the claim can be faulted. acids were also diluted out in sufficient quantities to be re-
crystallized. Methylmalonic acid was recrystallized and then diluted with pure, inactive, methylmalonic
to constant activity (3-4 times) acid, and the measured activity
compared with the calculated. By these means, which were repeated on several occasions, activity was shown to be tenaciously associated with methylmalonic acid in the order of increasing unsaturation of the long chain acid incubated, as shown in TABLE
V
Acid incubated
Final disint.lmin ix methylmalonate
~~ ~.~ [U-14C]Palmitate [U-%]Oleate [U-‘*C]Linoleate [U-“C]Linolenate
TABLE
per mg
~_
Ete;fl;;&%al
.~ 0.09 0.22 r.o7 7.04
3.0 9.8 66.3 311.4
VI
UEPEiS‘DENCE OF FATTY ACID OXIDATIONON CARNITINEAND ALBUMIN Swollen mitochondria used in Medium A with the indicated were used in each incubation.
omissions.
.__.__
~~
Fatty acid
Albumin
Carnitine ____.
[U-‘%]Linolenatc [U-%]Linolenate [U-‘*C]Linolenate [U-14C]Linolenate r *-%I Palmitate H&him.
+ + _
+
+
;
Biophys. Acta, 239 (1971) 168-177
120 nmoles of fatty acid
WO, produced (countslmin) 250 6100 16100 2 000 33200
MITOCHONDRIAL
OXIDATION
OF FATTY ACIDS
17.5
Table V. However, this result is misleading, and is not in agreement with the work described above. Recrystallization to constant activity is not an absolute criterion of radioc~emical purity, and in this case it appears that the reactive unsaturated acids have given oxidation products, resulting from the drastic work-up procedure, which are soluble in ethyl acetate but precipitate out in the presence of non-polar benzene.
ofcarnitirte Oxidation of fatty acids was found to be strongly dependent on the presence of carnitine and albumin, as shown in Table VI. If either carnitine or albumin was omitted from Medium A, the oxidation of [G14C]linolenate fell to a fraction of what it was in Medium A. The rate of oxidation was found to be propo~ion~ to carnitine concentration over the range o-16 mM, as shown in Fig. 2. Since the concentrations of carnitine used exceeds the buffering capacity of the medium, the buffer concentration was increased. Even with this increased phosphate concentration, a similar dependency of linolenate oxidation on carnitine was observed. However, the rate of oxidation became less strongly dependent, and higher concentrations of carnitine were needed to produce the same amount of oxidation (Fig. 2). Possibly, the higher level of phosphate present in the buffered medium inhibits the carnitine dependent oxidation. This effect was not investigated further. Effect
Fig. 2, Dependence of [U-i*C]linolenate oxidation on carnitine. IZO mM [U-*dC]linolenic acid used per incubation; 3.6 mg of protein was added per flask, and incubations proceeded for so min. “Unbuffered” incubations: carnitine added directly to Medium A. “Buffered” incubations: carnitine solution adjusted to pH 7.0 prior to addition to Medium A. l ----•, unbuffered incubations: O-O, buffered incubations. Fig. 3. Dependence of [r-i*Cjpalmitate oxidation on energy charge and chondria (2-4 mg) [r-%]palmitate (140 nmoles) were used and incubation A used, with nucleotide and carnitine concentrations as follows: O---O, nucleotides; o-----e 16 m&l carnitine, 2 mM nucleotides; A-A, 4 mM tides.
carnitine. Whole mitowas for zo min. Medium 16 mM carnitine, j m&l carnitine, 5 mM nucleo-
Oxidation of fatty acids in mitochondria is tightly coupled to oxidative phosphorylation. As Medium A contains no ADP apart from that endogenous to the mitochondria, it could be that an insufficiency of ADP to act as a phosphate acceptor is rate limiting for the oxidation. The effect of adenine nucleotide ratios (“energy charge”)22 was studied together w&h the effect of altering carnitine to nucleotide-ratio. Results are shown in Fig. 3. With whole mitochondria, using [r-**C]palmitate as the fatty acid, when a high concentration of nucleotide and a low concentration of carnitine are used, over-all oxidation is low. With a low level of nucleotide but a high Biochim.
Biophys.
Acta,
239
jr971)
168-177
176
R. J. HUXTABLE,
level of carnitine,
oxidation
is much faster. When high concentrations
S. J. WAKIL
of both are used,
the rate of oxidation is extremely high. The rate is maximal at an energy charge of around 0.65. Energy charge is calculated as half the number of high energy phosphate bonds per unit mole of nucleotide 22. Thus, ATP has a charge of I, ADP of 0.5, and AMP of 0.0. One feature of these oxidations in the presence of high concentrations of carnitine and adenosine nucleotide, is that oxidation occurs as an intense burst which last for some z-10 min. It then drops to a low, steady rate. DISCUSSION
The relative
rates
of oxidation
of long chain fatty
acids is depenent
incubation conditions to some extent (Table I). In Medium A, activity linoleate appears at a higher rate in CO, than that from [I-X]palmitate. with U-labeled
acids, as the unsaturation
increases,
the rate of oxidation
on the
from [I-'"ClHowever, falls off. Most
of the work previously published on the faster rate of oxidation of unsaturated acids have used r-labeled acids only. When exogenous Krebs cycle acids are added to Medium A, the rates of oxidation of all acids drop and becomes roughly constant. This is because passage of fatty acid-derived acetyl-CoA through the Krebs cycle becomes the rate limiting step, rather than b-oxidation of the fatty acid. Since making the observations on the combined effects of ATP and carnitine on the rate of mitochondrial oxidation, we have become aware of similar observations made by other workers. DRAHOTA et uZ.~~~~@ showed that brown fat mitochondria supplemented by carnitine and ATP gave a sudden burst of respiration. In our experiments the addition of carnitine increased the rate of oxidation up to very high concentration of carnitine, up to 40 mM in the presence of 48 mM phosphate. Optimum conditions found by DRAHOTA et al.zs~zs were z mM carnitine and 4 mM ATP. However, our experiments were carried out in the presence of bovine serum albumin, which decreases ATP-carnitine activated respiration. The difference in source of the mitochondria
may also be an important
factor in the different
and ATP. It has been claimed1 that a y-oxidation
pathway
response
for unsaturated
to carnitine fatty
acids
exists in rats, and the isolation of labeled methylmalonic acid following incubation of labeled unsaturated fatty acids with heart and muscle mitochondria was quoted in support. Using beef heart mitochondria, we have been unable to find evidence for such an oxidation route. Following incubation of I-%- and /or U-Y-labeled palmitic, oleic, linoleic, and linolenic acids with mitochondria, the acid-extractable products were analyzed by gas chromatography. Labeled methylmalonic acid was detected only from palmitate and, to a lesser extent, oleate. When the same acids were incubated in the presence of avidin, which should inhibit all biotin-dependent reactions, such as the carboxylation of propionyl-CoA, only palmitate showed any depression in the amount of oxidation. In the presence of fluorocitrate, none of the fatty acids produced carbon dioxide, succinate, or methylmalonate. If the y-oxidation pathway was operating, succinate should be formed directly, without interference from the action of Auorocitrate on aconitase. It must be concluded that y-oxidation is not occurring, at least not by a propionate + methylmalonate + succinate pathway, under the incubation conditions we are employing. The claim that the pathway exists in rats’ relies heavily on thin-layer chromatoBzochim.
Bzophys.
Acta,
239 (1971)
168-177
MITOCHONDRIAL
OXIDATION
OF FATTY
ACIDS
177
graphy of radioactive acids, by a procedure similar to the one described above. DuPont and Mathias have not succeeded in unequivocally demonstrating with rats unsaturated fatty acids can be oxidized via methylmalonic and propionic acids. We have found no evidence for such a pathway in beef heart mitochondria. It is possible that the pathway occurs in other tissues, though more evidence is needed than is at present available. ACKNOWLEDGEMENTS
This investigation was supported in part by Grant GM-06242-12 from the National Institutes of Health, United States Public Health Service, by a grant from the Life Insurance Medical Research Fund, by Grant 70-1030 from the American Heart Association, and by a grant from the American Cancer Society. The authors are grateful to Prof. D. E. Green and Dr. D. Almann (Institute for Enzyme Research, University of Wisconsin) for a generous gift of the heavy beef heart mitochondria. REFERENCES I 2
3
J.
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21 22
401.
G. GORANSSON AND T. OLIVECRONA,Acta Physiol. Stand.. 63 (1965) 424. G. GORAXSSON AND T. OLIVECRONA,Acta Physiol. Stand., 63 (1965) 428. G. GORANSSON AND T. OLIVECRONA,Acta Physiol. Stand., 63 (1965) 204. W. S. LYNN AND R. H. BROWN, Arch. Biochem. Biophys., 81 (1959) 353. J. DUPONT, Lipids, I (1966) 415. R. BRESSLER AND S. J. FRIEDBERG,Arch. Biochem. Biophys., 104 (1964) 427. P. BJ~RNTORP, I.Biol.Chem.. 24~,(1968) 2130. R. BLOMSTRAN;, Acta Physiol. &id:, 32 (1454) 99. K. BERNHARD, M. ROTHLIN, J. P. VUILLEUMIER ASD R. WYSS, Helv. Chim. Acta, 4r (1958) 1017. K. BERNHARD, M. ROTHIN AND H. WAGNER, Helv. Chim. Acta, 41 (1958)11.55. D.ENGELMANN,I.STORCK, H.-D. EISEXBARTH, A.NXHER,D.HEYSEASD K.SCHREIER,C~~~. Chim. Acta, 9 (1964) 126. G. ANTONY AND B. LANDAU, J. Lipid Res., 9 (1968) 267. R. ANDERSON, Biochim. Biophys. Acta, 152 (1968) 531. D. E. ATKINSON,in T. W. GOODWIN, Metabolic Roles ofcitrate, Academic Press, London and
New York, 1968. 2.3H. J. PETROWITZ AND G. PASTUSKA,J. Chromatog., 7 (1962) 128. 7 (1964) 56. 24 D. BRAUN AND H. GEENEN, J. Chromatog., Sot., 64(1942) 469. 25 D. PENNINGTON, E. E. SNELL AND R. E. EAKIN,J. Am.Chem. 26 N. M. GREENE, Biochem. J., 89 (1963) 585. 27 N. M. GREENE, Biochem. J., 89 (1963) 599. 28 2. DRAHOTA, E. HONOVA AND P. HAHN, Experienta, 24 (1968) 431. 29 2. DRAHOTA, E.Ho~ov.4,P.HAHN AND P.GAZZOTTI,in L. ERNSTER AND 2. DRAHOTA, A/lib chondria:Structure andFunction, Academic Press, London and New York, 1969. Chem., 177 (1949) 751. 30 A. G. GORNALL, C. J. BARDAWELL AND M. A. DAVID, J.Biol. Vol. VI, Interscience, 31 N. D. CHEROWIS,in A. WEISSBERGER, Technique of Organic Chemistry, New York and London, 1954,p. 49.
u’iochim.
Biophys.
Acta,
239 (1971)168-177
168 BB* 55875
COMPARATIVE
MITOCHONDRIAL
K. J. HUXTABLE*
AND S.4LIH
Department
of Biochemistry,
(Received
December
Duke
OXIDATION
OF FATTY
ACIDS
J. WAKIL** University
Medical
Center, Duvham,
N.C.
27706 (U.S.A.)
just, 1970)
SUMMARY
The comparative oxidation of I-%and U-14C-labeled fatty acids has been studied in beef heart mitochondria. The relative rates of oxidation depended on the conditions employed. Under certain conditions, the rate of oxidation of U-K-labeled acids decreased as unsaturation increased, whereas under the same conditions [I-%]linoleate was oxidized faster than [I-14C]palmitate, as measured by the production of radioactive carbon dioxide. Addition of L-(-)-carnitine and ATP together produced an intense pulse of oxidation. For [I-14C]palmitate, maximum oxidation under such conditions occurred at an energy charge of around 0.65. A search was made for evidence that oxidation of polyunsaturated acids could proceed by “yoxidation” to yield methylmalonic acid via propionic acid. No such evidence could be found, and it is concluded that in beef heart mitochondria, the oxidation of unsaturated fatty acids can be acounted conditions used here.
for solely
on the basis
of ,&oxidation
under
the
INTRODUCTION
While the mitochondrial oxidation of fatty acids has been well-studied, there is a paucity of work which has used uniformly labeled acids. Most work has used carboxyl labeled acids, and it has been assumed that the reactions of these, as followed by the label, are indicative of the complete fatty acid molecule. The /&oxidation pathway for fatty acids has been well established. However, there has been speculation as to the possible existence in mammals of a y-oxidation pathway in the catabolism of long chain unsaturated fatty acidsl-3. Unsaturated acids containing a 9,10 double bond can not undergo total /?-oxidation unless the site of unsaturation shifts. That this, indeed, happens has been demonstrated4+. Because of the possibility of gluconeogenesis from unsaturated acids, the existence of a yoxidation pathway remained an attractive speculation. y-Cleavage of the double * Present address, Department
of Pharmacology, University of Arizona, Tucson, Xriz. 8j72I (U.S.A.). ** Address reprint request to Salih J. Wakil, Duke University Medical Center, Durham, N.C. 27706 (U.S.A.). Biochim.
Biophys.
Acta,
239 (1971) 168-177
~ITOCHONDRIAL
OXIDATION
OF FATTY ACIDS
169
bond in a #I-oxidation pathway intermediate such as 3,6,9-dodecatrienoyl-CoA, derived from linolenic acid by the loss of three C, units, would give propionyl-CoAz. This could enter into gluconeogenesis via methylmalonyl-CoA, succinyl-CoA, and oxaloacetate6. The principle support for a y-oxidation theory has come from the supposedly faster rate of oxidation observed with unsaturated acid+-le. It has been argued that this could be explained by the “self-priming” effect of propionyl-Coil. production on the tricarboxylic acid cycle. By & novo production of tricarboxylic acid cycle intermediates, the flux through the cycle is increased. using rat livers, studied the relative distriHowever, ANTOXY AND LANDAPO, bution of activity in glucose following feeding of [2-%]acetate, [r6-14Cjpalmitate, and [ro-%]oleate. The labeling patterns of the glucose derived from all three were similar enough for the authors to conclude that [ro-14C]oleate underwent exclusive b-oxidation. ANDERSON 21 demonstrated that all the geometric isomers of 9,12octadecadienoic acid, in rat liver mitochondria, were oxidized to only CO, and ketone bodies. The amount of acetoacetate formed was directly proportional to the amount of acid oxidized, and all acids produced the same quantity of CO, under the same conditions. One can conclude that only /$-oxidation is occurring with all isomers. Here, we report some observations on the comparative oxidation of I-‘%- and I?-%-labeled acids by beef heart mitochondria. With U-“*C-labeled acids the rate of oxidation decreased as unsaturation increased as would be expected if B-oxidation was occurring4. No evidence was found for a different oxidation pathway. MATERIALS
AND
METHODS
Heavy beef heart mitochondria were obtained from the Institute for Enzyme Research, University of Wisconsin. They were kept in 0.25 M sucrose at -15" until used. Experiments were done within three months of isolation. A Barber-Coleman Selecta System Series 5000 gas-liquid chromatograp~ equipped with a radium ionization detector was used, with an 8-ft column packed with IS”/ diethylene glycol succinate on Chromosorb W, 60-80 mesh (Analytical Engineering Laboratories, Inc., Hamden, Conn.). For measurement of radioactivity, the eMuent gas was passed through a Nuclear Chicago proportional detector and the radioactivity automatically recorded. Scintillation counting was performed with a Tri-carb Model 314 EX liquid scintillation counter. All counts were corrected for efficiency using an internal standard. [%]Toluene (obtained from New England Nuclear Corp., Boston), measured volumetric~ly, was used to standardize activities of roooo countslmin or above. [14C]Hexadecane (prepared by dilution of a standard hex&cane obtained from the Radiochemical Centre Amersham), measured gravimetrically, was used to standardize activities below IO ooo counts/min. r-14C-labeled linolenic and U-%-labeled linolenic, linoleic, oleic, and palmitic acids were obtained from the Radiochemical Centre, England. I-‘K-labeled palmitic and linoleic acids were obtained from New England Nuclear Corp., Boston, and U14C-labeled linolenic and linoleic acids were additionally obtained from Applied Science Laboratories, Penn. Omnifluor was obtained from New England Nuclear Corp., Boston, carnitine from Nutritional Biochemicals, Cleveland, bovine serum albumin from Sigma, St.
R. J. HWXTABLE,S. J. WAKIL
‘70
Louis, and CoA, NAD+, ATP, ADP, and AMP from P-L Biochemicals, Milwaukee. Radioactive acids were diluted with inactive acids and made up as solutions in ethanol such that 0.01 ml contained the concentration of the acids needed per incubation. The purity of these solutions was checked at intervals by gas-liquid chromatography. The incubation media used were: Medium A: concentrations (mM) ; albumin 0.014,sucrose IOO, EDTA 0.4, KC1 80, sodium phosphate (pH 7.4) 8, M&l, 5, ATP I, NADf 2.5, CoA 0.04, carnitine I, NaHCO, 25. Total volume 2.5 ml. Medium B: as Medium A, with the further addition of maiic and citric acids in concentrations of 2 mM and 0.2 mM, respectively. Protein was determined by the biuret methodm. Where swollen mitochondria were used, these were suspended in water and homogenized by hand with a teflon homogenizer. Incubations were carried out at 37” in erlenmeyer flasks with a short piece of glass tubing welded to the bottom inside. In this tubing a plastic cup was placed. The flask was fitted with a serum cap. Just before the end of the incubation 0.2 ml of hyamine was injected into the cup, and the incubation then stopped by injection of 0.2 ml 3 M H,SO, into the medium. The CO, liberated was absorbed by the hyamine over a r-h period. The cup with the hyamine was then placed in a counting vial, and IO ml of scintillation solution added. The scintillaht used contained: dioxan 2.4 1, anisole 0.4 1, r,z-dimethoxyethane 0.4 1, omnifluor 25.6 g.
At the end of the incubation, 0.05 mg each of succinic and methylm~onic acids were added to the incubation mixture. The pH of this was then adjusted to II, and the solution was left at room temperature for 2 h. The pH was brought to 2 with HCI, and the solution was extracted with pentane until little further activity was removed. It was further extracted with ether (3 times). The ether extracts were reduced to a small volume, and spotted onto a precoated silica gel plate (obtained from Eastman Kodak Co., Rochester, N.Y.), oven-dried immediately before use. The solvent systems used were : benzene-methanol-acetic acid (45 : 8 : 4, by ~01.)~~.with RF values of 0.40 for methylmalonic acid, 0.58 for succinic acid, and 1.00 for fatty acids; benzene-dioxane-acetic acid (90: 25 : 4, by ~01.)~~with RF values of 0.24, 0.41, and 1.00 for methylmalonic, succinic, and fatty acids, respectively; and ethanolwater-25% ammonia (96: 16:16, by v01.)~~with RF values of 0.30, 0.37, and 1.00 for methylmalonic succinic and fatty acids, respectively. After being developed, the plate was oven-dried at 100’ for 0.5 h, and then sprayed with a solution made up of bromophenol blue (50 mg), andcitric acid (zoomg) in water (IOO m&The chromatographic plate was cut into equal sized portions, and each portion shaken with scintillator solution and counted. Isolation and crystallization
ofdicarboxylic
acids
At the end of the incubation, the mixture was divided into two equal halves. To one was added 50 mg succinic acid, and to the other 50 mg of methylmalonic acid. Each solution was worked up as described above. The reisolated succinic acid was recrystallized from acetic acid-chloroform, and methylmalonic acid from ethyl acetate-benzene. All recrystallizations were carried out in Craig tubes31. The activity of the acids (two samples each) were measured after each recrystallization. Biockim. Biapitys. Acta,
239 (1971) 168-177
MITOCIIONDRIAL OXIDATION OF FATTY ACIDS
171
The avidin-treated incubations contained 56 nmoles of avidin. Where biotinpretreated avidin was used, a biotin : avidin mole ratio of 5 : I was employed.
The incubation mixtures were basified to pi3 12, stored for 2 fr, then reacidified to pH. 2. The mixtures were ether extracted (4 times), and the extracts dried and reduced in volume to around 0.5 ml. A solution of diazomethane in ether was added until no more color was discharged. The methylated solutions were kept at 4” overnight before gaschromatographic analysis was carried out. Diazomethane was prepared by the action of base on bis(~-methyl-~-nitroso)terepht~~alamide in ether solution. The diazometh~e was distilled out, and trapped in ice-cold ether. TABLE Co,
I
PRODUCTION
UNDER
VARYING
CONDITIONS
OF INCUBATION
In each experiment, the first column gives the actual activity measured in the CO,, corrected for background and efficiency. The second column gives the nmoles of acid oxidized per mg of protein. All incubations contained 120 nmoles of the acid under study. Conditions used: (I) Whole mitochondtia (g mg) in Medium A, 30 min incubation. (II) Swollen mitochondria (13 mg) in Medium A, 30 min incubation. (III) Swollen mitocbondria (5 mg) in Medium A, 20 min incubation. (IV) Swollen mitochondria (3.5 mg) in Medium B, 30 min incubation.
Fatty acid
Expt. I ~-____ “CO,
Fatty acid
(disint. f oxidized
II-**C]Palmitate
min)
(nmolesjmg p&e+)
37 3oo
0.46
200
?J-W]Palmitate I-**C]Linoleate %-14C]Oleate U-rPC]Linoleate U-%]Linolenate
58 10500 14000 8300 4 7oo
Ex$x. II _-wo,
-._ Fatty acid
(disint. / min)
oxidized fnmoles/mg prorein)
423 9oo
3.6~
7300 IjS200
7-76 3.06 2.14 1‘34
Expt. III IWO, Fatty acid (disint./ oxidized min)
(rtmoleslmg
Expt. IV Fatty acid WO, (disint.j min)
oxidized (nmoleslmg @atein)
28400
0.89
18400 3 200 4 900 3 000 8 600
0.31 0.42 0.29 0.49
p?‘Otein)
1.26
0.40 0.38 0.23 0.13
1.02
II3400
68 300
56000 22400 15000 7 700
6.32 1.86
1.21 0.65
RESULTS
U-14C- and r-lGlabeled fatty acids were oxidized by mito~bo~dria, and the CO, produced trapped and counted. Results obtained under various conditions are shown in Table I. Swollen mitochondria gave much higher activities than whole mitochondria. In Medium A, the rate of oxidation of uniformly labeled fatty acids decreased as the unsaturation increased. However, higher activity was obtained from [I-%]linoleic acid than from [r-l%]palmitic acid. When exogenous Krebs cycle acids were added to the medium (tedium B), the mount of radio-labeled CO, being produced fell, and the relative rates of oxidation of the uniformly labeled acids altered to a near constant level for all the acids, This is presumably a reflection of the competing processes of faster flux through the Krebs cycle, but higher dilution of label into Krebs cycle intermediates. Fig. I shows the comparative rates of oxidation of [I-~%]- and [U-**C]linoleate at different acid concentrations. The curves are complex ones, reflecting the comphxity of the system, but they show that at low concentrations of acids the I-position is oxidized appreciably faster than the molecule, over-all. Since no free intermediary Biochim. Biophys. Acta, 239 (1971) 168-177
R. J. HUXTABLE, S. J. WAKIL
172
+otty Acid Added (n moles]
Fig. 1. Dependence of oxidation on concentration of acid for [I-%]and [U-“%]linoleate. Comparative rates of oxidation of [I-*%]- and [U-*4C]linoleate at different acid concentrations; 2.45 mg protein added per flask. Medium A used with swollen mitochondria.
chain length acids accumulate, the difference between the rate of oxidation of the [I-‘*Cl- and [U1*C]linoleate represents enzyme-bound moities undergoing active oxidation. ~-Oxidation is not an instantaneous process, and starting from the Iposition it takes a finite time to oxidize down to the eqosition. Every time a double bond is reached the process is slowed further due to the need for extra rearrangement steps, as shown by STOFFEL~~~. The noninvolvement of y-oxidation If part of the activity of the CO, produced from unsaturated acids was coming via propiol~ate-n~ethyl malonate-succinate pathway, this part should be biotin dependent. Propionyl-CoA carboxylase is a biotin-containing enzyme, and as such should be completely inhibited by treatment with the protein avidin25. Fatty acids were incubated with swollen mitochondria in the presence of avidin, and biotinpretreated avidin, with the results shown in Table II. Avidin of IO units/mgz6 and TABLE EFFECT
II OF AVIDIN
OK FATTY
ACID
OXIDATION
Medium A used with 120 nmoles of each acid and 3.46 mg mitochondria per flask. Incubation time was 20 min; percent activity is the percentage of activity originally present in the fatty acid which appears in the CO,.
[U-‘*C]Palmitate [U-‘*C]Oleate [U-14C]Linoleate [U-14C]Linolenate
7100 5500 2900 II00
2.01
5000
1.41
1.1.5 0.58
46~=~
0.95
2200 II00
0.45
0.22
0.23
7100 4500 1700
2.02 0.93 0.33
I200
0.24
commercial avidin of 2-5 units/mg were used, and no marked difference was observed between them. Only the oxidation of in-l~C]palmitate was appreciably affected by the presence of avidin. The reason for the effect of avidin on the oxidation of palmitate is not apparent. Nevertheless, the lack of effect of avidin on the oxidation of the unsaturated acids strongly indicates that a propionate-methyl malonate pathway is not involved in the oxidation of the unsaturated acids. Biochim. Biophys. A&a,
239 (x97x) 168-177
MITOCHONDRIAL OXIDATION OF FATTY ACIDS
r73
In order to measure directly the radioactivity in oxidation products apart from carbon dioxide, a gas chromatographic column combined with a proportional counter was used. The acid fractions were isolated from the incubation mixtures, and these fractions were methylated and subjected to gas chromatography. The column was held isothermal at 100’ for 30 min and then raised at 3”/min to 180’. Around 50 mass peaks were obtained, in a highly reproducible manner. The amount of radioactivity placed on the column was determined by liquid scintillation counting. Incubations were performed in Medium B in order to trap radioactivity in the intermediates, and prevent it passing through and appearing as radio-labeled CO, too rapidly. TABLE
III
ME~HYL~ALONIC
AND
SUCCINIC
ACIDS
FORMEU
ON FATTY
ACID
OXIDATION
Incubations performed in Medium B, with IZO nmoles of acid. Second column gives total percent activity found in CO,. Third column gives activity injected onto gas coIumn. Columns four and six give the actual measured activity on the column associated with methylmalonate and succinate, respectively. Acid
[U-i°C]Palmitate [U-‘*C]Oleate [U-W]Linoleate [U-1*C]Linolenate [I-i*C]Palmitate [I-%]Linoleate
Radioactivity in CO,
Disint. lmin placed on column
Radioactivity ~fethylmalonate Disint. /rnilz
-..---_Succinate -.. .-.._-_Total 0/0* Disint./min
0.9
29 100 76200 27 700 34000 109 900 105 700
2250 37.50
11.9 7.8
1.0
0.6 I.8 2.6 3.0
found
0
0
o 0
0 0
0
0
in
6 ooo 16700 8 700 10410 17200 xbooo
.Total
%*
18.9 28.5 16.5 48.5 12.5 27.5
* The total percentage activities associated with methyimalonate and succinate in the incubation. respectively, were caicuiated assuming that the exogenous methyhnalonate and succinate added (zoo yg per incubation) represent ail the acid present. The amount present in the column sample was measured from the mass peak.
The results of such incubations are shown in Table III. Large percentages of activity were found in succinic acid. Labeled methylmalonic acid, however, was only detected on incubation of palmitate and oleate. The activity in succinic acid is higher than, but parallels that found in CO,. The malic and citric acids present in the incubation mixture greatly dilute the pool of Krebs cycle intermediates available. Labeled acetyl-CoA can rapidly enter the pool, but can cycle only once, as the labeled carbons are diluted by the large amount of malate present. TABLE EFFECT
IV OF FLUOROCITRATE
ON FATTY
ACID
Medium B, with 1 wmole fluorocitrate Fatty acid
Activity
OXIDATION
per Aask added where indicated;
20 min incubation.
in CO, (disint. jmin) - Fluorocitrate
+ Fluovocitrate [U-W]Palmitate [U-*4C]Oleate [U-i*C]Linoleate [U-iK]Linolenate ;I-**ClLinolenate _ i
As a further
440 280 770 570 480
indication
8600 26400 18700 29 boo _~________
that
oxidation
of the polyunsaturated Biochim.
Biophys.
Acta,
acids was not 239 (x971) 168-177
R. J. HUXTABLE,
I74 proceeding
via methylmalonic
acid, incubations
were carried
S. J. WAKIL
out in the presence
of
sodium fluorocitrate. This prevented the production of any labeled CO,, as shown in Table IV. Fluorocitric acid would prevent any succinic acid being formed via ,8oxidation and the Krebs cycle. However, fluorocitric acid should not interfere with the generation of succinic acid via propionic acid, if such a pathway is in operation. On examination of the products of fluorocitrate incubation by gas chromatography, no labeled methylmalonic or succinic acid could be detected with any sample. experiments strongly militate against the existence of a y-oxidation pathway system under study.
These in the
An attempt was made to measure radioactivity associated with methylmalonic and succinic acids isolated from fatty acid oxidation. The dicarboxylic acids were diluted out following incubations,
as described
in the experimental
section.
The acid
extracts were subjected to thin-layer chromatography, and following development, the plates were cut into equal sized strips and counted by liquid scintillation. Activity was found to trail down the plate when unsaturated fatty acids had been incubated. No peak of activity was ever detected in a methyl malonate fraction, but activity was usually found associated with succinate. We find this to be an unreliable method for measuring activities in dicarboxylic acids in the presence of radio fatty acid. A recent claim’ to have detected radioactive methyl malonate following incubation of labeled unsaturated fatty acids with various preparations from rat tissue rests on a similar chromatographic The dicarboxylic
technique, and on these grounds the claim can be faulted. acids were also diluted out in sufficient quantities to be re-
crystallized. Methylmalonic acid was recrystallized and then diluted with pure, inactive, methylmalonic
to constant activity (3-4 times) acid, and the measured activity
compared with the calculated. By these means, which were repeated on several occasions, activity was shown to be tenaciously associated with methylmalonic acid in the order of increasing unsaturation of the long chain acid incubated, as shown in TABLE
V
Acid incubated
Final disint.lmin ix methylmalonate
~~ ~.~ [U-14C]Palmitate [U-%]Oleate [U-‘*C]Linoleate [U-“C]Linolenate
TABLE
per mg
~_
Ete;fl;;&%al
.~ 0.09 0.22 r.o7 7.04
3.0 9.8 66.3 311.4
VI
UEPEiS‘DENCE OF FATTY ACID OXIDATIONON CARNITINEAND ALBUMIN Swollen mitochondria used in Medium A with the indicated were used in each incubation.
omissions.
.__.__
~~
Fatty acid
Albumin
Carnitine ____.
[U-‘%]Linolenatc [U-%]Linolenate [U-‘*C]Linolenate [U-14C]Linolenate r *-%I Palmitate H&him.
+ + _
+
+
;
Biophys. Acta, 239 (1971) 168-177
120 nmoles of fatty acid
WO, produced (countslmin) 250 6100 16100 2 000 33200
MITOCHONDRIAL
OXIDATION
OF FATTY ACIDS
17.5
Table V. However, this result is misleading, and is not in agreement with the work described above. Recrystallization to constant activity is not an absolute criterion of radioc~emical purity, and in this case it appears that the reactive unsaturated acids have given oxidation products, resulting from the drastic work-up procedure, which are soluble in ethyl acetate but precipitate out in the presence of non-polar benzene.
ofcarnitirte Oxidation of fatty acids was found to be strongly dependent on the presence of carnitine and albumin, as shown in Table VI. If either carnitine or albumin was omitted from Medium A, the oxidation of [G14C]linolenate fell to a fraction of what it was in Medium A. The rate of oxidation was found to be propo~ion~ to carnitine concentration over the range o-16 mM, as shown in Fig. 2. Since the concentrations of carnitine used exceeds the buffering capacity of the medium, the buffer concentration was increased. Even with this increased phosphate concentration, a similar dependency of linolenate oxidation on carnitine was observed. However, the rate of oxidation became less strongly dependent, and higher concentrations of carnitine were needed to produce the same amount of oxidation (Fig. 2). Possibly, the higher level of phosphate present in the buffered medium inhibits the carnitine dependent oxidation. This effect was not investigated further. Effect
Fig. 2, Dependence of [U-i*C]linolenate oxidation on carnitine. IZO mM [U-*dC]linolenic acid used per incubation; 3.6 mg of protein was added per flask, and incubations proceeded for so min. “Unbuffered” incubations: carnitine added directly to Medium A. “Buffered” incubations: carnitine solution adjusted to pH 7.0 prior to addition to Medium A. l ----•, unbuffered incubations: O-O, buffered incubations. Fig. 3. Dependence of [r-i*Cjpalmitate oxidation on energy charge and chondria (2-4 mg) [r-%]palmitate (140 nmoles) were used and incubation A used, with nucleotide and carnitine concentrations as follows: O---O, nucleotides; o-----e 16 m&l carnitine, 2 mM nucleotides; A-A, 4 mM tides.
carnitine. Whole mitowas for zo min. Medium 16 mM carnitine, j m&l carnitine, 5 mM nucleo-
Oxidation of fatty acids in mitochondria is tightly coupled to oxidative phosphorylation. As Medium A contains no ADP apart from that endogenous to the mitochondria, it could be that an insufficiency of ADP to act as a phosphate acceptor is rate limiting for the oxidation. The effect of adenine nucleotide ratios (“energy charge”)22 was studied together w&h the effect of altering carnitine to nucleotide-ratio. Results are shown in Fig. 3. With whole mitochondria, using [r-**C]palmitate as the fatty acid, when a high concentration of nucleotide and a low concentration of carnitine are used, over-all oxidation is low. With a low level of nucleotide but a high Biochim.
Biophys.
Acta,
239
jr971)
168-177
176
R. J. HUXTABLE,
level of carnitine,
oxidation
is much faster. When high concentrations
S. J. WAKIL
of both are used,
the rate of oxidation is extremely high. The rate is maximal at an energy charge of around 0.65. Energy charge is calculated as half the number of high energy phosphate bonds per unit mole of nucleotide 22. Thus, ATP has a charge of I, ADP of 0.5, and AMP of 0.0. One feature of these oxidations in the presence of high concentrations of carnitine and adenosine nucleotide, is that oxidation occurs as an intense burst which last for some z-10 min. It then drops to a low, steady rate. DISCUSSION
The relative
rates
of oxidation
of long chain fatty
acids is depenent
incubation conditions to some extent (Table I). In Medium A, activity linoleate appears at a higher rate in CO, than that from [I-X]palmitate. with U-labeled
acids, as the unsaturation
increases,
the rate of oxidation
on the
from [I-'"ClHowever, falls off. Most
of the work previously published on the faster rate of oxidation of unsaturated acids have used r-labeled acids only. When exogenous Krebs cycle acids are added to Medium A, the rates of oxidation of all acids drop and becomes roughly constant. This is because passage of fatty acid-derived acetyl-CoA through the Krebs cycle becomes the rate limiting step, rather than b-oxidation of the fatty acid. Since making the observations on the combined effects of ATP and carnitine on the rate of mitochondrial oxidation, we have become aware of similar observations made by other workers. DRAHOTA et uZ.~~~~@ showed that brown fat mitochondria supplemented by carnitine and ATP gave a sudden burst of respiration. In our experiments the addition of carnitine increased the rate of oxidation up to very high concentration of carnitine, up to 40 mM in the presence of 48 mM phosphate. Optimum conditions found by DRAHOTA et al.zs~zs were z mM carnitine and 4 mM ATP. However, our experiments were carried out in the presence of bovine serum albumin, which decreases ATP-carnitine activated respiration. The difference in source of the mitochondria
may also be an important
factor in the different
and ATP. It has been claimed1 that a y-oxidation
pathway
response
for unsaturated
to carnitine fatty
acids
exists in rats, and the isolation of labeled methylmalonic acid following incubation of labeled unsaturated fatty acids with heart and muscle mitochondria was quoted in support. Using beef heart mitochondria, we have been unable to find evidence for such an oxidation route. Following incubation of I-%- and /or U-Y-labeled palmitic, oleic, linoleic, and linolenic acids with mitochondria, the acid-extractable products were analyzed by gas chromatography. Labeled methylmalonic acid was detected only from palmitate and, to a lesser extent, oleate. When the same acids were incubated in the presence of avidin, which should inhibit all biotin-dependent reactions, such as the carboxylation of propionyl-CoA, only palmitate showed any depression in the amount of oxidation. In the presence of fluorocitrate, none of the fatty acids produced carbon dioxide, succinate, or methylmalonate. If the y-oxidation pathway was operating, succinate should be formed directly, without interference from the action of Auorocitrate on aconitase. It must be concluded that y-oxidation is not occurring, at least not by a propionate + methylmalonate + succinate pathway, under the incubation conditions we are employing. The claim that the pathway exists in rats’ relies heavily on thin-layer chromatoBzochim.
Bzophys.
Acta,
239 (1971)
168-177
MITOCHONDRIAL
OXIDATION
OF FATTY
ACIDS
177
graphy of radioactive acids, by a procedure similar to the one described above. DuPont and Mathias have not succeeded in unequivocally demonstrating with rats unsaturated fatty acids can be oxidized via methylmalonic and propionic acids. We have found no evidence for such a pathway in beef heart mitochondria. It is possible that the pathway occurs in other tissues, though more evidence is needed than is at present available. ACKNOWLEDGEMENTS
This investigation was supported in part by Grant GM-06242-12 from the National Institutes of Health, United States Public Health Service, by a grant from the Life Insurance Medical Research Fund, by Grant 70-1030 from the American Heart Association, and by a grant from the American Cancer Society. The authors are grateful to Prof. D. E. Green and Dr. D. Almann (Institute for Enzyme Research, University of Wisconsin) for a generous gift of the heavy beef heart mitochondria. REFERENCES I 2
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