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Formation of branched chain acylcarnitines in mitochondria
372
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
26452
FORMATION OF B R A N C H E D CHAIN A C Y L C A R N I T I N E S IN M I T O C H O N D R I A H E L G E E R 1 K S O L B E R G AND J O N B R E M E R
Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet, Oslo (Norway) (Received J u l y 2oth, 197 o)
SUMMARY
i. The formation of acylcarnitines in isolated mitochondria incubated with L-EMe-3H]carnitine and the branched chain 2-oxoacids corresponding to valine, leucine and isoleucine has been studied. 2. Hitherto unknown acylcarnitines were formed and identified as isobutyrylcarnitine (from 2-oxoisovalerate), isovalerylcarnitine (from 2-oxoisocaproate) and 2-methylbutyrylcarnitine (from 2-oxo-3-methylvalerate). 3. The addition of D N P or ADP + P i to the incubation medium increased the formation of propionylcarnitine from 2-oxoisovalerate. 4. The carnitine acetyltransferase (EC 2.3.1.7) was found to transfer acyl groups from branched chain acylcarnitines, but the transfer rates were lower than those found when the corresponding straight chain acylcarnitines were the substrates. 5- Liver, kidney and heart mitochondria from the rat and liver mitochondria from the mouse were all found to have branched chain 2-oxoacid dehydrogenase activity.
INTRODUCTION
Several different acylcarnitines have been identified in mammalian tissues. BOHMER AND BREMER1 have shown that liver of normally fed rats contains significant amounts of acetylcarnitine, propionylcarnitine, and long-chain acylcarnitines. In addition, liver contains small amounts of at least one unidentified acylcarnitine 1, 2. We have found the same compound(s) in relatively greater concentrations in mouse liver (unpublished results). Since the substrate specificities of carnitine acetyltransferase (acetyl-CoA : carnitine 0-acetyltransferase, EC 2.3.1.7)1, 3 as well as carnitine palmityltransferase (palmityl-CoA:carnitine 0-palmityltransferase, EC 2.3.I.-) 4 are rather broad with respect to the acyl group, it is possible that several hitherto unknown acylcarnitines exist in animal tissues. This will depend on the concentrations of the corresponding coenzyme A thioesters in the tissues. Since the carnitine acyltransferases are mitochondrial enzymes we have started the search for new acylcarnitines in experiments in vitro by incubating mitochondria from different tissues with different substrates. In the present communication the formation of branched chain acylcarnitines from the 2-oxoacids corresponding to valine, leucine, and isoleucine is reported. Biochim. Biophys. Acta, 222 (197 o) 372-380
BRANCHED CHAIN ACYLCARNITINES
373
MATERIALS AND METHODS
Chemicals The following commercial chemicals were used: L-carnitine hydrochloride (Otsuka Pharmaceutical Company, Osaka, Japan); acylchlorides, straight and branched chain carboxylic acids, and Dowex 5o W-X8, 200-400 mesh (Fluka AG, Buchs, Switzerland) ; ATP, ADP, and sodium salts of 2-oxoacids (Sigma Chemical Company, St. Louis, Mo., U.S.A.) ; coenzyme A and carnitine acetyltransferase (Boehringer and Soehne GmbH, Mannheim, Germany); silicic acid H (after stahl) and thionylchloride (Merck AG, Darmstadt, Germany). The purity of the acids and acylchlorides was tested by gas-liquid chromatography. DL- and L-EMe-SHlcarnitine were synthesized essentially by the method of BREMER AND NORUM5 from EaHJmethyliodide (Radiochemical Centre, Amersham, England) and DL- and L-3-hydroxy-4-(dimethylamino)butyric acid, and diluted with unlabelled L-carnitine hydrochloride to a specific activity of 30 and 5 mC/mmole, respectively. The radiochemical purity of each compound was greater than 99 % when tested by thin-layer chromatography (System A, described below). L-Acylcarnitine hydrochlorides were synthesized from L-carnitine hydrochloride and acylchlorides I or by a modified method of ZIEGLER et al.6:2 mmoles of thionylchloride were added to approx. IO mmoles of the acid and left for 2-12 h at 80 °. Moisture was kept out by Molecular Sieve 3A pellets (British Drug Houses Ltd., Poole, England). Then 2 mmoles of L-carnitine hydrochloride dissolved in 0.8 ml trifluoroacetic acid was added. The mixture was left at 80 ° for a couple of hours. After cooling the reaction mixture to room temperature, the acylcarnitines were isolated according to the method of BOHMER AND BREMER1. The purity of each compound was tested by thin-layer chromatography (System A), and the identity was verified by mass spectrometry. It was impossible to obtain pure methacrylylcarnitine by these methods. A sticky compound with RE approx. 0.06 (System A) was always produced (polymerization product?). The presence of o.I mmole of hydroquinone in the reaction mixture, however, reduced the formation of this by-product, possibly by preventing polymerization reactions. The mass spectrum of methacrylylcarnitine was recorded after purification by thinlayer chromatography (System A). All other chemicals were commercial products of high purity.
Chromatography Carnitine and acylcarnitines were usually separated on thin-layer silicic acid plates developed with methanol-chloroform-water-conc, ammonia-formic acid (55 : 50: Io : 7.5:2.5, by vol.) 7 (System A). The RF values showed some variation mainly dependent on loading, but the order of peaks was always as shown in Fig. I. In some experiments another solvent was used for developing thin-layer chromatograms: methanol-acetoue-conc. HC1 (9o:1o:Io, by vol.) 8 (System B). The following RF values were obtained with Solvent B: butyrobetaine 0.32-0.35, carnitine o.41-o.43, and acylcarnitines 0.43-o.5 o. The solvent front was always run approx. 14 cm. To localize radioactivity on the plates the silicic acid was cut into bands 3-6 mm wide and scraped into counting vials. Ion-exchange chromatography of carnitines 1 was performed on a I cm × 5o cm column filled with Dowex 5 ° W-X8, 200-400 mesh, NH4+, eluted with 0.2 M amBiochim. Biophys. Acta, 222 (197 o) 372-38o
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H . E . SOLBERG, J. BREMER
monium formate buffer (pH 4.2). The peaks were localized by measuring the radioactivity in o.2-ml aliquots of each fraction or by HESTRIN'S hydroxamate reaction 9 as modified by BREMER~°. The elution volume of carnitine was 230-280 ml. The following elution volumes relative to that of carnitine were found: valine, 0.03; acetylcarnitine, 0.7-0.8; propionylcarnitine was hidden in the carnitine peak; isobutyrylcarnitine, 1.1-1.3; 2-methylbutyrylcarnitine, 1.7; isovalerylcarnitine, 1.9; and methacrylylcarmtine, 3.3. Radioactive samples were measured in a Packard Tri-Carb scintillation spectrometer Model 331o with 5 or IO ml of a scintillator solution consisting of: dimethyl1,4-bis-(5-phenyloxazolyl-2)-benzene (dimethyl POPOP), 0.25 g; 2,5-diphenyloxazole (PPO), 25 g; naphthalene, 400 g; ethanol, 115o ml; dioxane, 1925 ml; and xylol, 1925 ml. The counting efficiency was about 35 % for tritium. The presence of silicic acid reduced the efficiency approximately by three-fourth.
Mass spectrometry Carnitine and carnitine esters were introduced into a Varian CH 7 mass spectrometer through the direct inlet on a probe equipped with temperature-controlled heating. The spectra of carnitine and acylcarnitines are complex and are produced by the combined effect of pyrolysis and electron impact 11. Close supervision of the heating was necessary to obtain reproducible spectra. The standard conditions used were probe temperature (lOO-12o°); ion source temperature (approx. 16o°); ionizing energy (7 ° eV); and cathodic emission current (IOO or 300/~A). Carnitine hydrochloride itself has a base peak at m/e 58 and peaks at m/e 59, 42 and 30 caused by fragmentation and rearrangement of trimethylamine 12 eliminated by pyrolysis. The remaining skeleton of carnitine rearranges to the 3-hydroxy7-1actone n which has a molecular peak at role lO2 and prominent fragments at m/e 84, 74, 55, 44 and 43- Peaks at m/e 36 and 38 in a 3 : i ratio are caused by HC1. The short-chain acylcarnitines show pyrolytic elimination of the acid esterified at the 3-hydroxy group leaving an unsaturated 7-1actone (m/e 84) after the pyrolytic deamination ~a. The peaks at m/e lO2, 74, 44 and 43 observed in the free carnitine spect r u m are therefore absent or of low intensity. Longer chain acylcarnitines (straight chain: C8 or above, branched chain: C 5 or above) have peaks from both the free acid and the 3-O-acyl-7-1actone. A carnitine ester m a y thus be identified by the superimposed spectra of the trimethylamine, the unsaturated or esterified ~-lactone and the eliminated free carboxylic acid.
Incubation of mitochondria with 2-oxoacids Female mice of N M R I / B o m strain (18-22 g) or maleWistar/Moll rats (15o-2oo g) were used. Liver, kidney, or heart mitochondria were isolated by the method of SCHNEIDER AND HOGEBOOM 13 essentially as described by MYERS AND SLATER 14. T h e particles were washed once in o.15 M KC1 and resuspended in the same medium. The protein concentrations were determined by the micro-Kjeldahl procedure on duplicate samples of the suspensions. The incubations were performed in a shaking bath at 37 ° with air as the gaseous phase. The reaction mixtures were, as specified in the legends of tables and figures, made up to a final volume of i ml with o.15 M KC1. The reactions were started by the addition of mitochondrial suspension and stopped by I ml of IO % trichloroacetic Biochim. Biophys. Aata, 222 (i97 o) 372-380
375
BRANCHED CHAIN ACYLCARNITINES
acid. After the centrifugation the trichloroacetic acid in the supernatant was removed b y extracting twice with diethyl ether. The water phase was lyophilized or blown to dryness under an air current. Before application on thin-layer silicic acid plates the residues were redissolved in 50 #1 water followed by the addition of 50 #1 methanol. In some experiments the water phase was directly applied on the top of the Dowex column. RESULTS AND DISCUSSION
Formation of acylcarnitines in mitochondria Preliminary experiments showed that mouse liver mitochondria did not produce acylcarnitines when branched chain DL-amino acids were substrates. R a t liver mitochondria can metabolize D-amino acids 1~. In rat kidney mitochondria DAWSON AND HIRDTM have shown that the activities of L-valine or L-leucine aminotransferases were limited by the availability of amino group acceptor (i.e. 2-oxoglutarate). To avoid complications introduced by variations in the deamination step, the 2-oxoacids corresponding to the branched chain amino acids were chosen as substrates in the experiments reported below. Table I shows that mitochondria produced acetylcarnitine and propionylcarnitine when the metabolic pathways of the substrates included acetyl-CoA or propionyl-CoA as intermediates. There was only one exception: the acetylcarnitine formation was low from 2-oxoisocaproate. This probably shows that the rate of production of acetyl-CoA from 2-oxoisocaproate is slow compared with the capacity of the acetyl-CoA consuming processes in mitochondria (mainly acetoacetate and citrate formation). In addition, unknown acylcarnitines with RF values 0.40-0.50 (Fig. i) were produced in significant amounts from the branched chain 2-oxoacids. TABLE I FORMATION O F
ACYLCARN1TINES
FROM
2-OXOACIDS IN MITOCHONDRIA FROM MOUSE AND RAT TISSUES
T h e i n c u b a t i o n m i x t u r e c o n t a i n e d in a final v o l u m e of i m l : m i t o c h o n d r i a (as s t a t e d be l ow ); s u b s t r a t e (sodium salt), 5 raM; DL- c r L-EMe-3H~carnitine ( E x p t . I or ti, r e s p e c t i v e l y ) , i m M ; E D T A , O. lO-O.15 raM; N - t r i s ( h y d r o x y m e t h y l ) m e t h y l - 2 - a m i n o e t h a n e sulfonic acid buffer (TES buffer) (pH 7.4), 20 mM. The r e a c t i o n w a s r u n for 20 r a i n ( E x p t . I) or 3 ° ra i n ( E x p t . II) a t 37 °. AC, a c e t y l c a r n i t i n e ; PC, p r o p i o n y l c a r n i t i n e ; MC, m e d i u m c h a i n a c y l c a r n i t i n e s : i s o b u t y r y l c a r n i t i n e (from 2 - 0 x o i s o v a l e r a t e ) , i s o v a l e r y l c a r n i t i n e (from 2-0 xoi s oc a proa t e ), 2 - m e t h y l b u t y r y l c a r n i t i n e (from 2 - o x o - 3 - m e t h y l v a l e r a t e ) , or u n i d e n t i f i e d (from e n d o g e n o u s s u b s t r a t e s ) . The r e s u l t s from r a t l i v e r a n d k i d n e y are m e a n s of d u p l i c a t e s .
Substrate
A cylcarnitines produced (nmoles/mg mitochondrial protein) * Expt. I Mouse liver (2.1 rng protein)
Expt. I I Rat liver (1.2 mg protein)
Rat kidney (2. 7 mg protein)
Rat heart (L8 mg protein)
AC PC MC
AC PC MC
AC PC MC
AC PC MC
9 4 3 14
4 3 8 62
I 2 2 29
Endogenous 16 7 2-Oxoisovalerate 9 58 2-Oxoisocaproate 5 5 2 - O x o - 3 - m e t h y l - n - v a l e r a t e 38 66 2-Oxobutyrate 6 173
2 5° 2 4 2
3 37 6 66
II 19 io IO
2 6 83 lO 5 i 26 91 23
I io 23 i o o i 35 46 35
* V a l u e s < 5 n m o l e s / m g p r o t e i n are o n l y a p p r o x i m a t e .
Biochim. Biophys. Acta, 222 (I97 o) 372-380
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H.E. SOLBERG, J. BREMER
Identification of products The unknown products were identified as the acylcarnitines corresponding to the coenzyme A thioesters formed in the branched chain 2-oxoacid dehydrogenase reaction. This conclusion is based on the following evidence. Firstly, chromatography of incubates with carrier amounts of synthetic acylcarnitines on thin-layer plates (System A) gave RF values of the radioactive peaks of the products formed from 2-oxoisovalerate, 2-oxoisocaproate, and 2-oxo-3-methyl-n-valerate identical with those of the iodine spots of isobutyrylcarnitine, isovalerylcarnitine, and 2-methylbutyrylcarnitine, respectively. Secondly, the radioactive peaks of the products coincided with the hydroxamate peaks of the synthetic acylcarnitine carriers after chromatography on the Dowex 5° NH4+ column. The possibility remained, however, that the products were the acylcarnitines corresponding to the unsaturated coenzyme A thioesters formed in the dehydrogenation step following the oxidative decarboxylation of the 2-oxoacids. These unsaturated carnitine esters might not be separated ha our chromatographic systems. One unsaturated compound, methacrylylcarnitine, was accordingly synthesized. Isobutyrylcarnitine and methacrylylcarnitine had, in fact, nearly identical RF values on the thin-layer plates (System A), but the elution volumes from the Dowex 50 column
2O
10
D
t/'I 4
C,
ilL, 40 27
P
_~B
55
88
dl 1,, d. LI., I
,
0
? ¢. ~_
50
361
/
,..b
43
E
B
rn •
m• 5
36
84
20
55
,
73
I,, .oL ,, ...I ..... 30
40
50
60
rn/e
I.... 70
80
3,~ 90
Fig. I. T h i n - l a y e r c h r o m a t o g r a p h y (silicic acid, s o l v e n t s y s t e m A) of c a r n i t i n e a n d t h e acylc a r n i t i n e s p r o d u c e d in r a t k i d n e y m i t o c h o n d r i a from 2-oxoisovalerate (upper part), 2-oxoisoc a p r o a t e (middle part), or 2 - o x o - 3 - m e t h y l v a l e r a t e (lower part). T h e c h r o m a t o g r a m s are f r o m t h e e x p e r i m e n t r e p o r t e d in T a b l e I. F r a c t i o n s 3 m m wide. A, carnitine; B, a c e t y l c a r n i t i n e ; C, prop i o n y l c a r n i t i n e ; D, i s o b u t y r y l c a r n i t i n e ; E, i s o v a l e r y l c a r n i t i n e ; F, 2 - m e t h y l b u t y r y l c a r n i t i n e . I n t h i s c h r o m a t o g r a p h i c s y s t e m o t h e r b r a n c h e d or s t r a i g h t m e d i u m c h a i n a c y l c a r n i t i n e s also m o v e d to t h e position of t h e P e a k s D - F (RF o.4o-o.5o ). Fig. 2. Mass s p e c t r a of (B) s y n t h e t i c i s o b u t y r y l c a r n i t i n e h y d r o c h l o r i d e a n d of (A) t h e a c y l c a r n i t i n e w i t h RF 0.40-o.50 (thin-layer s y s t e m A) p r o d u c e d b y m o u s e liver m i t o c h o n d r i a (79 m g protein) w h e n i n c u b a t e d for 60 m i n a t 37 ° in t h e presence of s o d i u m 2-oxoisovalerate, 5 raM; L-~Me-SH] c a r n i t i n e , i raM; E D T A , o . i o raM; T E S buffer (pH 7-4), 20 m M ; a n d o.15 M KC1 u p to a final v o l u m e of 25 ml. A f t e r isolation on t h i n - l a y e r silicic acid p l a t e s ( S y s t e m A), t h e c o m p o u n d w a s f u r t h e r purified b y c h r o m a t o g r a p h y on a D o w e x 5o NH4+ c o l u m n eluted w i t h o.2 M a m m o n i u m f o r m a t e buffer (pH 4.2) a n d on t h i n - l a y e r silicic acid ( S y s t e m B). T h e m a s s s p e c t r a were recorded a s described in METHODS. P r o b e t e m p e r a t u r e , 117 ° (A) a n d lO4 ° (B); electron emission current, i o o t,A. O r d i n a t e : relative i n t e n s i t y (%). m ~ m e t a s t a b l e p e a k s a t m/e 39.2 a n d 60.6. P e a k s a t role 5 ° a n d 52 are p r o b a b l y d u e to c o n t a m i n a n t s from t h e last c h r o m a t o g r a p h i c step.
Biochim. Biophys. Acta, 222 (197 o) 372-380
377
BRANCHED CHAIN ACYLCARNITINES
differed considerably (approx. 1.2 and 3.3, respectively, relative to the elution volume of carnitine). As it has also been shown 1 that the elution volume of crotonylcarnitine is greater than that of butyrylcarnitine, it is probable that all unsaturated medium chain acylcarnitines are more strongly bound to the Dowex 50 NH4+ column than the corresponding saturated compounds. No unsaturated acylcarnitines were detected in the column chromatograms. When the product formed from 2-oxoisovalerate was isolated and purified it was identified as isobutyrylcarnitine on mass spectrometry (Fig. 2). The peaks of free isobutyric acid were at m/e 88 (molecular ion), 73, 43 (base peak), 41 and 27. In contrast, the mass spectrum of methacrylylcarnitine had peaks from methacrylic acid at m/e 86 (molecular ion), 69, 41 (base peak of the acid), and 39. The acylcarnitines produced from 2-oxoisocaproate and 2-oxo-3-methylvalelate were not isolated for mass spectrometry. Analogous to the isobutyrylcarnitine, however, these were also considered to be the saturated compounds. The lack of unsaturated or 3-hydroxy acylcarnitines in our chromatograms might be caused by too low a concentration of the corresponding CoA-bound intermediates in the mitochondria metabolizing branched chain 2-oxoacids. It is also possible that these CoA-esters are poor substrates for the carnitine acyltransferases.
The specificity of carnitine acetyltransferase The substrate specificity of the acetyltransferase was examined by the exchange of carnitine according to the reaction: L-Acylcarnitine -- CoASH ~- L-carnitine + acyl-CoA When acylcarnitines were incubated with tritium-labelled carnitine in the presence of carnitine acetyltransferase and catalytic amounts of CoA, radioactivity was incorporated into the acylcarnitines at different rates according to the substrate specificity of the enzyme with respect to the acyl groups. In this system propionylcarnitine was the most reactive substrate (Table II), confirming the observation of BOHMER TABLE
II
SUBSTRATE
SPECIFICITY
OF
COMMERCIAL
ACETYLTRANSFERASE
T h e r e a c t i o n m i x t u r e c o n t a i n e d in a final v o l u m e of i m l ( a d j u s t e d w i t h d i s t i l l e d w a t e r) : a c e t y l t r a n s f e r a s e , a p p r o x , o , I u n i t ; s u b s t t a t e ' ( L - a c y l c a r n i t i n e h y d r o c h l o r i d e ) , i m M ; L-[Me-3H]c a r n i t i n e , I m M ; CoA, o.i m M ; Tris buffer (pH 7.5), o. i M. The r e a c t i o n w a s s t a r t e d b y a d d i t i o n of l a b e l l e d c a r n i t i n e a n d r u n for i o m i n a t 37 °. A f t e r s t o p p i n g w i t h 2 m l e t h a n o l , t h e m i x t u r e w a s e v a p o r a t e d to d r y n e s s u n d e r a n a i r c u r r e n t . The p r o c e d u r e c o n t i n u e d as d e s c r i b e d for t h e e x p e r i m e n t s w i t h m i t o c h o n d r i a . T h e r e s u l t s s h o w n are m e a n s of d u p l i c a t e s .
Substrate
Radioactivity incorporated in the substrate (%)
L-Acetylcarnitine L-Propionylcarnitine L-Butyrylcarnitine L-Valerylcarnitine L-Isobutyrylcarnitine L-Isovalerylcarnitine L-2-Methylbutyrylcarnitine L-Crotonylcarnitine L-Methacrylcarnitine
7-3 12.8 8.7 2.3 1.5 o. 8 * o.7" o.6" o. 5 *
* Values
Relative exchange rate (related to that of acetylearnitine) i.o I. 8 1.2 o. 3 o.2 o. 1 < o.I < o. I < o. i
< 1 % are o n l y a p p r o x i m a t e .
Bioehim. Biophys. Acta, 222 (197 o) 3 7 2 - 3 8 o
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H.E. SOLBERG, J. BREMER
AND BREMER1. Branching of the acyf group reduced the reactivity. That isobutyrylcarnitine was the most reactive of the branched acylcarnitines tested is probably one of the reasons why mitochondria produced mole isobutyrylcarnitine than isovalery|carnitine or 2-methylbutyrylcarnitine (Table 5). Introduction of a hydroxyl gioup in 3-position of butyrylcarnitine reduced its reactivity 1as did dehydrogenation (Table II). Relatively high concentrations of the unsaturated or 3-hydroxy acyl-CoA intermediates in the metabolic pathways of the branched chain amino acids would probably be necessary to produce detectable amounts of the corresponding acylcarnitines in the mitochondria.
Comparison of acylcarnitine production in mitochondria from various sources In some experiments the acylcarnitine formation in mitochondria from various organs of rats and mice were compared. Table I shows representative results. The amount of an acyl-CoA intermediate trapped as the corresponding acylcarnitine is the result of at least four factors: the capacities of (I) the acyl-CoA producing and (2) the acyl-CoA consuming steps; (3) the amount of carnitine acetyltransferase present, and (4) its capacity for transferring the acyl group in question. Quantitative intelpretations of the results are accordingly difficult. It is evident, however, that liver, kidney, and heart mitochondria all have branched chain 2-oxoacid dehydrogenases and all the following enzymes of the branched chain amino acid metabolic pathways since both acetyl- and propionyl-carnitine were formed. Other tissues were not examined. GOEDDE et al. 17found branched chain 2-oxoacid dehydrogenase activity in other tissues and used the activity of the dehydrogenases in human leucocytes in diagnostic studies on the branched chain 2-oxoaciduria ("maple syrup urine disease"). BREMER7 observed oxidation of some straight chain 2-oxoacids in rat heart, brain, kidney, and liver. It is thus probable that most tissues have the capacity for oxidative decarboxylation of straight or branched chain 2-oxoacids. The reason why BOHMER15, 18 did not observe medium chain acylcarnitines in his studies on the propionylcarnitine formation from amino acids and 2-oxoacids, is probably that he used rat liver mitochondria. Our results indicate that these generally produce less branched chain acylcarnitines than mitochondria from other tissues tested. One reason for this m a y be the low level of carnitine acetyltransferase in rat liver 19. It is not known whether there is relatively more of the acetyltransferase in mouse liver. Effect of incubation time and additions on acylcarnitine formation from 2-0xoisovalerate 2-Oxoisovalerate was chosen as substrate for some further studies since the isobutyrylcarnitine formation was greater than the production of other branched chain acylcarnitines in mitochondria (Table I). Fig. 3 (upper part) shows the effect of incubation time on acylcarnitine production. The formation of isobutyrylcarnitine started immediately, while the propionylcarnitine production showed a lag. In other experiments the curve of propionylcarnitine formation rose steadily after the initial lag and crossed that of isobutyrylcarnitine formation after 30-60 min of incubation. Addition of 2,4-dinitrophenol to the incubation mixture (Fig. 3, lower part, and Table I I I ) resulted in accelerated propionylcarnitine production without an initial lag period. BOHMER15 also observed formation of propionylcarnitine from 2-oxoisovalerate in the presence of 2,4-dinitrophenol and used this as an indication of the Biochim. t3iophys. Acta, 222 (197o) 372 38o
379
BRANCHED CHAIN ACYLCARNITINES
absence of ATP-dependent steps (e.g. activation of free carboxylic acids) between initial 2-oxoacid decarboxylation and propionyl-CoA formation. However, the increased formation of propionylcarnitine in the presence of 2,4-dinitrophenol requires an additional explanation. 2,4-Dinitrophenol may accelerate the oxidation steps leading to propionyl-CoA, and it will at the same time inhibit the ATP-dependent propionyl-CoA carboxylase (EC 6.4.1.3) by lowering the ATP levels in mitochondria. Since carboxylation probably is the main pathway of propionyl-CoA in mammalian tissues 2°, its inhibition will cause a rise in propionyl-CoA. The acetylcarnitine formation is probably caused by oxidation of endogenous substrates (fatty acids?) since acetyl-CoA is not an intermediate in the metabolism of 2-oxoisovalerate. The very
tooI noo0ton
c~ DNP added
c
c -; --e o
5
15 Incubation t i m e (rain)
?
45
Fig. 3. Effect of i n c u b a t i o n t i m e a n d 2 , 4 - d i n i t r o p h e n o l (DNP) on t h e p r o d u c t i o n of a c y l c a r n i t i n e s f r o m 2 - o x o i s o v a l e r a t e in m o u s e l i v e r m i t o c h o n d r i a . Th e i n c u b a t i o n m i x t u r e c o n t a i n e d in a final v o l u m e of I m l : m i t o c h o n d r i a , 3.4 m g p r o t e i n ; s o d i u m 2 - o x o i s o v a l e r a t e , 5 m M ; L-[Me-aH] c a r n i t i n e , i mM; E D T A , o.15 m M ; a n d T E S buffer (pH 7.4), 20 raM. One s e t of ve s s e l s c o n t a i n e d in a d d i t i o n 2 , 4 - d i n i t r o p h e n o l , o.I mM (lower figure). Th e r e a c t i o n w a s r u n a t 37 °. Q - - O , a c e t y l carnitine. []--O, propionylcarnitine. &--&, isobutyrylcarnitine. TABLE
III
EFFECT OF 2,4-DINITROPHENOL AND A D P + Pi ON THE PRODUCTION OF ACYLCARNITINES FROM 2-OXOISOVALERATE
IN
MOUSE
LIVER
MITOCHONDRIA
T h e i n c u b a t i o n m i x t u r e c o n t a i n e d in a final v o l u m e of I m l : m o u s e l i v e r m i t o c h o n d r i a , 3.7 m g p r o t e i n ; s o d i u m 2 - o x o i s o v a l e r a t e , 5 m M ; L- [ M e - 3 H ] c a r n i t i n e , I mM ; E D T A , o. 15 mM ; T E S buffer ( p H 7.4), 2o raM; a n d a d d i t i o n s as s t a t e d . The r e a c t i o n w a s r u n for 3 ° rain a t 37 °. T h e r e s u l t s s h o w n are m e a n s of d u p l i c a t e s .
Additions
A cylcarnitines produced (nmoles/mg mitochondrial protein)
None 2,4-Dinitrophenol
(o,I mM)
A D P (5 mM) a n d Pl (5 mM)
A cetylcarnitine
Propionylcarnitine
lsobutyrylcarnitine
19
45 9° 96
54 66 34
i
31
* V a l u e s < 5 n m o l e s / m g p r o t e i n are o n l y a p p r o x i m a t e .
Biochim. Biophys. Acta, 222 (197 o) 3 7 2 - 3 8 0
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H . E . SOLBERG, J. BREMER
low l e v e l of a c e t y l c a r n i t i n e in t h e p r e s e n c e of 2 , 4 - d i n i t r o p h e n o l (Fig. 3, T a b l e IFI) m i g h t t h e n be e x p l a i n e d b y i n h i b i t i o n of t h e A T P - d e p e n d e n t a c y l - C o A s y n t h e t a s e s t e p (EC 6.2.1.3) i n i t i a t i n g t h e f l - o x i d a t i o n of f a t t y acids, a l t h o u g h s t i m u l a t i o n of a c e t y l - C o A c o n s u m i n g r e a c t i o n s c a n n o t be e x c l u d e d . A D P a n d p h o s p h a t e also s t i m u l a t e d p r o p i o n y l c a r n i t i n e f o r m a t i o n (Table III), as d i d 2 , 4 - d i n i t r o p h e n o l , p r o b a b l y b y s i m i l a r m e c h a n i s m s . A D P a n d P i g i v e a p r o d u c t i n h i b i t i o n of t h e p r o p i o n y l - C o A c a r b o x y l a s e 21. T h e i n c r e a s e d p r o d u c t i o n of a c e t y l c a r n i t i n e in t h e p r e s e n c e of A D P + P i m a y be c a u s e d b y s t i m u l a t e d a c t i v a t i o n of f a t t y acids on t h e o u t e r m i t o c h o n d r i a l m e m b r a n e ~2 b y t h e a c c e l e r a t e d A T P s y n t h e s i s . T h i s results in i n c r e a s e d f l - o x i d a t i o n of e n d o g e n o u s f a t t y acids a n d i n c r e a s e d f o r m a t i o n of a c e t y l - C o A a n d a c e t y l c a r n i t i n e . ACKNOWLEDGMENTS T h e skilled t e c h n i c a l a s s i s t a n c e of Miss W. T h o r b j 0 r n s e n a n d Mrs. B. H a g a is greatly appreciated. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 2I 22
T. BOHMER AND J. BREMER, Biochim. Biophys. Acta, 152 (1968) 559. T. BOHMER AND J. BREMER, Biochim. Biophys. Acta, 152 (1968) 44 o. J. F. A. CHASE, Biochem. J., lO4 (1967) 51o. K. R. NORUM, Biochim. Biophys. Acta, 89 (1964) 95. J. BREMER AND K. R. NORUM, J. Biol. Chem., 242 (1967) 1744. H. J. ZIEGLER, P. BRUCKNER AND F. BINON, J. Org. Chem., 32 (1967) 3989. J. BREMER, European J. Biochem., 8 (1969) 535. P. ENEROTH AND G. LINDSTEDT, Anal. Biochem., IO (1965) 479S. HESTRIN, ft. Biol. Chem., 18o (1949) 249. J. BREMER, Biochim. Biophys. Acta, 116 (1966) i. G. HVISTENDAHL, K. UNDHEIM AND J. t3REMER, Organic Mass Spectrometry, in the press. G. HVISTENDAHLAND K. UNDHEIM, Org. Mass Spectry., 3 (197o) 821. W. C. SCHNEIDER AND G. H. HOGEBOOM,J. Biol. Chem., 183 (195 o) 123. D. 1K. MYERS AND E. C. SLATER, Biochem. J., 67 (1957) 558. T. BOHMER, Biochim. Biophys. Acta, 2o8 (197 o) 167. A. G. DAWSON AND F. J. R. HIRD, Arch. Biochem. Biophys., 127 (1968) 622. H. W. GOEDDE, IV[. Hi)FNER, V. M~HLENBECK AND K. G. BLUME, Biochim. Biophys. Acta, 132 (1967) 524 . T. BOHMER, Biochim. Biophys. Acta, 164 (1968) 487 . K. IR. NORUM AND J. BREMER, J. Biol. Chem., 242 (1967) 407 . Y. KAZlRO AND S. OCHOA,Advan. Enzymol., 26 (1964) 283. M. J. WEIDEMANN AND H. A. KREBS, Biochem. J., I I I (1969) 69. K. R. NORUM, M. FARSTAD AND J. BREMER, Biochem. Biophys. Res. Commun., 24 (1966) 797
Biochim. Biophys. Acta, 222 (197 o) 372-380
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
26452
FORMATION OF B R A N C H E D CHAIN A C Y L C A R N I T I N E S IN M I T O C H O N D R I A H E L G E E R 1 K S O L B E R G AND J O N B R E M E R
Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet, Oslo (Norway) (Received J u l y 2oth, 197 o)
SUMMARY
i. The formation of acylcarnitines in isolated mitochondria incubated with L-EMe-3H]carnitine and the branched chain 2-oxoacids corresponding to valine, leucine and isoleucine has been studied. 2. Hitherto unknown acylcarnitines were formed and identified as isobutyrylcarnitine (from 2-oxoisovalerate), isovalerylcarnitine (from 2-oxoisocaproate) and 2-methylbutyrylcarnitine (from 2-oxo-3-methylvalerate). 3. The addition of D N P or ADP + P i to the incubation medium increased the formation of propionylcarnitine from 2-oxoisovalerate. 4. The carnitine acetyltransferase (EC 2.3.1.7) was found to transfer acyl groups from branched chain acylcarnitines, but the transfer rates were lower than those found when the corresponding straight chain acylcarnitines were the substrates. 5- Liver, kidney and heart mitochondria from the rat and liver mitochondria from the mouse were all found to have branched chain 2-oxoacid dehydrogenase activity.
INTRODUCTION
Several different acylcarnitines have been identified in mammalian tissues. BOHMER AND BREMER1 have shown that liver of normally fed rats contains significant amounts of acetylcarnitine, propionylcarnitine, and long-chain acylcarnitines. In addition, liver contains small amounts of at least one unidentified acylcarnitine 1, 2. We have found the same compound(s) in relatively greater concentrations in mouse liver (unpublished results). Since the substrate specificities of carnitine acetyltransferase (acetyl-CoA : carnitine 0-acetyltransferase, EC 2.3.1.7)1, 3 as well as carnitine palmityltransferase (palmityl-CoA:carnitine 0-palmityltransferase, EC 2.3.I.-) 4 are rather broad with respect to the acyl group, it is possible that several hitherto unknown acylcarnitines exist in animal tissues. This will depend on the concentrations of the corresponding coenzyme A thioesters in the tissues. Since the carnitine acyltransferases are mitochondrial enzymes we have started the search for new acylcarnitines in experiments in vitro by incubating mitochondria from different tissues with different substrates. In the present communication the formation of branched chain acylcarnitines from the 2-oxoacids corresponding to valine, leucine, and isoleucine is reported. Biochim. Biophys. Acta, 222 (197 o) 372-380
BRANCHED CHAIN ACYLCARNITINES
373
MATERIALS AND METHODS
Chemicals The following commercial chemicals were used: L-carnitine hydrochloride (Otsuka Pharmaceutical Company, Osaka, Japan); acylchlorides, straight and branched chain carboxylic acids, and Dowex 5o W-X8, 200-400 mesh (Fluka AG, Buchs, Switzerland) ; ATP, ADP, and sodium salts of 2-oxoacids (Sigma Chemical Company, St. Louis, Mo., U.S.A.) ; coenzyme A and carnitine acetyltransferase (Boehringer and Soehne GmbH, Mannheim, Germany); silicic acid H (after stahl) and thionylchloride (Merck AG, Darmstadt, Germany). The purity of the acids and acylchlorides was tested by gas-liquid chromatography. DL- and L-EMe-SHlcarnitine were synthesized essentially by the method of BREMER AND NORUM5 from EaHJmethyliodide (Radiochemical Centre, Amersham, England) and DL- and L-3-hydroxy-4-(dimethylamino)butyric acid, and diluted with unlabelled L-carnitine hydrochloride to a specific activity of 30 and 5 mC/mmole, respectively. The radiochemical purity of each compound was greater than 99 % when tested by thin-layer chromatography (System A, described below). L-Acylcarnitine hydrochlorides were synthesized from L-carnitine hydrochloride and acylchlorides I or by a modified method of ZIEGLER et al.6:2 mmoles of thionylchloride were added to approx. IO mmoles of the acid and left for 2-12 h at 80 °. Moisture was kept out by Molecular Sieve 3A pellets (British Drug Houses Ltd., Poole, England). Then 2 mmoles of L-carnitine hydrochloride dissolved in 0.8 ml trifluoroacetic acid was added. The mixture was left at 80 ° for a couple of hours. After cooling the reaction mixture to room temperature, the acylcarnitines were isolated according to the method of BOHMER AND BREMER1. The purity of each compound was tested by thin-layer chromatography (System A), and the identity was verified by mass spectrometry. It was impossible to obtain pure methacrylylcarnitine by these methods. A sticky compound with RE approx. 0.06 (System A) was always produced (polymerization product?). The presence of o.I mmole of hydroquinone in the reaction mixture, however, reduced the formation of this by-product, possibly by preventing polymerization reactions. The mass spectrum of methacrylylcarnitine was recorded after purification by thinlayer chromatography (System A). All other chemicals were commercial products of high purity.
Chromatography Carnitine and acylcarnitines were usually separated on thin-layer silicic acid plates developed with methanol-chloroform-water-conc, ammonia-formic acid (55 : 50: Io : 7.5:2.5, by vol.) 7 (System A). The RF values showed some variation mainly dependent on loading, but the order of peaks was always as shown in Fig. I. In some experiments another solvent was used for developing thin-layer chromatograms: methanol-acetoue-conc. HC1 (9o:1o:Io, by vol.) 8 (System B). The following RF values were obtained with Solvent B: butyrobetaine 0.32-0.35, carnitine o.41-o.43, and acylcarnitines 0.43-o.5 o. The solvent front was always run approx. 14 cm. To localize radioactivity on the plates the silicic acid was cut into bands 3-6 mm wide and scraped into counting vials. Ion-exchange chromatography of carnitines 1 was performed on a I cm × 5o cm column filled with Dowex 5 ° W-X8, 200-400 mesh, NH4+, eluted with 0.2 M amBiochim. Biophys. Acta, 222 (197 o) 372-38o
374
H . E . SOLBERG, J. BREMER
monium formate buffer (pH 4.2). The peaks were localized by measuring the radioactivity in o.2-ml aliquots of each fraction or by HESTRIN'S hydroxamate reaction 9 as modified by BREMER~°. The elution volume of carnitine was 230-280 ml. The following elution volumes relative to that of carnitine were found: valine, 0.03; acetylcarnitine, 0.7-0.8; propionylcarnitine was hidden in the carnitine peak; isobutyrylcarnitine, 1.1-1.3; 2-methylbutyrylcarnitine, 1.7; isovalerylcarnitine, 1.9; and methacrylylcarmtine, 3.3. Radioactive samples were measured in a Packard Tri-Carb scintillation spectrometer Model 331o with 5 or IO ml of a scintillator solution consisting of: dimethyl1,4-bis-(5-phenyloxazolyl-2)-benzene (dimethyl POPOP), 0.25 g; 2,5-diphenyloxazole (PPO), 25 g; naphthalene, 400 g; ethanol, 115o ml; dioxane, 1925 ml; and xylol, 1925 ml. The counting efficiency was about 35 % for tritium. The presence of silicic acid reduced the efficiency approximately by three-fourth.
Mass spectrometry Carnitine and carnitine esters were introduced into a Varian CH 7 mass spectrometer through the direct inlet on a probe equipped with temperature-controlled heating. The spectra of carnitine and acylcarnitines are complex and are produced by the combined effect of pyrolysis and electron impact 11. Close supervision of the heating was necessary to obtain reproducible spectra. The standard conditions used were probe temperature (lOO-12o°); ion source temperature (approx. 16o°); ionizing energy (7 ° eV); and cathodic emission current (IOO or 300/~A). Carnitine hydrochloride itself has a base peak at m/e 58 and peaks at m/e 59, 42 and 30 caused by fragmentation and rearrangement of trimethylamine 12 eliminated by pyrolysis. The remaining skeleton of carnitine rearranges to the 3-hydroxy7-1actone n which has a molecular peak at role lO2 and prominent fragments at m/e 84, 74, 55, 44 and 43- Peaks at m/e 36 and 38 in a 3 : i ratio are caused by HC1. The short-chain acylcarnitines show pyrolytic elimination of the acid esterified at the 3-hydroxy group leaving an unsaturated 7-1actone (m/e 84) after the pyrolytic deamination ~a. The peaks at m/e lO2, 74, 44 and 43 observed in the free carnitine spect r u m are therefore absent or of low intensity. Longer chain acylcarnitines (straight chain: C8 or above, branched chain: C 5 or above) have peaks from both the free acid and the 3-O-acyl-7-1actone. A carnitine ester m a y thus be identified by the superimposed spectra of the trimethylamine, the unsaturated or esterified ~-lactone and the eliminated free carboxylic acid.
Incubation of mitochondria with 2-oxoacids Female mice of N M R I / B o m strain (18-22 g) or maleWistar/Moll rats (15o-2oo g) were used. Liver, kidney, or heart mitochondria were isolated by the method of SCHNEIDER AND HOGEBOOM 13 essentially as described by MYERS AND SLATER 14. T h e particles were washed once in o.15 M KC1 and resuspended in the same medium. The protein concentrations were determined by the micro-Kjeldahl procedure on duplicate samples of the suspensions. The incubations were performed in a shaking bath at 37 ° with air as the gaseous phase. The reaction mixtures were, as specified in the legends of tables and figures, made up to a final volume of i ml with o.15 M KC1. The reactions were started by the addition of mitochondrial suspension and stopped by I ml of IO % trichloroacetic Biochim. Biophys. Aata, 222 (i97 o) 372-380
375
BRANCHED CHAIN ACYLCARNITINES
acid. After the centrifugation the trichloroacetic acid in the supernatant was removed b y extracting twice with diethyl ether. The water phase was lyophilized or blown to dryness under an air current. Before application on thin-layer silicic acid plates the residues were redissolved in 50 #1 water followed by the addition of 50 #1 methanol. In some experiments the water phase was directly applied on the top of the Dowex column. RESULTS AND DISCUSSION
Formation of acylcarnitines in mitochondria Preliminary experiments showed that mouse liver mitochondria did not produce acylcarnitines when branched chain DL-amino acids were substrates. R a t liver mitochondria can metabolize D-amino acids 1~. In rat kidney mitochondria DAWSON AND HIRDTM have shown that the activities of L-valine or L-leucine aminotransferases were limited by the availability of amino group acceptor (i.e. 2-oxoglutarate). To avoid complications introduced by variations in the deamination step, the 2-oxoacids corresponding to the branched chain amino acids were chosen as substrates in the experiments reported below. Table I shows that mitochondria produced acetylcarnitine and propionylcarnitine when the metabolic pathways of the substrates included acetyl-CoA or propionyl-CoA as intermediates. There was only one exception: the acetylcarnitine formation was low from 2-oxoisocaproate. This probably shows that the rate of production of acetyl-CoA from 2-oxoisocaproate is slow compared with the capacity of the acetyl-CoA consuming processes in mitochondria (mainly acetoacetate and citrate formation). In addition, unknown acylcarnitines with RF values 0.40-0.50 (Fig. i) were produced in significant amounts from the branched chain 2-oxoacids. TABLE I FORMATION O F
ACYLCARN1TINES
FROM
2-OXOACIDS IN MITOCHONDRIA FROM MOUSE AND RAT TISSUES
T h e i n c u b a t i o n m i x t u r e c o n t a i n e d in a final v o l u m e of i m l : m i t o c h o n d r i a (as s t a t e d be l ow ); s u b s t r a t e (sodium salt), 5 raM; DL- c r L-EMe-3H~carnitine ( E x p t . I or ti, r e s p e c t i v e l y ) , i m M ; E D T A , O. lO-O.15 raM; N - t r i s ( h y d r o x y m e t h y l ) m e t h y l - 2 - a m i n o e t h a n e sulfonic acid buffer (TES buffer) (pH 7.4), 20 mM. The r e a c t i o n w a s r u n for 20 r a i n ( E x p t . I) or 3 ° ra i n ( E x p t . II) a t 37 °. AC, a c e t y l c a r n i t i n e ; PC, p r o p i o n y l c a r n i t i n e ; MC, m e d i u m c h a i n a c y l c a r n i t i n e s : i s o b u t y r y l c a r n i t i n e (from 2 - 0 x o i s o v a l e r a t e ) , i s o v a l e r y l c a r n i t i n e (from 2-0 xoi s oc a proa t e ), 2 - m e t h y l b u t y r y l c a r n i t i n e (from 2 - o x o - 3 - m e t h y l v a l e r a t e ) , or u n i d e n t i f i e d (from e n d o g e n o u s s u b s t r a t e s ) . The r e s u l t s from r a t l i v e r a n d k i d n e y are m e a n s of d u p l i c a t e s .
Substrate
A cylcarnitines produced (nmoles/mg mitochondrial protein) * Expt. I Mouse liver (2.1 rng protein)
Expt. I I Rat liver (1.2 mg protein)
Rat kidney (2. 7 mg protein)
Rat heart (L8 mg protein)
AC PC MC
AC PC MC
AC PC MC
AC PC MC
9 4 3 14
4 3 8 62
I 2 2 29
Endogenous 16 7 2-Oxoisovalerate 9 58 2-Oxoisocaproate 5 5 2 - O x o - 3 - m e t h y l - n - v a l e r a t e 38 66 2-Oxobutyrate 6 173
2 5° 2 4 2
3 37 6 66
II 19 io IO
2 6 83 lO 5 i 26 91 23
I io 23 i o o i 35 46 35
* V a l u e s < 5 n m o l e s / m g p r o t e i n are o n l y a p p r o x i m a t e .
Biochim. Biophys. Acta, 222 (I97 o) 372-380
376
H.E. SOLBERG, J. BREMER
Identification of products The unknown products were identified as the acylcarnitines corresponding to the coenzyme A thioesters formed in the branched chain 2-oxoacid dehydrogenase reaction. This conclusion is based on the following evidence. Firstly, chromatography of incubates with carrier amounts of synthetic acylcarnitines on thin-layer plates (System A) gave RF values of the radioactive peaks of the products formed from 2-oxoisovalerate, 2-oxoisocaproate, and 2-oxo-3-methyl-n-valerate identical with those of the iodine spots of isobutyrylcarnitine, isovalerylcarnitine, and 2-methylbutyrylcarnitine, respectively. Secondly, the radioactive peaks of the products coincided with the hydroxamate peaks of the synthetic acylcarnitine carriers after chromatography on the Dowex 5° NH4+ column. The possibility remained, however, that the products were the acylcarnitines corresponding to the unsaturated coenzyme A thioesters formed in the dehydrogenation step following the oxidative decarboxylation of the 2-oxoacids. These unsaturated carnitine esters might not be separated ha our chromatographic systems. One unsaturated compound, methacrylylcarnitine, was accordingly synthesized. Isobutyrylcarnitine and methacrylylcarnitine had, in fact, nearly identical RF values on the thin-layer plates (System A), but the elution volumes from the Dowex 50 column
2O
10
D
t/'I 4
C,
ilL, 40 27
P
_~B
55
88
dl 1,, d. LI., I
,
0
? ¢. ~_
50
361
/
,..b
43
E
B
rn •
m• 5
36
84
20
55
,
73
I,, .oL ,, ...I ..... 30
40
50
60
rn/e
I.... 70
80
3,~ 90
Fig. I. T h i n - l a y e r c h r o m a t o g r a p h y (silicic acid, s o l v e n t s y s t e m A) of c a r n i t i n e a n d t h e acylc a r n i t i n e s p r o d u c e d in r a t k i d n e y m i t o c h o n d r i a from 2-oxoisovalerate (upper part), 2-oxoisoc a p r o a t e (middle part), or 2 - o x o - 3 - m e t h y l v a l e r a t e (lower part). T h e c h r o m a t o g r a m s are f r o m t h e e x p e r i m e n t r e p o r t e d in T a b l e I. F r a c t i o n s 3 m m wide. A, carnitine; B, a c e t y l c a r n i t i n e ; C, prop i o n y l c a r n i t i n e ; D, i s o b u t y r y l c a r n i t i n e ; E, i s o v a l e r y l c a r n i t i n e ; F, 2 - m e t h y l b u t y r y l c a r n i t i n e . I n t h i s c h r o m a t o g r a p h i c s y s t e m o t h e r b r a n c h e d or s t r a i g h t m e d i u m c h a i n a c y l c a r n i t i n e s also m o v e d to t h e position of t h e P e a k s D - F (RF o.4o-o.5o ). Fig. 2. Mass s p e c t r a of (B) s y n t h e t i c i s o b u t y r y l c a r n i t i n e h y d r o c h l o r i d e a n d of (A) t h e a c y l c a r n i t i n e w i t h RF 0.40-o.50 (thin-layer s y s t e m A) p r o d u c e d b y m o u s e liver m i t o c h o n d r i a (79 m g protein) w h e n i n c u b a t e d for 60 m i n a t 37 ° in t h e presence of s o d i u m 2-oxoisovalerate, 5 raM; L-~Me-SH] c a r n i t i n e , i raM; E D T A , o . i o raM; T E S buffer (pH 7-4), 20 m M ; a n d o.15 M KC1 u p to a final v o l u m e of 25 ml. A f t e r isolation on t h i n - l a y e r silicic acid p l a t e s ( S y s t e m A), t h e c o m p o u n d w a s f u r t h e r purified b y c h r o m a t o g r a p h y on a D o w e x 5o NH4+ c o l u m n eluted w i t h o.2 M a m m o n i u m f o r m a t e buffer (pH 4.2) a n d on t h i n - l a y e r silicic acid ( S y s t e m B). T h e m a s s s p e c t r a were recorded a s described in METHODS. P r o b e t e m p e r a t u r e , 117 ° (A) a n d lO4 ° (B); electron emission current, i o o t,A. O r d i n a t e : relative i n t e n s i t y (%). m ~ m e t a s t a b l e p e a k s a t m/e 39.2 a n d 60.6. P e a k s a t role 5 ° a n d 52 are p r o b a b l y d u e to c o n t a m i n a n t s from t h e last c h r o m a t o g r a p h i c step.
Biochim. Biophys. Acta, 222 (197 o) 372-380
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BRANCHED CHAIN ACYLCARNITINES
differed considerably (approx. 1.2 and 3.3, respectively, relative to the elution volume of carnitine). As it has also been shown 1 that the elution volume of crotonylcarnitine is greater than that of butyrylcarnitine, it is probable that all unsaturated medium chain acylcarnitines are more strongly bound to the Dowex 50 NH4+ column than the corresponding saturated compounds. No unsaturated acylcarnitines were detected in the column chromatograms. When the product formed from 2-oxoisovalerate was isolated and purified it was identified as isobutyrylcarnitine on mass spectrometry (Fig. 2). The peaks of free isobutyric acid were at m/e 88 (molecular ion), 73, 43 (base peak), 41 and 27. In contrast, the mass spectrum of methacrylylcarnitine had peaks from methacrylic acid at m/e 86 (molecular ion), 69, 41 (base peak of the acid), and 39. The acylcarnitines produced from 2-oxoisocaproate and 2-oxo-3-methylvalelate were not isolated for mass spectrometry. Analogous to the isobutyrylcarnitine, however, these were also considered to be the saturated compounds. The lack of unsaturated or 3-hydroxy acylcarnitines in our chromatograms might be caused by too low a concentration of the corresponding CoA-bound intermediates in the mitochondria metabolizing branched chain 2-oxoacids. It is also possible that these CoA-esters are poor substrates for the carnitine acyltransferases.
The specificity of carnitine acetyltransferase The substrate specificity of the acetyltransferase was examined by the exchange of carnitine according to the reaction: L-Acylcarnitine -- CoASH ~- L-carnitine + acyl-CoA When acylcarnitines were incubated with tritium-labelled carnitine in the presence of carnitine acetyltransferase and catalytic amounts of CoA, radioactivity was incorporated into the acylcarnitines at different rates according to the substrate specificity of the enzyme with respect to the acyl groups. In this system propionylcarnitine was the most reactive substrate (Table II), confirming the observation of BOHMER TABLE
II
SUBSTRATE
SPECIFICITY
OF
COMMERCIAL
ACETYLTRANSFERASE
T h e r e a c t i o n m i x t u r e c o n t a i n e d in a final v o l u m e of i m l ( a d j u s t e d w i t h d i s t i l l e d w a t e r) : a c e t y l t r a n s f e r a s e , a p p r o x , o , I u n i t ; s u b s t t a t e ' ( L - a c y l c a r n i t i n e h y d r o c h l o r i d e ) , i m M ; L-[Me-3H]c a r n i t i n e , I m M ; CoA, o.i m M ; Tris buffer (pH 7.5), o. i M. The r e a c t i o n w a s s t a r t e d b y a d d i t i o n of l a b e l l e d c a r n i t i n e a n d r u n for i o m i n a t 37 °. A f t e r s t o p p i n g w i t h 2 m l e t h a n o l , t h e m i x t u r e w a s e v a p o r a t e d to d r y n e s s u n d e r a n a i r c u r r e n t . The p r o c e d u r e c o n t i n u e d as d e s c r i b e d for t h e e x p e r i m e n t s w i t h m i t o c h o n d r i a . T h e r e s u l t s s h o w n are m e a n s of d u p l i c a t e s .
Substrate
Radioactivity incorporated in the substrate (%)
L-Acetylcarnitine L-Propionylcarnitine L-Butyrylcarnitine L-Valerylcarnitine L-Isobutyrylcarnitine L-Isovalerylcarnitine L-2-Methylbutyrylcarnitine L-Crotonylcarnitine L-Methacrylcarnitine
7-3 12.8 8.7 2.3 1.5 o. 8 * o.7" o.6" o. 5 *
* Values
Relative exchange rate (related to that of acetylearnitine) i.o I. 8 1.2 o. 3 o.2 o. 1 < o.I < o. I < o. i
< 1 % are o n l y a p p r o x i m a t e .
Bioehim. Biophys. Acta, 222 (197 o) 3 7 2 - 3 8 o
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H.E. SOLBERG, J. BREMER
AND BREMER1. Branching of the acyf group reduced the reactivity. That isobutyrylcarnitine was the most reactive of the branched acylcarnitines tested is probably one of the reasons why mitochondria produced mole isobutyrylcarnitine than isovalery|carnitine or 2-methylbutyrylcarnitine (Table 5). Introduction of a hydroxyl gioup in 3-position of butyrylcarnitine reduced its reactivity 1as did dehydrogenation (Table II). Relatively high concentrations of the unsaturated or 3-hydroxy acyl-CoA intermediates in the metabolic pathways of the branched chain amino acids would probably be necessary to produce detectable amounts of the corresponding acylcarnitines in the mitochondria.
Comparison of acylcarnitine production in mitochondria from various sources In some experiments the acylcarnitine formation in mitochondria from various organs of rats and mice were compared. Table I shows representative results. The amount of an acyl-CoA intermediate trapped as the corresponding acylcarnitine is the result of at least four factors: the capacities of (I) the acyl-CoA producing and (2) the acyl-CoA consuming steps; (3) the amount of carnitine acetyltransferase present, and (4) its capacity for transferring the acyl group in question. Quantitative intelpretations of the results are accordingly difficult. It is evident, however, that liver, kidney, and heart mitochondria all have branched chain 2-oxoacid dehydrogenases and all the following enzymes of the branched chain amino acid metabolic pathways since both acetyl- and propionyl-carnitine were formed. Other tissues were not examined. GOEDDE et al. 17found branched chain 2-oxoacid dehydrogenase activity in other tissues and used the activity of the dehydrogenases in human leucocytes in diagnostic studies on the branched chain 2-oxoaciduria ("maple syrup urine disease"). BREMER7 observed oxidation of some straight chain 2-oxoacids in rat heart, brain, kidney, and liver. It is thus probable that most tissues have the capacity for oxidative decarboxylation of straight or branched chain 2-oxoacids. The reason why BOHMER15, 18 did not observe medium chain acylcarnitines in his studies on the propionylcarnitine formation from amino acids and 2-oxoacids, is probably that he used rat liver mitochondria. Our results indicate that these generally produce less branched chain acylcarnitines than mitochondria from other tissues tested. One reason for this m a y be the low level of carnitine acetyltransferase in rat liver 19. It is not known whether there is relatively more of the acetyltransferase in mouse liver. Effect of incubation time and additions on acylcarnitine formation from 2-0xoisovalerate 2-Oxoisovalerate was chosen as substrate for some further studies since the isobutyrylcarnitine formation was greater than the production of other branched chain acylcarnitines in mitochondria (Table I). Fig. 3 (upper part) shows the effect of incubation time on acylcarnitine production. The formation of isobutyrylcarnitine started immediately, while the propionylcarnitine production showed a lag. In other experiments the curve of propionylcarnitine formation rose steadily after the initial lag and crossed that of isobutyrylcarnitine formation after 30-60 min of incubation. Addition of 2,4-dinitrophenol to the incubation mixture (Fig. 3, lower part, and Table I I I ) resulted in accelerated propionylcarnitine production without an initial lag period. BOHMER15 also observed formation of propionylcarnitine from 2-oxoisovalerate in the presence of 2,4-dinitrophenol and used this as an indication of the Biochim. t3iophys. Acta, 222 (197o) 372 38o
379
BRANCHED CHAIN ACYLCARNITINES
absence of ATP-dependent steps (e.g. activation of free carboxylic acids) between initial 2-oxoacid decarboxylation and propionyl-CoA formation. However, the increased formation of propionylcarnitine in the presence of 2,4-dinitrophenol requires an additional explanation. 2,4-Dinitrophenol may accelerate the oxidation steps leading to propionyl-CoA, and it will at the same time inhibit the ATP-dependent propionyl-CoA carboxylase (EC 6.4.1.3) by lowering the ATP levels in mitochondria. Since carboxylation probably is the main pathway of propionyl-CoA in mammalian tissues 2°, its inhibition will cause a rise in propionyl-CoA. The acetylcarnitine formation is probably caused by oxidation of endogenous substrates (fatty acids?) since acetyl-CoA is not an intermediate in the metabolism of 2-oxoisovalerate. The very
tooI noo0ton
c~ DNP added
c
c -; --e o
5
15 Incubation t i m e (rain)
?
45
Fig. 3. Effect of i n c u b a t i o n t i m e a n d 2 , 4 - d i n i t r o p h e n o l (DNP) on t h e p r o d u c t i o n of a c y l c a r n i t i n e s f r o m 2 - o x o i s o v a l e r a t e in m o u s e l i v e r m i t o c h o n d r i a . Th e i n c u b a t i o n m i x t u r e c o n t a i n e d in a final v o l u m e of I m l : m i t o c h o n d r i a , 3.4 m g p r o t e i n ; s o d i u m 2 - o x o i s o v a l e r a t e , 5 m M ; L-[Me-aH] c a r n i t i n e , i mM; E D T A , o.15 m M ; a n d T E S buffer (pH 7.4), 20 raM. One s e t of ve s s e l s c o n t a i n e d in a d d i t i o n 2 , 4 - d i n i t r o p h e n o l , o.I mM (lower figure). Th e r e a c t i o n w a s r u n a t 37 °. Q - - O , a c e t y l carnitine. []--O, propionylcarnitine. &--&, isobutyrylcarnitine. TABLE
III
EFFECT OF 2,4-DINITROPHENOL AND A D P + Pi ON THE PRODUCTION OF ACYLCARNITINES FROM 2-OXOISOVALERATE
IN
MOUSE
LIVER
MITOCHONDRIA
T h e i n c u b a t i o n m i x t u r e c o n t a i n e d in a final v o l u m e of I m l : m o u s e l i v e r m i t o c h o n d r i a , 3.7 m g p r o t e i n ; s o d i u m 2 - o x o i s o v a l e r a t e , 5 m M ; L- [ M e - 3 H ] c a r n i t i n e , I mM ; E D T A , o. 15 mM ; T E S buffer ( p H 7.4), 2o raM; a n d a d d i t i o n s as s t a t e d . The r e a c t i o n w a s r u n for 3 ° rain a t 37 °. T h e r e s u l t s s h o w n are m e a n s of d u p l i c a t e s .
Additions
A cylcarnitines produced (nmoles/mg mitochondrial protein)
None 2,4-Dinitrophenol
(o,I mM)
A D P (5 mM) a n d Pl (5 mM)
A cetylcarnitine
Propionylcarnitine
lsobutyrylcarnitine
19
45 9° 96
54 66 34
i
31
* V a l u e s < 5 n m o l e s / m g p r o t e i n are o n l y a p p r o x i m a t e .
Biochim. Biophys. Acta, 222 (197 o) 3 7 2 - 3 8 0
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H . E . SOLBERG, J. BREMER
low l e v e l of a c e t y l c a r n i t i n e in t h e p r e s e n c e of 2 , 4 - d i n i t r o p h e n o l (Fig. 3, T a b l e IFI) m i g h t t h e n be e x p l a i n e d b y i n h i b i t i o n of t h e A T P - d e p e n d e n t a c y l - C o A s y n t h e t a s e s t e p (EC 6.2.1.3) i n i t i a t i n g t h e f l - o x i d a t i o n of f a t t y acids, a l t h o u g h s t i m u l a t i o n of a c e t y l - C o A c o n s u m i n g r e a c t i o n s c a n n o t be e x c l u d e d . A D P a n d p h o s p h a t e also s t i m u l a t e d p r o p i o n y l c a r n i t i n e f o r m a t i o n (Table III), as d i d 2 , 4 - d i n i t r o p h e n o l , p r o b a b l y b y s i m i l a r m e c h a n i s m s . A D P a n d P i g i v e a p r o d u c t i n h i b i t i o n of t h e p r o p i o n y l - C o A c a r b o x y l a s e 21. T h e i n c r e a s e d p r o d u c t i o n of a c e t y l c a r n i t i n e in t h e p r e s e n c e of A D P + P i m a y be c a u s e d b y s t i m u l a t e d a c t i v a t i o n of f a t t y acids on t h e o u t e r m i t o c h o n d r i a l m e m b r a n e ~2 b y t h e a c c e l e r a t e d A T P s y n t h e s i s . T h i s results in i n c r e a s e d f l - o x i d a t i o n of e n d o g e n o u s f a t t y acids a n d i n c r e a s e d f o r m a t i o n of a c e t y l - C o A a n d a c e t y l c a r n i t i n e . ACKNOWLEDGMENTS T h e skilled t e c h n i c a l a s s i s t a n c e of Miss W. T h o r b j 0 r n s e n a n d Mrs. B. H a g a is greatly appreciated. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 2I 22
T. BOHMER AND J. BREMER, Biochim. Biophys. Acta, 152 (1968) 559. T. BOHMER AND J. BREMER, Biochim. Biophys. Acta, 152 (1968) 44 o. J. F. A. CHASE, Biochem. J., lO4 (1967) 51o. K. R. NORUM, Biochim. Biophys. Acta, 89 (1964) 95. J. BREMER AND K. R. NORUM, J. Biol. Chem., 242 (1967) 1744. H. J. ZIEGLER, P. BRUCKNER AND F. BINON, J. Org. Chem., 32 (1967) 3989. J. BREMER, European J. Biochem., 8 (1969) 535. P. ENEROTH AND G. LINDSTEDT, Anal. Biochem., IO (1965) 479S. HESTRIN, ft. Biol. Chem., 18o (1949) 249. J. BREMER, Biochim. Biophys. Acta, 116 (1966) i. G. HVISTENDAHL, K. UNDHEIM AND J. t3REMER, Organic Mass Spectrometry, in the press. G. HVISTENDAHLAND K. UNDHEIM, Org. Mass Spectry., 3 (197o) 821. W. C. SCHNEIDER AND G. H. HOGEBOOM,J. Biol. Chem., 183 (195 o) 123. D. 1K. MYERS AND E. C. SLATER, Biochem. J., 67 (1957) 558. T. BOHMER, Biochim. Biophys. Acta, 2o8 (197 o) 167. A. G. DAWSON AND F. J. R. HIRD, Arch. Biochem. Biophys., 127 (1968) 622. H. W. GOEDDE, IV[. Hi)FNER, V. M~HLENBECK AND K. G. BLUME, Biochim. Biophys. Acta, 132 (1967) 524 . T. BOHMER, Biochim. Biophys. Acta, 164 (1968) 487 . K. IR. NORUM AND J. BREMER, J. Biol. Chem., 242 (1967) 407 . Y. KAZlRO AND S. OCHOA,Advan. Enzymol., 26 (1964) 283. M. J. WEIDEMANN AND H. A. KREBS, Biochem. J., I I I (1969) 69. K. R. NORUM, M. FARSTAD AND J. BREMER, Biochem. Biophys. Res. Commun., 24 (1966) 797
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