- Email: [email protected]
In vitro studies on the metabolism of hexadecanedioic acid and its mono-l -carnitine- ester
Biochimiccr et Biopkysiccr Arto, e3: Elsevier
Scientific
Publishing
306 (1973)
Company,
r-14
Amsterdam
- Printed
in The Netherlands
RBA 56224
IN VITRO STUDIES ACID
JON
ON THE METABOLISM
AND ITS MONO-L-CARNITINE:
ELLTNG
PETTERSEN
fnstitr~ie c/f Chid
(Received
OF HEXADECANEDiOlC
ESTER
October
Bi(}cjzef~tisfr~, U~i~le~sit~~ vf Oslo, Rjks~los~ifffIet,
23rd,
Oslo I [iVor~v~_v)
t97z)
SUMMARY
T. A method for the synthesis of the mono-~-carnitine ester of h~xadecanedioic acid (hexadecanedioylcarilitine~ is described. A thin-fayer chromatography system for the separation of long-chain monocarboxylic and long-chain dicarboxylic acylcarnitines was developed. 2. Hexadecanedioic acid can be activated by rat liver mitochondria in the presence of CoA, ATP, and Mg2+. Since Triton X- 100 does not increase the activation capacity, the enzyme(s) activating long-chain dicarboxylic acids seems to be localized outside the inner mitochondrial compartment. 3. HexadecanedioyIcarnitine is substrate for a solubilized preparation of hexadecanoyl-CoA:carnitine 0-hexadecanoyltransferase (EC 2.3. I .-), but the reaction rate is only about r/to of that with palmitylcarnitine. Accordingly, mitochondriat CoA can be acylated by hexadecanoylcarnitine, but much slower than by palmitylcarnitine. 4. Hexadecanedioylcarnitine competes with palmitylcarnitine in the reaction catalyzed by the enzyme. 5. In fasted, ketotic streptozotocin-diabetic, and clofibrate-fed rats the carnitine acyItransferase activity was significantly increased. The relative increases were about the same whether hexadecanedioylcarnitine or palmitylcarnitine were used as substrates. 6. The observations under points 4 and 5 indicate that the same enzyme (hexadecanoyl-CoA:carnitine 0-hexadecanoyltransferase, EC 2.3. I .-) may transport activated hexadecanedioic as well as palmitic acid across the inner mitochondrial membrane. 7. The 0, uptake of rat heart and liver mitochondria with hexadecanedioyicarnitine as substrate was very low and transitory. Hexadecanedioylcarnitine did not inhibit the oxidation of palmitylcarnitine. 8. The present in vitro studies demonstrate a possible mechanism for the degradation of long-chain dicarboxylic acids, viz. activation by CoA and transportation into the inner mitochondrial compartment as carnitine esters followed by /Ioxidation.
J. E. PETTERSEN
Recently we have found that ketotic patients excrete considerable amounts of t?-hexanedioic and n-octanedioic acid in the urine’.2. Peroral intake of hexadecanedioic acid in healthy humans induced an increased urinary excretion of hexanedioic and octanedioic acid’. J/Z rig experiments with radioactively labelled hexadecanoic acid given to ketotic streptozotocin-diabetic rats indicated that the two short-chain dicarboxylic acids may bc formed from long-chain mono~arboxyl~c acids by an initial w-oxidation followed by /I-oxidations of the fang-chain dicarboxylic acids thus formed4. In ketosis there is an increased liberation of fatty acids from the fat depots and, moreover, an increased w-oxidation capacity has been demonstrated in liver microsomes from ketotic rats’.‘. These two findings may alone explain the increased urinary excretion of hexanedioic and octanedioi~ acid in ketosis. However, the exact pathways involved in the short-chain dicarboxylic acid formation is not known, and this Ied us to investigate the metabolism of long-chain dicarboxyhc acids. The present paper describes the synthesis of the nlo~lo-L-~arllitiIle ester of hexadecanedioi~ acid, and it? t?irfo studies on the ~etabol~sln of this carnitine ester and of hexadecanedioic acid itself. MATERIALS
AND
METHODS
I_-Carnitine was a gift from Otsuka Pharmaceutical Factory, Osaka, Japan. L-[~~,-‘H]Cnrnitine and palmityl-L-carnitine (palmitylcarnitine) were kindly supplied by Professor .I. Bren~er7.8. In the activation experiments 0.08 icl r.-[~fe-3H]carnitir~e was used (~OrrespoIlding to I 22 ’ IO” cpni~~~l~ole under the conditions used), and in the was used (corresponding to isotope exchange assays 0.0 1 M r_-[Me-“Hlcarnitine 935 10~ cpm,/jrmole). Hexadecanoyl-CoA:carnitine O-hexadecanoyltransferase (carnitine palmityltransferase. EC 2.3. I .-) was prepared according to Norum’ with the modifications described by Farstad er LI~.‘O.Oleylcarnitine, linoleyl-carnitine, and l~nole~~yl~ar~~itine were gifts from Idr B. 0. Christophersen”. ~exade~anedioi~ acid (more than 99 li;, pure when controlled by combined gas--liquid chromatographymass spectrometry) was obtained from Fiuka AG. Buchs, Switzerland, and CoA was delivered by C. F. Boehringer und Siihne GmBH, ~ailnt~eirn, Germany. Streptozototin (a gift from the Upjohn Company, Michigan, BISA) was dissolved jrn~lled~~itely before use in 0.9 ofi saline acidified to pH 4.5 with citric acid”““. Clofibrate (Atromidin) was a gift from ICI Ltd, Cheshire, England. Other chetnicals were commercial products of high purity.
Male rats of Wistar-M&l strain weighing 220-280 g were placed in metabolic cages and adapted for one week on the stock diet. The normal rats (5 animals) had free access to water and the stock diet. The fasted rats (5 animals) were deprived of food for 48 h prior to death. Diabetes meilitus was induced in IO rats as described earlier” by the intravenous injection of streptozotocin’2.‘” ( IOOmg/kg body weight). 5 animals with glucosuria and ketonuria were selected for further studies and killed 48 h after the in_jection. Blood glucose value for this group was 312 i_ 55 (mean .& S.D.), range 23738X mgj IOO ml. The amount of ketone bodies in blood was for the fasted rats I .2 k 0.2.
HEXADECANEDIOIC
ACID
METABOLISM
3
range 0.8-1.3 mg/ IOO ml, and for the diabetic rats, 3.8 &- 2.4, range I .9-7.9 mg/ IOO ml. 5 animals were fed 0.3 % clofibrate in the stock diet for one week. These animals have been described elsewhere14. There were no significant changes in body weight or liver protein concentration, whereas an increase in mean liver weight as first described by Best and Duncan” was observed. Preparation qf’ homogenates and mitochondric Rat liver and heart mitochondria were isolated in 0.25 M sucrose containing I mM EDTA according to Myers and Slater16; they were washed twice and resuspended in o. I 5 tvl KC1 in concentrations correspotlding to I g of fresh tissue per ml. Total liver homogenates contained I g of liver per 60 ml final solution. In the studies on fatty acid activation 5 . IO-~ M ATP was added to counteract enzyme inactivation (ref. 17). Preparation qf the mono-t-cnmitine ester qf hexadecanedioic acid (hexadecanerlioqIcarnitke 1 A modification of the method described by Bremer’ for the synthesis of longchain acylcarnitines was used. 15 mmoles of hexadecanedioic acid and 60 mmoles of oxalylchloride were mixed and kept at 55 “C for 2 h yielding hexadecanedioylchloride. Moisture was kept out by Molecular Sieve 3A pellets (British Drug Houses Ltd, Poole, England). The excess of unreacted oxalylchloride was removed under reduced pressure with the water pump at 55 “C. 2.5 mmoles of L-carnitine was dissolved in 4 ml of trifluoroacetic acid. This mixture was mixed thoroughly with the hexadecanedioylchloride. A large excess of the acid chloride was used to obtain the mono- and not the dical.nitine ester. The reaction was carried out on a water bath (40 “C) for 8 h under continuous stirring. Then light petroleum (b.p. 40-60 “C), ice chips and water were added. To assure that all acid chlorides were hydrolyzed, the mixture was made slightly alkaline (pH 8) by the addition of NaHCO,. The mixture was acidified to pH I with HCI, and was repeatediy extracted with diethyl ether to remove the free dicarboxylic acid.The hexadecanedioylcarnitine wasextracted with butanol. The butanol phase was washed with water to remove any free carnitine present, and was then taken to dryness on a rotovapor. By precipitation with diethyi ether from methanol the hexadecaj?edioylcarnitine was obtained as a white a~norphous material.
Thin-layer chromatography was performed at room temperature with Kieselgel H (Merck) plates. Solvent systems were either chloroform-methanol-cont. ammoniawater (50:30:8:2.5, by vol.) or hexane--diethyl ether-acetic acid (60:40: I, by vol.). The spots were made visible with iodine vapour. Combined gas-liquid chronlato~raph)/-nlass spectrometry was performed in an ~nstrl~nlent consisting of a Varian 1400 gas chromatograph (column material IO:! OV- I 7 on Gas Chrom Q (80-too mesh) or 8 %, BDS on Chromosorb W (80-r oo mesh) : temperature programmed from 80-300 or 80-180 “C, respectively, at a rate of 8 “C per min; ion current detector; helium as carrier gas with a flow of 30 ml/min) combined with a Varian CH 7 mass spectrometer (ionisation energy 70 eV>. The hexadecanedioic acid was methylated with diazomethane liberated from N-nitrosomethylurea. Mass spectrometric analyses were also undertaken in an AEI double focusing
4
J. E. PETTERSEN
instrument, Model MS 902 (source temperature 220 C, 70 eV ionizing energy, IOO ;rA ionisation current). The samples were introduced by the heated direct insertion probe. Perfluorotributylamine was used as internal standard. Radioactivity was measured in a Packard Tri-Carb liquid scintillation counter Model 3310 with a counting efciency ofabout 25 “; for “H. The scintillation solution consisted of 5 g PPO, 0.05 g ~iin~ethyl-POPOP, 385 ml xylol, 385 ml dioxanc, 230 ml ethanol, and 80 g llaphthalene. Mito~hondrial 0, uptake was measured polarograp~~i~aily with a Clark oxygen electrode, essentially as described by Chappel18. Protein was measured by the method of Lowry et al.‘“.
The CoA-dependent incorporation of “H-labelled free carnitine into ncylwas assayed by an isotope exchange tarnitines catalyzed by carnitine acyltransferase2’ assay as described by Norum9. Because of the complex kinetics of the exchange reaction” the activity was expressed in arbitrary units (cpm). Acyl-CoA synthetase activity was measured by trapping the acyl-CoA formed reaction products were subjected as acylcarnitines ‘o-‘7.22 . The butanol-extractable to thin-layer chromatography for the separation of hexadecanedioylcarnitine and acylcarnitines formed from monocarboxylic endogenous fatty acids. In a few experiments Triton X-loo was added to the incubation medium to determine whether the hexadccanedioic acid was activated outside and/or inside the inner mitochondrial membrane22. The acylation of m~tochondrial CoA was determined by Illeasuri~~g the amount of HClO,-insoluble CoA formed when mitochondria were incubated with acylcarnitines. The mitochondria were preincubated with a,.+dinitrophenol to remove preformed a.cyl-CoA before the addition of cyanide and acylcarnitines. The incubation was stopped with HCIO,, and the precipitate was washed twice with one volume of o. I M HCIO,. Long-chain acyl-CoA present in the precipitate was hydrolyzed with I M KOH23, and the CoA liberated was assayed by its ability to catalyze the incorporation of [~~e-3H~carnitin~ into ~lmityl~arnitine by carnitine palmityltra~~s~ ferase”. RESULTS
By thin-layer chromatography with the chloroform-methanol-ammonia-water system the preparation of l~exade~~lned;oyl~arrlitine showed only one spot when made visible with iodine vapour (R, value of 0.25). A sample from a synthesis where only 2 equivalents of acid (and I equiv of carnitine) had been used, showed a major spot with R,. 0.25, and in addition a small spot with R, 0.20. This spot probably represents the dicarnitine ester of hexadecanedioic acid. On thin-layer chromatograms v,ith hexaneediethyl ether-acetic acid as solvent system only one spot with R, o was seen; thus no free fatty acids were present. Kjeldahl analysis of the preparation of hexadecanedioylcarnitine showed a nitrogen content of 3.10 y:, (theoretical value 3.00 T#:).
HEXADECANEDIOIC
ACID
METABOLlSM
5
Mass spectra of various acylcarnjtines have been described by others’*,“s.26. Fig. ra shows the mass spectrum of hexad~canedioylcarnitine (as hydrochIoride). The major pyrolytic reaction is elimination of the acid caused by a-proton activation by the quarternary nitrogen, leaving an unsaturated y-lactone (m/e 84) after the liberation of trimethylaminez5. The whole series of peaks corresponding to the fragmentation of hexadecanedioic acid (Fig. I b) can be seen. The base peak at m/e 98 is due to the fragment C,H,,CO ” . The peaks at m/e 269 and nlje 268 seen in the spectrum of hexadecanedioglcarnitine are also present in the spectrum of free hexadecanedioic
120
140
160
180
200
220
240
260
280
300
mle
‘:‘ljslb; 120
140
160
(
,I
180
200
,!1,
220
240
260
280
3;O
m/e Fig. I. Mass spectra of hexadecanedioylcarniiine relative intensity less than z 7.; have been omitted.
(a) and
hexadecanedioic
acid
(b).
Peaks
with
acid, but with an intensity of only 0.6 and 1.6%~ of the base peak, respectively. The increase in the heights of the peaks at m/e 84 and m/e 55 in the mass spectrum of hexadecanedioylcarnitine (compared to that of hexadecanedioic acid) are due to the carnitine moiety2”. The y-lactone ring fragment with I?r/e 84 yields a peak at /?r/e 55 by loss of CHO. Peaks at nzje 59, m/e $3, m/e 42, and m/e 32 are due to trimethylamine25.28. Peaks at mje 36 and m/e 38 are due to the Cl- evaporating as HCI. The molecule ion (which is seen in the spectra of unsaturated long-chain acyl~arnitines”) is not seen in the hexadecanedioy~carnitine spectrum, neither are a peak at M-59 (due to loss of trimethylami~~e1i~25) or a peak at nz,/e 144 (corresponding to a McLafferty rearrangement fragment seen in the mass spectra of saturated long-chain acylcarnitinesz5). Peaks at m/e 102 and m/e 74, which are prominent in the spectrum of free carnitine’“, are also absent.
J. E. PETTEKSEN
6
Thin-1aJw chron~atograph,v of‘ carnitine esters of long-chain monoand dicarhox~Vic acids Kieselgel H plates and the chloroform-~methanol-ammonia-water solvent system were used. R, values obtained for palmitylcarnitine, tetradecanoylcarnitine, oleylcarnitine, linoleylcarnitine, and linolenylcarnitine were about 0.50. The R, value for hexadecanedioylcarnitine was 0.25-0.32. Carnitine had R, about o. I o. Thus, in this system a satisfactory separation of the carnitine esters of hexadecanedioic and of long-chain monocarboxylic fatty acids was obtained. Carnitii7e ac~ltratwj~rase actiritJ3 )2litll llexac~eeatie~iio~~l~.al~t~itineas mhstrate Fig. 2 shows the thin-layer chromatographic separation of reaction products after isotope exchange assays with hexadecanedioylcarnitine and palmitylcarnitine as substrates. When carnitine palmityltransferase prepared from calf liver and either palmitylcarnitine (Fig. 2a) or hexadecanedioylcarnitine (Fig. zb) were used as substrates, only one peak containing radioactivity was seen in either case. The peaks had
b
100
E 8
0
Fig. 2. Thin-layer chromatographic separation of reaction products from three carnitine acyltransferase isotope exchange assays. The incubation mixture (5 ml) contained: 50 mM TrissHCl buffer (pH 7.4) 5 mM GSH, 0.13 mM CoA, 0.5 mM t_-[Me-3HIcarnitine; and in addition in Expt a:o.z mM palmitylcarnitine and carnitine palmityltransferase (0.63 mg protein); in Expt b: 0.03 mM hexadecanedioylcarnitine and carnitine palmityltransferase (1.26 mg protein); and in Expt c: 0.03 mM hexadecanedioylcarnitine and total liver homogenate (1.67 mg wet wt of tissue). The incubation was run for IO min at 35 “C and stopped with HCI, and the incubation mixture was extracted with butanol. An aliquot
of the butanol
phase was subjected
to thin-layer
chromatography
(see legend to Fig. 4).
R, values of 0.50 and 0.25, respectively. and cochromatographed with synthetic palmitylcarnitine and hexadecanedioylcarnitine. Also when a total liver homogenate was used as enzyme source, and hexadecanedioylcarnitine as substrate, only one
r~~j~a~tiye peak was seen (RF 0.25, co~h~omato~r~phed with syI~tbe~ic hexadecaIled~o~lcar~itj~e). Thus, ~a~niti~e esters of endo~e~ous long-chain mollocarboxyli~ acids were presentaniy in ~nsi~ni~cant remounts, or totally jacking. furthermore, the s~ntbesized ~~exa~~e~anedio~~carniti~edid not ~o~ta~~ contaminations like ~~rn~ti~~ esterr of going-chain ~tono~a~boxy~ic acids in detectable a~~ou~~s.
Fig. 3. The enzymic exchange of the carnitine maiety of different aeyic~r~jtines with ~-[iw+~H]~~rn~t~[~e as a f~r~ct~on of acyi~rnitit~o concentrations. Carnitine ~lm~ty~trallsf~rase prepared according to Norum with the modi~catior~s of Farstad et n/J” (0.21 mg of protein) was used. The incubation mixture of I ml also contained 0.5 mM ~-~~~~-3H]car~iti~e, o.13 mM Co& 5 mM CSM, and 50 mM Tris-HCI buffer (pi-i 7.4). The reaction was run for 5 min at 35 “C. ~~rnity~~~rn~t~~~. ( 1: hexadecanedioylcarnitine, (e---e).
Fig. 3 shows the rate of exchange as a function of the concentration of the a~yl~arn~tiues, The maxima1 reaction rate was about 10 times as high with carnitine as substrate at all Co~~eI~t~tions as when hexadeca~edioy~carnitj~e An ~nhibit~oI1of the reaction rate can be seen with higher concentrations hexadecalledioylcamitirle and paImilyIcan?ititle. This substrate inhibition TABLE
substrate palmitylwas used. of both at higher
I
The reaction mixture (5 ml) contail~~d carnitinc palrnit~ltransf~r~lsc (0.79 my of protein), and ncytcarnitines as stated in the table. The incubation mixture also contained: 50 mM Tris-MCI buffer (pH 7.4). 5 mM GSH; 0.13 mM CaA, and 0.5 mM L-[Mc-3WJcarnitine. The reaction was rim for 6 min at 35 ‘C and stopped with I-ICI, and the incubation mixture was extracted with ~~II~I?~I. An aliquot of the butanof phase was assayed for radioactivity at once, and another aliyttot was tirst subjected to thin-layer chromatography for separation of hexadecanedioylcarnitine and palmitylcarnitine (see Fig. 4). _ _. .~_. _ ._l-““.l~l ~[~(~~~~u~f~~e ~c.~icff~t~ir~t~~.~fkwrrred (tpm j Sff~~lr~~f~ innmlesl __. ____,_.l_l,” ___. ~___~ _^..~~-_l_______l ._: - .-.-.“~-.~ Pdmii,dNesrrcleccr,lediuJI-II-l~.~~~~~~eennr~i~)~~lP~i//llif~.iiwrlrititie ~~rrI~ti~~ crrrffititw cmttilinc ,.” _,__._.,“_“” “.l.“._^“l”__l_____~_“” -_.-- . ---I_-~-“-__^~-~^-~_. ..
0
2200
0
200
205
2000 zoo
0
g 730
ZO$jlO
216
200
21 gro 9820
1.56 345
0 _-_.__
I180
250
2500
200 2200 _ I ____._.
0
0 -__~.-.“‘--“.
0
22605 .--
2.2 440 _” ._.,.
.
..I^-
._.-
0 _-_,-__
-....
.
_-i”, “. “__
-
a
J. E. PETTERSEN
acylcarnitine concentrations has previously been observed in experiments with palmitylcarnitine’. Table I shows the results of isotope exchange assays with a carnitine palmityltransferase preparation (see Materials and Methods) as enzyme source. When a constant amount of hexadecanedioyic~lrniti~e was used and increasing amounts of paimitylcarnitine were added, decreasing amounts of radioactively Iabelled hexadcIabeiled pa~n~~ty~carnititie caIledioylcarn~tii?e and increasin, u c‘amounts of radioactively were formed. When the amount of ~ll~it~lcarnjtine was kept constant and the amounts of hexadecanedioyic~lrnitiiie added were increased, a decreased formation of labelled palmitylcarnitine accompanied by an increased formation of labelled hexadecanedioylcarnitine was found. These findings may indicate that there is a competition between the two acylcarnitines for the same enzyme, and that palmitylcarnitine is the preferred substrate.
Both with hexadecanedioylcarnitine and paln~itylcarnitine as substrate, the activity in the exchange reaction (for experimental conditions, see Table II) was proportional to the amounts of liver homogenate added, at least up to 0.7 mg of protein. As we expected an increased enzyme activity in the treated animals, amounts of total liver homogenate corresponding to only about 0.2 mg of protein were used in the experiments shown in Tables II and 111. TABLE
11
ACTIVITY OF CARNITINE ACYLTRANSFERASE WITH HEXADECANEDIOYLCARNITINE AND PALMiTYLCARNITlNE AS SUBSTRATES IN LIVERS OF NORMAL, FASTED, AND STREPTOZOTOCIN-DIABETIC RATS The
of
hexadecanedioylcarnitine and palmitylcarnitine as is therefore given in arbitrary units (see Materials and Methods). The reaction mixture ii ml) contained 50 mM Tris-HCI buffer (pH 7.4). 5 mM GSH, o. I 3 mM CoA, 0.5 mM L-[Me-“Hlcarnitine, 50 111total liver homogenate. and 0.03 mM hexadecanedioylcarnitine or 0.2 mM palmitylcarnitine. The results are given as mean SD.** means statistically significant from tbc control group. P _ 0.01. Asterisks in parentheses signify statistical difTercnces from the fasted group. Grollp cp,,1 lo-3 PET l-pm /o-3 per rprtt per Suhsfrcltr activity
substrates
s:ifs
carnitine
assayed
acyltransferase
8s an exchange
with
reaction
and
too g rot _. Pdlmitylcarnitinc
~lrnitylcar~~itille P~im~t~lc~r~litiile Hexadccanedioylcarrlitine Hexadecanedioylcarnitine Hexadecalicdioyicarnitine -
.-
-” -
..
--..
Normal Fasted Diabetic Normal Fasted Diabetic -
5286
827s
/ 636
7x** I 1055 f397**f**i 530 .I 67 724 I 103** 953 : I74**(*)
fi wet li’t of’liwr
,171 2904 3725 II8
251 323
, II9 : 255x* 485**(*) 15
8. 35** / 70**
wtg protein
i 633 1 1065** : 3008**(**) 5.59 * 66 1019 , 160** $434 ! ;or **t*j
5576 11776 I6669
Table II shows the results of isotope exchange assays with hexadecanedioylcarnitine or palmitylcarnitine as substrate, and as enzyme source total liver homogenates from normal, fasted, and diabetic rats. The acyltransferase activity was significantly increased in the fasted animals, and the increase waseven more pr-onounced in the diabetic group. In fasting and diabetes the liver weight decreases relatively
HEXADECANEDIOIC ACLD METABOLISM TABLE 111 RELATIVE
ACTIVITIES
DLOYLCARNITINE NORMAL, FASTED,
OF CARNITINE
The values given are the means (IOO",,).The fasted and diabetic conditions, see Table Il.
Diabetic
Clofibrate-fed
HEXADECANEIN
LIVERS
OF
_ Acticit?
pw
Activity
per
of iitw
Activity
100 g roi
g wet wf
Palmitylcarnitine Hexadecanedioylcarnitine
IS8
248 2.13
ztt
Palmitylcarnitine Hexadecanedioylcarnitine
209 I80
318 274
299 257
Palmitylcarnitine Hcxadecanedioylcarnitine
353 346
274 265
282
_
Fasted
WITH
SUBSTRATES RATS
of the different groups calculated as per cent of the normal group groups are identical with the groups in Table II. For experimental
.%hsrmrr
Grmp
AC~LTRANSF~RAS~
AND PALMITYLCARNITINE AS DIABETIC, AND CLOFIBRATE-FED
‘37
per
iu,q proteiir
182
274
more than the body weight of the animals, and therefore the differences between the normal and the two other groups are least when the results are related to body weight. Table 111 shows that the relative increases above the normal group are of the same magnitude with hexadecanedioylcarnitine and palmitylcarnitine as substrate. Table III also contains results from another feeding experiment. In liver homogenates from rats fed clofibrate for one week there was a significantly increased transferase activity (P < 0.01). The increases were of the same magnitude both with hexadecanedioylcartlitine and palmityl~~rnitine as substrates, and they were about the same as in the diabetic rats when the results were related to mg protein (specific activity) or to g wet weight of liver. However, when the results were related to body weight, the increase was much larger in the clofibrate-fed rats. This is due to the large increase in liver weight in these animals.
Activation of hexadecanedioic acid Fig. 4 shows the thin-layer chromatographic separation of the butanolextractable products of the fatty acid activation reaction with (bottom) and without (top) hexadecanedioic acid added as substrate. From the top chromatogram it can be seen that a product with RF 0.50 was obtained when no substrate was added. This product cochromatographed with the carnitine esters of several long-chain monocarboxylic acids (palmitic, tetradecanoic. oleic, linoleic, and linolenic acid) in both of the chromatographic systems used. This product therefore most probably represents activated endogenous long-chain fatty acids trapped as acylcarnitines. The bottom cl~romatogran~ shows that when hexadecanedioic acid was added as substrate two products were obtained, one which cochromatographed with synthetic hexadecanedioylcarnitine (R, 0.25). and one representing activated endogenous fatty acids (RF 0.50). Jn parallel experiments (incubation mixtures as shown in legend to Fig. 4) the thin-layer chromatographic step was omitted. The butanol phases from the experiment without and with hexadecanedioic acid added as substrate contained 95 and 198 nmoles of acylcarnitines, respectively (formed per 6 min). In the experiment where hexadecanedioic acid was added, r/6 of the total amount of ac~lcarnitines was acti-
.I. E. PETTERSEN
Fig.
4. Thin-layer
ncrivation
chromatographis
reactions
where
separation
the activated
fatty
of the
butanol-extractable
acids have
chro~~~to~~n~
shows the reaction
products
when Ile~~Ide~l~e~lioic
chromatogram
shows the reaction
products
when no substrate
substrates).
The reaction
5 mM ATP,
4 mM
mixture
of carnitine
palmilyltransf~rasc,
(5 ml) contained:
t.-[,~~e-3H]car!litine,
sium hexadecanedioate.
5 mM GSH, {wjv)
0.85”,,
The amount
of total
incubation
was started
by the addition
of tissue. and the reaction
was stopped
by the addition
of 0.3 ml of cont.
formed
Lvcrc extracted
with 7.5 ml of butanol-saturalel! dissolved
in methrtool
added, carnilinc
with 5 ml of butanol.
ci~ronl~t~~~r~~phy on Kicselgel
ammonia-water
radioactivity
standards
cailedioylc3rilitinc.
was measured
were localized
by esposurc
Tri-Garb
to iodine
CoA,
of potas-
added before
phnsc wzs washed once The a~ylcarnitiws
H (Merck)
vapour.
vials. scintillation
liquid The
were
plates with solvent
(50: 30:8: 2.5, by vol.). The front was
in a Packard
o.25: (2) palmiryl;art~itine.
I mhl
of water nas
The butanol
(0.5 cm wide) of the silicic acid were scraped into counting
and
7.4)> 0.1 mM
about 0.5 mg protein
~.ns run for 6 min at 35 ‘C. The
and 4.5 ml
water and taken to dryness on a rotovapor.
before thin-layer
system chloroforrc-methal~ol--cont. Fractions
KCN,
Top
of tndogenous
(wet wt of tissue) was 4. I 5 mg. The
incubation
the acylrarnitines
buRer (pH
~lburnil~, o. I 3 M KCI, and either no or
HCI,
acid
Bottom
acid was added its substrate.
MgC12, 5 mM
liver homogenate
of fatty
as acylcarnitines.
was added (activation
0.1 M Tris-HCI 5 mM
products
been trapped
scintillation
~LUI
14 cm.
solution counter.
RF values ~vcrc: ii)
\VX Acyi-
hcude
0.50.
vated hexadecanedioic acid (33 nmoles), and 5/h (165 nmoles) wcrc activated endogenous fatty acids. Thus, of the me:tsured increase of 103 nmoles of activated acid with hexadecanedioic acid added as substrate, only 33 nmolcs (or about 1i3) was activated flexadecanedioic acid, and the rest was activated endogenous fatty acids. This corresponds to an activation of hexadecanedioic acid of about / .J /moles/g wet weight of liver per min, i.e. about t/lo ofthe activation capacity for palmitic acid”.““. Isolated m~tochon~~ria were used as enzyme source in a few ex~~r~l?l~nts. In some of these Triton X-loo was added (final concentration o.o~:‘;,), otherwise the incubation conditions were as described in legend to Fig. 4. Essentially the same amounts of activated hexadecanedioic acid was found with Triton X-100 added about 95(jo) as without this detergent present (100”:). This indicates that the activation of long-chain dicarboxylic acids takes place outside the inner mito~hondri~~1 compartment, as is also the case with the long-chain monocarboxylic acids”,23-3”.
HEXADECANEDlOlC TABLE
ACID
METABOLISM
II
IV
ACYLATION
OF
M~TOCHOND~IAL
COENZYME
A BY HEXAD~C~ANEDIOYLCA~NI-
TINE OR PALMITYL~ARNITINE Mitochondria (7 mg of protein) were preincubated for 3 min with 15 mM N-tris(hydroxymethyl)methyl-z-aminoethanesulfonic acid buffer (pH 7.4). about 0.1 M KCI, and 0.2 mM z,4-dinitrophenol in a total volume of 3.5 ml. Then 5 /4moles KCN and acylcarnitine were added. The temperature was 15 C. The mitochondria were precipitated by I M HCIO, (3.5 ml) after 3 min. .._ .._ _~ _ _.. ..-. Additior7 ijlid~~Sl
_____-. None Palmitylcarnitine, 0.2 Hexadecanedioylcarnitine, Hexadecanedioylcarnitine,
CoA puesmt in die tiClO,i~7s~i~~bt~~ prrcipifnte i fl~?~uies~,~~~ protein) . _______.. .__..__
0.2 1.0
0.39 1.67 0.62 0.84
Table TV shows the amounts of ~ClO~-insoluble CoA (long-chajn acyl-CoA) formed when mitochondria were incubated with hexadecanedioylcari~itine or paimitylcarnitine. Nearly 6 times as much long-chain acyl-CoA was obtained with palmitylcarnitine as substrate as with the same amounts of hexadecanedioylcarnitine. When larger amounts of hexadecanedioylcarnitine were used, more long-chain acylCoA was formed.
Fig. 5 shows the O2 uptake of rat liver mitochondria uncoupled with dinitrophenol, and with malate added to stimulate the tricarboxylic acid cycle. A but significant, increase in O1 uptake was seen when hexadecanedioylcarnitine added (Trace a). However, the 0, uptake decreased rapidly with time. When
2,4low, was pal-
Fig. 5. Oxygraph traces showing the 0, uptake of rat liver mitochondria with hexadecanedioylcarnitine (H) and/or palmitylcarnitine (P) as substrates. The incubation medium (2.5 ml) contained 0.1 M KCI, 6 mM MgC12. IO mM N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid buffer (pH 7.4), 0.5% (w/v) bovine serum albumin, 2 mM malate, 0.08 mM z,q-dinitrophenol, and mitochondria (3.5 mg of protein). The acylcarnitines were added in amounts corresponding to 0.04 mM of each. The temperature was 25 “C.
I2
J. E. Pt.TTEIISEN
mitylcarnitine then was added the 0, uptake reached immediately the same level as with palmitylcarnitine added as the sole substrate. Thus, no inhibition of the oxidation of palmitylcarnitine by hexadecanedioylcarnitine was observed. Neither did any decrease in 0, uptake occur when hexadecanedioylcarnitine was added to mitochondria oxidizing palmitylcarnitine (Trace b). With rat heart mitochondria similar results were obtained. When malonate was added, the 0, uptake was lower, but the results were otherwise similar to those in Fig. 5. DISCUSSION
The present results show that hexadecanedioic acid can be activated, but at a lower rate than palmitic acid. The activation mainly occurs outside the inner mitochondrial compartment, as is also the case with monocarboxylic long-chain fatty acids ‘7.24.30. We have shown that when dicarboxylic acids are added to the incubation medium, there is an increased activation of endogenous monocarboxylic acids in addition to the activation of dicarboxylic acids. This may be due to a release of endogenous fatty acids, bound for instance to proteins, in exchange of the added dicarboxylic acids. Therefore specific methods are necessary in studies on dicarboxylic acid activation to ascertain that the measured increase of activated acids really represents activated dicarboxylic acids. The carnitine acyltransferases in the inner mitochondrial membrane3’ catalyze the reversible transfer of acyl groups from acyl-CoA to t_-carnitine and thereby mediate the transport of activated fatty acids through that membrane32.33. Our results show that hexadecanedioic acid can use this transport mechanism. Hexadecanedioylcarnitine is substrate for the enzyme in the isotope exchange assay, and mitochondrial CoA may be acylated by hexadecanedioylcarnitine. The isotope exchange assays with hexadecanedioylcarnitine and palmitylcarnitine added together as substrates indicated that the two acylcarnitines used the same enzyme. Solberg et ~1.“~ have shown that clofibrate feeding increases significantly the activities of all carnitine acyltransferases (i.e. short-chain (EC 2.3. I .7), medium-chain, and long-chain (EC 2.3. I .-) carnitine acyltransferase) in rat liver, but not to the same extent. In the present studies we found that in fasted, diabetic, and clofbrate-fed animals the carnitine acyltransferase activity showed a parallel increase with hexadecanedioylcarnitine and with palmitylcarnitine as substrate (Table III). This further supports our suggestion that the same enzyme, pi:. hexadecanoyl-Co/i: carnitine O-hexadecanoyltransferase (EC 2.3. I .-), is responsible for the transport both of activated long-chain monocarboxylic and dicarboxylic acids across the inner mitochondrial membrane. An increased activity of this enzyme has been described previously in fat-fed”s.36, alloxan-diabetic3’, fasted3”,36, and clofibrate-fed’4.34 rats. An increased urinary excretion of short-chain dicarboxylic acids may be caused by increased amounts of precursors, 1li7. long-chain dicarboxylic acids (see Introduction), and/or by an increased activity of the enzymes participating in the degradation of long-chain dicarbocylic acids. The well-known increased liberation of fatty acids in ketosis, combined with the increased w-oxidation capacity which has been reported by Wada c’t al.‘.“, may yield increased amounts of long-chain dicarboxylic acids. Moreover, we have shown that the carnitine acyltransferase activity with hexadecanedioylcarnitine as substrate is increased in this metabolic condition. Thus, both factors,
HEXADECANEDIOlC
ACID
METABOLISM
‘3
viz. a formation of increased amounts of long-chain dicarboxylic acids and an augmented degradation of these acids, may be of importance for the urinary excretion of short-chain dicarboxylic acids in ketosis. We have shown that the short-chain dicarboxylic acids may be formed from long-chain monocarboxylic acids by a primary o-oxidation, followed by /i-oxidations of the long-chain dicarboxylic acids thus formed4. III viva experiments performed by various other groups3.37m39 also indicate that long-chain dicarboxylic acids are degraded by the P-oxidation mechanism. Following the ingestion of acids with oddnumbered carbon-chains, odd-numbered short-chain dicarboxylic acids are excreted in the urine; and following the ingestion of acids with an even-numbered carbonchain, even-numbered acids are excreted. However, the low 0, uptake of mitochondria with hexadecanedioylcarnitine as substrate necessitates further studies to clarify if other chain-shortening processes than ordinary fi-oxidation are of importance in the degradation of long-chain dicarboxylic acids. ACKNOWLEDGEMENTS
The author is indebted to Professor .J. Bremer, Dr M. Aas, and Dr S. Skrede for helpful suggestions and discussions. The technical assistance of Mrs Kari Frogner is gratefully acknowledged. Through the kind cooperation of the Chemistry Department (Mr K. Undheim and Mr G. Hvistendahl), University of Oslo, we had access to the high resolution mass spectrometer. REFERENCES I Pettersen,
J. E., Jellum, E. and Eldjarn, L. (1971) &and. J. C/in. Lab. Invest. 2.7, Suppl. I 18. 69 J. E., Jellum, E. and Eldjarn, L. (1972) C/in. C/rim. Acfa 38, I 7-24 3 Verkade, P. E.. Van der Lee, J. and Van der Alphen, A. J. S. (1937) Hoppe-.Se_r/ers Z. Phj,sio/. Chrm. 250, 47756 4 PetterSen. J. E. (1972) Clin. Chim. Acta, 41, 2-j-237 5 Wada. F.. Hirata, K., Shibata, H., Higashi, K. and Sakamoto, Y. (1967) J. Eiochem. Tokyo 62, 2 Pettersen,
134-136 6 Wada, F. (1966) J. Jap. Biochern. Sot. 38, 818-825 7 Stokke. 0. and Bremer, J. (1970) Biochim. Bioph_vs. Acta 218, 552-554 8 Brcmer. J. (1968) Biochem Prep. 12, 69973 9 Norum. K. R. (1964) Biochitn. Biophys. Acra 89, 95-108 IO Farstad, M., Bremer, J. and Norum, K. R. (1967) Biochim. Biaphys. Acra 132. 492-502 I I Christophersen, B. 0. and Bremer, J. (I 972) Biochim. Biophys. Acta 260, 5 15-526 12 Junod, A., Lambert, A. E., Orci, L., Pictet, R., Gonet, A. E. and Renold, A. E. (1967) Proc. Sac. Esp. Biol. Med. I 26, 201~205 I 3 Junod. A.. Lambert, A. E., Stauffacher, W. and Renold. A. E. (1969) J. C/in. rmest. 48, 2129-2139 14 Daae, L. N. W. and Aas, M. Atherusckrusis, in the press 15 Best. M. M. and Duncan, C. H. (1964) J. Lab. C/in. Med. 64, 634-642 16 Myers, D. K. and Slater, E. C. (1957) Biochrm. J. 67, 558-572 I 7 Aas, M. (1971) Biuchirn. BiophJx Acta 23 I, 32-47 18 Chappel, J. B. (1964) Bioc/rern. J. 90, 225-237 19 Lowry, 0. H., Rosebrough, N. J., Farr, A. 1.. and Randall, R. J. (195 I) J. Biol. Chem 193, 26520 2I 22 23
275 Bremer, Bremer, Aas. M. Bremer.
J. (1962) J. Biol. Chew. 237, 2228-2231 J. and Norum, K. R. (I 967) J. Biol. Chern. 242, I 74991755 and Bremer, J. (1968) Biochim. Biophvs. Acta 164, I 57-166 J. and Wojtczak, A. B. (1972) Biochim. Biophys. Acta 280, 515-530
J. E. PETTERSEN
is
S. and Bremer, J. (1970) Eur. J. Biuclre~~r. 14, 465-472 Hvistendahl. G., Undheim. K. and Bremer. J. (1970) Org. .&cfr~ss &wcf~~~u. 3. x433-1438 Solberg, H. E. and Eremer. J. (1970) Biocftiuz. Biopk_vs. Acfrr 222, 372-380 Holmes. J. L. and Jean, T. St. (1970) Oyr. Moss Specfrrtm. 3. 1y+r~r8 Hvistendahl, G. and Undheim, K. (1970) Org. Muss S~CITO~~I.3, 82i...824 Van Tot, A. and Hiilsmann, W. C. (1969) Biochi~u. BioplrJx. Acts I&, 342-353 Norum. K. R., Farstad, M. and Bremer, J. (1966) Biochr,r/. Bioph)~s. RPS. Comm~,/r. 24. 797-804 Norum, K. R. and Bremer. J. (1967) J. Bio/. Clwur. 242, 407-41 I l3remer. J. (196.3) J. Biul. Clxw. 238. 2774-2779 Fritz, 1. B. and Yue. K. T. N. (1963) J. Lipici’Res. 4, 279-288 Solberg. H. E., Aas, M. and Daae, L. N. W. ( 1972) Biocltiw. Biopfr.~,s.Actri z.80, 434-439 Norum, K. R. (1965) Biacizin~. Biuph_u. Acfrr 98. 652-654 Aas, M. and Daae, L. N. W. (1971) Biochim. Bioph,tx Artrr 239, zo%rr6 Bergstrclm, S., Borgstr@m, B.. Tryding, N. and Westnn, G. (1954) Biorheitr. j. 58, 604-608 Tryding. N. and Westetr, G. (1956) A&I Clwt~. Scwfd. IO, 1234-1242 Yamakawa, T. (1950) J. Biochr~u. 37, 343-353
24 Skrede, 25
26 27 28 29
30 31 32 33 34 35 36 37 38 39
Scientific
Publishing
306 (1973)
Company,
r-14
Amsterdam
- Printed
in The Netherlands
RBA 56224
IN VITRO STUDIES ACID
JON
ON THE METABOLISM
AND ITS MONO-L-CARNITINE:
ELLTNG
PETTERSEN
fnstitr~ie c/f Chid
(Received
OF HEXADECANEDiOlC
ESTER
October
Bi(}cjzef~tisfr~, U~i~le~sit~~ vf Oslo, Rjks~los~ifffIet,
23rd,
Oslo I [iVor~v~_v)
t97z)
SUMMARY
T. A method for the synthesis of the mono-~-carnitine ester of h~xadecanedioic acid (hexadecanedioylcarilitine~ is described. A thin-fayer chromatography system for the separation of long-chain monocarboxylic and long-chain dicarboxylic acylcarnitines was developed. 2. Hexadecanedioic acid can be activated by rat liver mitochondria in the presence of CoA, ATP, and Mg2+. Since Triton X- 100 does not increase the activation capacity, the enzyme(s) activating long-chain dicarboxylic acids seems to be localized outside the inner mitochondrial compartment. 3. HexadecanedioyIcarnitine is substrate for a solubilized preparation of hexadecanoyl-CoA:carnitine 0-hexadecanoyltransferase (EC 2.3. I .-), but the reaction rate is only about r/to of that with palmitylcarnitine. Accordingly, mitochondriat CoA can be acylated by hexadecanoylcarnitine, but much slower than by palmitylcarnitine. 4. Hexadecanedioylcarnitine competes with palmitylcarnitine in the reaction catalyzed by the enzyme. 5. In fasted, ketotic streptozotocin-diabetic, and clofibrate-fed rats the carnitine acyItransferase activity was significantly increased. The relative increases were about the same whether hexadecanedioylcarnitine or palmitylcarnitine were used as substrates. 6. The observations under points 4 and 5 indicate that the same enzyme (hexadecanoyl-CoA:carnitine 0-hexadecanoyltransferase, EC 2.3. I .-) may transport activated hexadecanedioic as well as palmitic acid across the inner mitochondrial membrane. 7. The 0, uptake of rat heart and liver mitochondria with hexadecanedioyicarnitine as substrate was very low and transitory. Hexadecanedioylcarnitine did not inhibit the oxidation of palmitylcarnitine. 8. The present in vitro studies demonstrate a possible mechanism for the degradation of long-chain dicarboxylic acids, viz. activation by CoA and transportation into the inner mitochondrial compartment as carnitine esters followed by /Ioxidation.
J. E. PETTERSEN
Recently we have found that ketotic patients excrete considerable amounts of t?-hexanedioic and n-octanedioic acid in the urine’.2. Peroral intake of hexadecanedioic acid in healthy humans induced an increased urinary excretion of hexanedioic and octanedioic acid’. J/Z rig experiments with radioactively labelled hexadecanoic acid given to ketotic streptozotocin-diabetic rats indicated that the two short-chain dicarboxylic acids may bc formed from long-chain mono~arboxyl~c acids by an initial w-oxidation followed by /I-oxidations of the fang-chain dicarboxylic acids thus formed4. In ketosis there is an increased liberation of fatty acids from the fat depots and, moreover, an increased w-oxidation capacity has been demonstrated in liver microsomes from ketotic rats’.‘. These two findings may alone explain the increased urinary excretion of hexanedioic and octanedioi~ acid in ketosis. However, the exact pathways involved in the short-chain dicarboxylic acid formation is not known, and this Ied us to investigate the metabolism of long-chain dicarboxyhc acids. The present paper describes the synthesis of the nlo~lo-L-~arllitiIle ester of hexadecanedioi~ acid, and it? t?irfo studies on the ~etabol~sln of this carnitine ester and of hexadecanedioic acid itself. MATERIALS
AND
METHODS
I_-Carnitine was a gift from Otsuka Pharmaceutical Factory, Osaka, Japan. L-[~~,-‘H]Cnrnitine and palmityl-L-carnitine (palmitylcarnitine) were kindly supplied by Professor .I. Bren~er7.8. In the activation experiments 0.08 icl r.-[~fe-3H]carnitir~e was used (~OrrespoIlding to I 22 ’ IO” cpni~~~l~ole under the conditions used), and in the was used (corresponding to isotope exchange assays 0.0 1 M r_-[Me-“Hlcarnitine 935 10~ cpm,/jrmole). Hexadecanoyl-CoA:carnitine O-hexadecanoyltransferase (carnitine palmityltransferase. EC 2.3. I .-) was prepared according to Norum’ with the modifications described by Farstad er LI~.‘O.Oleylcarnitine, linoleyl-carnitine, and l~nole~~yl~ar~~itine were gifts from Idr B. 0. Christophersen”. ~exade~anedioi~ acid (more than 99 li;, pure when controlled by combined gas--liquid chromatographymass spectrometry) was obtained from Fiuka AG. Buchs, Switzerland, and CoA was delivered by C. F. Boehringer und Siihne GmBH, ~ailnt~eirn, Germany. Streptozototin (a gift from the Upjohn Company, Michigan, BISA) was dissolved jrn~lled~~itely before use in 0.9 ofi saline acidified to pH 4.5 with citric acid”““. Clofibrate (Atromidin) was a gift from ICI Ltd, Cheshire, England. Other chetnicals were commercial products of high purity.
Male rats of Wistar-M&l strain weighing 220-280 g were placed in metabolic cages and adapted for one week on the stock diet. The normal rats (5 animals) had free access to water and the stock diet. The fasted rats (5 animals) were deprived of food for 48 h prior to death. Diabetes meilitus was induced in IO rats as described earlier” by the intravenous injection of streptozotocin’2.‘” ( IOOmg/kg body weight). 5 animals with glucosuria and ketonuria were selected for further studies and killed 48 h after the in_jection. Blood glucose value for this group was 312 i_ 55 (mean .& S.D.), range 23738X mgj IOO ml. The amount of ketone bodies in blood was for the fasted rats I .2 k 0.2.
HEXADECANEDIOIC
ACID
METABOLISM
3
range 0.8-1.3 mg/ IOO ml, and for the diabetic rats, 3.8 &- 2.4, range I .9-7.9 mg/ IOO ml. 5 animals were fed 0.3 % clofibrate in the stock diet for one week. These animals have been described elsewhere14. There were no significant changes in body weight or liver protein concentration, whereas an increase in mean liver weight as first described by Best and Duncan” was observed. Preparation qf’ homogenates and mitochondric Rat liver and heart mitochondria were isolated in 0.25 M sucrose containing I mM EDTA according to Myers and Slater16; they were washed twice and resuspended in o. I 5 tvl KC1 in concentrations correspotlding to I g of fresh tissue per ml. Total liver homogenates contained I g of liver per 60 ml final solution. In the studies on fatty acid activation 5 . IO-~ M ATP was added to counteract enzyme inactivation (ref. 17). Preparation qf the mono-t-cnmitine ester qf hexadecanedioic acid (hexadecanerlioqIcarnitke 1 A modification of the method described by Bremer’ for the synthesis of longchain acylcarnitines was used. 15 mmoles of hexadecanedioic acid and 60 mmoles of oxalylchloride were mixed and kept at 55 “C for 2 h yielding hexadecanedioylchloride. Moisture was kept out by Molecular Sieve 3A pellets (British Drug Houses Ltd, Poole, England). The excess of unreacted oxalylchloride was removed under reduced pressure with the water pump at 55 “C. 2.5 mmoles of L-carnitine was dissolved in 4 ml of trifluoroacetic acid. This mixture was mixed thoroughly with the hexadecanedioylchloride. A large excess of the acid chloride was used to obtain the mono- and not the dical.nitine ester. The reaction was carried out on a water bath (40 “C) for 8 h under continuous stirring. Then light petroleum (b.p. 40-60 “C), ice chips and water were added. To assure that all acid chlorides were hydrolyzed, the mixture was made slightly alkaline (pH 8) by the addition of NaHCO,. The mixture was acidified to pH I with HCI, and was repeatediy extracted with diethyl ether to remove the free dicarboxylic acid.The hexadecanedioylcarnitine wasextracted with butanol. The butanol phase was washed with water to remove any free carnitine present, and was then taken to dryness on a rotovapor. By precipitation with diethyi ether from methanol the hexadecaj?edioylcarnitine was obtained as a white a~norphous material.
Thin-layer chromatography was performed at room temperature with Kieselgel H (Merck) plates. Solvent systems were either chloroform-methanol-cont. ammoniawater (50:30:8:2.5, by vol.) or hexane--diethyl ether-acetic acid (60:40: I, by vol.). The spots were made visible with iodine vapour. Combined gas-liquid chronlato~raph)/-nlass spectrometry was performed in an ~nstrl~nlent consisting of a Varian 1400 gas chromatograph (column material IO:! OV- I 7 on Gas Chrom Q (80-too mesh) or 8 %, BDS on Chromosorb W (80-r oo mesh) : temperature programmed from 80-300 or 80-180 “C, respectively, at a rate of 8 “C per min; ion current detector; helium as carrier gas with a flow of 30 ml/min) combined with a Varian CH 7 mass spectrometer (ionisation energy 70 eV>. The hexadecanedioic acid was methylated with diazomethane liberated from N-nitrosomethylurea. Mass spectrometric analyses were also undertaken in an AEI double focusing
4
J. E. PETTERSEN
instrument, Model MS 902 (source temperature 220 C, 70 eV ionizing energy, IOO ;rA ionisation current). The samples were introduced by the heated direct insertion probe. Perfluorotributylamine was used as internal standard. Radioactivity was measured in a Packard Tri-Carb liquid scintillation counter Model 3310 with a counting efciency ofabout 25 “; for “H. The scintillation solution consisted of 5 g PPO, 0.05 g ~iin~ethyl-POPOP, 385 ml xylol, 385 ml dioxanc, 230 ml ethanol, and 80 g llaphthalene. Mito~hondrial 0, uptake was measured polarograp~~i~aily with a Clark oxygen electrode, essentially as described by Chappel18. Protein was measured by the method of Lowry et al.‘“.
The CoA-dependent incorporation of “H-labelled free carnitine into ncylwas assayed by an isotope exchange tarnitines catalyzed by carnitine acyltransferase2’ assay as described by Norum9. Because of the complex kinetics of the exchange reaction” the activity was expressed in arbitrary units (cpm). Acyl-CoA synthetase activity was measured by trapping the acyl-CoA formed reaction products were subjected as acylcarnitines ‘o-‘7.22 . The butanol-extractable to thin-layer chromatography for the separation of hexadecanedioylcarnitine and acylcarnitines formed from monocarboxylic endogenous fatty acids. In a few experiments Triton X-loo was added to the incubation medium to determine whether the hexadccanedioic acid was activated outside and/or inside the inner mitochondrial membrane22. The acylation of m~tochondrial CoA was determined by Illeasuri~~g the amount of HClO,-insoluble CoA formed when mitochondria were incubated with acylcarnitines. The mitochondria were preincubated with a,.+dinitrophenol to remove preformed a.cyl-CoA before the addition of cyanide and acylcarnitines. The incubation was stopped with HCIO,, and the precipitate was washed twice with one volume of o. I M HCIO,. Long-chain acyl-CoA present in the precipitate was hydrolyzed with I M KOH23, and the CoA liberated was assayed by its ability to catalyze the incorporation of [~~e-3H~carnitin~ into ~lmityl~arnitine by carnitine palmityltra~~s~ ferase”. RESULTS
By thin-layer chromatography with the chloroform-methanol-ammonia-water system the preparation of l~exade~~lned;oyl~arrlitine showed only one spot when made visible with iodine vapour (R, value of 0.25). A sample from a synthesis where only 2 equivalents of acid (and I equiv of carnitine) had been used, showed a major spot with R,. 0.25, and in addition a small spot with R, 0.20. This spot probably represents the dicarnitine ester of hexadecanedioic acid. On thin-layer chromatograms v,ith hexaneediethyl ether-acetic acid as solvent system only one spot with R, o was seen; thus no free fatty acids were present. Kjeldahl analysis of the preparation of hexadecanedioylcarnitine showed a nitrogen content of 3.10 y:, (theoretical value 3.00 T#:).
HEXADECANEDIOIC
ACID
METABOLlSM
5
Mass spectra of various acylcarnjtines have been described by others’*,“s.26. Fig. ra shows the mass spectrum of hexad~canedioylcarnitine (as hydrochIoride). The major pyrolytic reaction is elimination of the acid caused by a-proton activation by the quarternary nitrogen, leaving an unsaturated y-lactone (m/e 84) after the liberation of trimethylaminez5. The whole series of peaks corresponding to the fragmentation of hexadecanedioic acid (Fig. I b) can be seen. The base peak at m/e 98 is due to the fragment C,H,,CO ” . The peaks at m/e 269 and nlje 268 seen in the spectrum of hexadecanedioglcarnitine are also present in the spectrum of free hexadecanedioic
120
140
160
180
200
220
240
260
280
300
mle
‘:‘ljslb; 120
140
160
(
,I
180
200
,!1,
220
240
260
280
3;O
m/e Fig. I. Mass spectra of hexadecanedioylcarniiine relative intensity less than z 7.; have been omitted.
(a) and
hexadecanedioic
acid
(b).
Peaks
with
acid, but with an intensity of only 0.6 and 1.6%~ of the base peak, respectively. The increase in the heights of the peaks at m/e 84 and m/e 55 in the mass spectrum of hexadecanedioylcarnitine (compared to that of hexadecanedioic acid) are due to the carnitine moiety2”. The y-lactone ring fragment with I?r/e 84 yields a peak at /?r/e 55 by loss of CHO. Peaks at nzje 59, m/e $3, m/e 42, and m/e 32 are due to trimethylamine25.28. Peaks at mje 36 and m/e 38 are due to the Cl- evaporating as HCI. The molecule ion (which is seen in the spectra of unsaturated long-chain acyl~arnitines”) is not seen in the hexadecanedioy~carnitine spectrum, neither are a peak at M-59 (due to loss of trimethylami~~e1i~25) or a peak at nz,/e 144 (corresponding to a McLafferty rearrangement fragment seen in the mass spectra of saturated long-chain acylcarnitinesz5). Peaks at m/e 102 and m/e 74, which are prominent in the spectrum of free carnitine’“, are also absent.
J. E. PETTEKSEN
6
Thin-1aJw chron~atograph,v of‘ carnitine esters of long-chain monoand dicarhox~Vic acids Kieselgel H plates and the chloroform-~methanol-ammonia-water solvent system were used. R, values obtained for palmitylcarnitine, tetradecanoylcarnitine, oleylcarnitine, linoleylcarnitine, and linolenylcarnitine were about 0.50. The R, value for hexadecanedioylcarnitine was 0.25-0.32. Carnitine had R, about o. I o. Thus, in this system a satisfactory separation of the carnitine esters of hexadecanedioic and of long-chain monocarboxylic fatty acids was obtained. Carnitii7e ac~ltratwj~rase actiritJ3 )2litll llexac~eeatie~iio~~l~.al~t~itineas mhstrate Fig. 2 shows the thin-layer chromatographic separation of reaction products after isotope exchange assays with hexadecanedioylcarnitine and palmitylcarnitine as substrates. When carnitine palmityltransferase prepared from calf liver and either palmitylcarnitine (Fig. 2a) or hexadecanedioylcarnitine (Fig. zb) were used as substrates, only one peak containing radioactivity was seen in either case. The peaks had
b
100
E 8
0
Fig. 2. Thin-layer chromatographic separation of reaction products from three carnitine acyltransferase isotope exchange assays. The incubation mixture (5 ml) contained: 50 mM TrissHCl buffer (pH 7.4) 5 mM GSH, 0.13 mM CoA, 0.5 mM t_-[Me-3HIcarnitine; and in addition in Expt a:o.z mM palmitylcarnitine and carnitine palmityltransferase (0.63 mg protein); in Expt b: 0.03 mM hexadecanedioylcarnitine and carnitine palmityltransferase (1.26 mg protein); and in Expt c: 0.03 mM hexadecanedioylcarnitine and total liver homogenate (1.67 mg wet wt of tissue). The incubation was run for IO min at 35 “C and stopped with HCI, and the incubation mixture was extracted with butanol. An aliquot
of the butanol
phase was subjected
to thin-layer
chromatography
(see legend to Fig. 4).
R, values of 0.50 and 0.25, respectively. and cochromatographed with synthetic palmitylcarnitine and hexadecanedioylcarnitine. Also when a total liver homogenate was used as enzyme source, and hexadecanedioylcarnitine as substrate, only one
r~~j~a~tiye peak was seen (RF 0.25, co~h~omato~r~phed with syI~tbe~ic hexadecaIled~o~lcar~itj~e). Thus, ~a~niti~e esters of endo~e~ous long-chain mollocarboxyli~ acids were presentaniy in ~nsi~ni~cant remounts, or totally jacking. furthermore, the s~ntbesized ~~exa~~e~anedio~~carniti~edid not ~o~ta~~ contaminations like ~~rn~ti~~ esterr of going-chain ~tono~a~boxy~ic acids in detectable a~~ou~~s.
Fig. 3. The enzymic exchange of the carnitine maiety of different aeyic~r~jtines with ~-[iw+~H]~~rn~t~[~e as a f~r~ct~on of acyi~rnitit~o concentrations. Carnitine ~lm~ty~trallsf~rase prepared according to Norum with the modi~catior~s of Farstad et n/J” (0.21 mg of protein) was used. The incubation mixture of I ml also contained 0.5 mM ~-~~~~-3H]car~iti~e, o.13 mM Co& 5 mM CSM, and 50 mM Tris-HCI buffer (pi-i 7.4). The reaction was run for 5 min at 35 “C. ~~rnity~~~rn~t~~~. ( 1: hexadecanedioylcarnitine, (e---e).
Fig. 3 shows the rate of exchange as a function of the concentration of the a~yl~arn~tiues, The maxima1 reaction rate was about 10 times as high with carnitine as substrate at all Co~~eI~t~tions as when hexadeca~edioy~carnitj~e An ~nhibit~oI1of the reaction rate can be seen with higher concentrations hexadecalledioylcamitirle and paImilyIcan?ititle. This substrate inhibition TABLE
substrate palmitylwas used. of both at higher
I
The reaction mixture (5 ml) contail~~d carnitinc palrnit~ltransf~r~lsc (0.79 my of protein), and ncytcarnitines as stated in the table. The incubation mixture also contained: 50 mM Tris-MCI buffer (pH 7.4). 5 mM GSH; 0.13 mM CaA, and 0.5 mM L-[Mc-3WJcarnitine. The reaction was rim for 6 min at 35 ‘C and stopped with I-ICI, and the incubation mixture was extracted with ~~II~I?~I. An aliquot of the butanof phase was assayed for radioactivity at once, and another aliyttot was tirst subjected to thin-layer chromatography for separation of hexadecanedioylcarnitine and palmitylcarnitine (see Fig. 4). _ _. .~_. _ ._l-““.l~l ~[~(~~~~u~f~~e ~c.~icff~t~ir~t~~.~fkwrrred (tpm j Sff~~lr~~f~ innmlesl __. ____,_.l_l,” ___. ~___~ _^..~~-_l_______l ._: - .-.-.“~-.~ Pdmii,dNesrrcleccr,lediuJI-II-l~.~~~~~~eennr~i~)~~lP~i//llif~.iiwrlrititie ~~rrI~ti~~ crrrffititw cmttilinc ,.” _,__._.,“_“” “.l.“._^“l”__l_____~_“” -_.-- . ---I_-~-“-__^~-~^-~_. ..
0
2200
0
200
205
2000 zoo
0
g 730
ZO$jlO
216
200
21 gro 9820
1.56 345
0 _-_.__
I180
250
2500
200 2200 _ I ____._.
0
0 -__~.-.“‘--“.
0
22605 .--
2.2 440 _” ._.,.
.
..I^-
._.-
0 _-_,-__
-....
.
_-i”, “. “__
-
a
J. E. PETTERSEN
acylcarnitine concentrations has previously been observed in experiments with palmitylcarnitine’. Table I shows the results of isotope exchange assays with a carnitine palmityltransferase preparation (see Materials and Methods) as enzyme source. When a constant amount of hexadecanedioyic~lrniti~e was used and increasing amounts of paimitylcarnitine were added, decreasing amounts of radioactively Iabelled hexadcIabeiled pa~n~~ty~carnititie caIledioylcarn~tii?e and increasin, u c‘amounts of radioactively were formed. When the amount of ~ll~it~lcarnjtine was kept constant and the amounts of hexadecanedioyic~lrnitiiie added were increased, a decreased formation of labelled palmitylcarnitine accompanied by an increased formation of labelled hexadecanedioylcarnitine was found. These findings may indicate that there is a competition between the two acylcarnitines for the same enzyme, and that palmitylcarnitine is the preferred substrate.
Both with hexadecanedioylcarnitine and paln~itylcarnitine as substrate, the activity in the exchange reaction (for experimental conditions, see Table II) was proportional to the amounts of liver homogenate added, at least up to 0.7 mg of protein. As we expected an increased enzyme activity in the treated animals, amounts of total liver homogenate corresponding to only about 0.2 mg of protein were used in the experiments shown in Tables II and 111. TABLE
11
ACTIVITY OF CARNITINE ACYLTRANSFERASE WITH HEXADECANEDIOYLCARNITINE AND PALMiTYLCARNITlNE AS SUBSTRATES IN LIVERS OF NORMAL, FASTED, AND STREPTOZOTOCIN-DIABETIC RATS The
of
hexadecanedioylcarnitine and palmitylcarnitine as is therefore given in arbitrary units (see Materials and Methods). The reaction mixture ii ml) contained 50 mM Tris-HCI buffer (pH 7.4). 5 mM GSH, o. I 3 mM CoA, 0.5 mM L-[Me-“Hlcarnitine, 50 111total liver homogenate. and 0.03 mM hexadecanedioylcarnitine or 0.2 mM palmitylcarnitine. The results are given as mean SD.** means statistically significant from tbc control group. P _ 0.01. Asterisks in parentheses signify statistical difTercnces from the fasted group. Grollp cp,,1 lo-3 PET l-pm /o-3 per rprtt per Suhsfrcltr activity
substrates
s:ifs
carnitine
assayed
acyltransferase
8s an exchange
with
reaction
and
too g rot _. Pdlmitylcarnitinc
~lrnitylcar~~itille P~im~t~lc~r~litiile Hexadccanedioylcarrlitine Hexadecanedioylcarnitine Hexadecalicdioyicarnitine -
.-
-” -
..
--..
Normal Fasted Diabetic Normal Fasted Diabetic -
5286
827s
/ 636
7x** I 1055 f397**f**i 530 .I 67 724 I 103** 953 : I74**(*)
fi wet li’t of’liwr
,171 2904 3725 II8
251 323
, II9 : 255x* 485**(*) 15
8. 35** / 70**
wtg protein
i 633 1 1065** : 3008**(**) 5.59 * 66 1019 , 160** $434 ! ;or **t*j
5576 11776 I6669
Table II shows the results of isotope exchange assays with hexadecanedioylcarnitine or palmitylcarnitine as substrate, and as enzyme source total liver homogenates from normal, fasted, and diabetic rats. The acyltransferase activity was significantly increased in the fasted animals, and the increase waseven more pr-onounced in the diabetic group. In fasting and diabetes the liver weight decreases relatively
HEXADECANEDIOIC ACLD METABOLISM TABLE 111 RELATIVE
ACTIVITIES
DLOYLCARNITINE NORMAL, FASTED,
OF CARNITINE
The values given are the means (IOO",,).The fasted and diabetic conditions, see Table Il.
Diabetic
Clofibrate-fed
HEXADECANEIN
LIVERS
OF
_ Acticit?
pw
Activity
per
of iitw
Activity
100 g roi
g wet wf
Palmitylcarnitine Hexadecanedioylcarnitine
IS8
248 2.13
ztt
Palmitylcarnitine Hexadecanedioylcarnitine
209 I80
318 274
299 257
Palmitylcarnitine Hcxadecanedioylcarnitine
353 346
274 265
282
_
Fasted
WITH
SUBSTRATES RATS
of the different groups calculated as per cent of the normal group groups are identical with the groups in Table II. For experimental
.%hsrmrr
Grmp
AC~LTRANSF~RAS~
AND PALMITYLCARNITINE AS DIABETIC, AND CLOFIBRATE-FED
‘37
per
iu,q proteiir
182
274
more than the body weight of the animals, and therefore the differences between the normal and the two other groups are least when the results are related to body weight. Table 111 shows that the relative increases above the normal group are of the same magnitude with hexadecanedioylcarnitine and palmitylcarnitine as substrate. Table III also contains results from another feeding experiment. In liver homogenates from rats fed clofibrate for one week there was a significantly increased transferase activity (P < 0.01). The increases were of the same magnitude both with hexadecanedioylcartlitine and palmityl~~rnitine as substrates, and they were about the same as in the diabetic rats when the results were related to mg protein (specific activity) or to g wet weight of liver. However, when the results were related to body weight, the increase was much larger in the clofibrate-fed rats. This is due to the large increase in liver weight in these animals.
Activation of hexadecanedioic acid Fig. 4 shows the thin-layer chromatographic separation of the butanolextractable products of the fatty acid activation reaction with (bottom) and without (top) hexadecanedioic acid added as substrate. From the top chromatogram it can be seen that a product with RF 0.50 was obtained when no substrate was added. This product cochromatographed with the carnitine esters of several long-chain monocarboxylic acids (palmitic, tetradecanoic. oleic, linoleic, and linolenic acid) in both of the chromatographic systems used. This product therefore most probably represents activated endogenous long-chain fatty acids trapped as acylcarnitines. The bottom cl~romatogran~ shows that when hexadecanedioic acid was added as substrate two products were obtained, one which cochromatographed with synthetic hexadecanedioylcarnitine (R, 0.25). and one representing activated endogenous fatty acids (RF 0.50). Jn parallel experiments (incubation mixtures as shown in legend to Fig. 4) the thin-layer chromatographic step was omitted. The butanol phases from the experiment without and with hexadecanedioic acid added as substrate contained 95 and 198 nmoles of acylcarnitines, respectively (formed per 6 min). In the experiment where hexadecanedioic acid was added, r/6 of the total amount of ac~lcarnitines was acti-
.I. E. PETTERSEN
Fig.
4. Thin-layer
ncrivation
chromatographis
reactions
where
separation
the activated
fatty
of the
butanol-extractable
acids have
chro~~~to~~n~
shows the reaction
products
when Ile~~Ide~l~e~lioic
chromatogram
shows the reaction
products
when no substrate
substrates).
The reaction
5 mM ATP,
4 mM
mixture
of carnitine
palmilyltransf~rasc,
(5 ml) contained:
t.-[,~~e-3H]car!litine,
sium hexadecanedioate.
5 mM GSH, {wjv)
0.85”,,
The amount
of total
incubation
was started
by the addition
of tissue. and the reaction
was stopped
by the addition
of 0.3 ml of cont.
formed
Lvcrc extracted
with 7.5 ml of butanol-saturalel! dissolved
in methrtool
added, carnilinc
with 5 ml of butanol.
ci~ronl~t~~~r~~phy on Kicselgel
ammonia-water
radioactivity
standards
cailedioylc3rilitinc.
was measured
were localized
by esposurc
Tri-Garb
to iodine
CoA,
of potas-
added before
phnsc wzs washed once The a~ylcarnitiws
H (Merck)
vapour.
vials. scintillation
liquid The
were
plates with solvent
(50: 30:8: 2.5, by vol.). The front was
in a Packard
o.25: (2) palmiryl;art~itine.
I mhl
of water nas
The butanol
(0.5 cm wide) of the silicic acid were scraped into counting
and
7.4)> 0.1 mM
about 0.5 mg protein
~.ns run for 6 min at 35 ‘C. The
and 4.5 ml
water and taken to dryness on a rotovapor.
before thin-layer
system chloroforrc-methal~ol--cont. Fractions
KCN,
Top
of tndogenous
(wet wt of tissue) was 4. I 5 mg. The
incubation
the acylrarnitines
buRer (pH
~lburnil~, o. I 3 M KCI, and either no or
HCI,
acid
Bottom
acid was added its substrate.
MgC12, 5 mM
liver homogenate
of fatty
as acylcarnitines.
was added (activation
0.1 M Tris-HCI 5 mM
products
been trapped
scintillation
~LUI
14 cm.
solution counter.
RF values ~vcrc: ii)
\VX Acyi-
hcude
0.50.
vated hexadecanedioic acid (33 nmoles), and 5/h (165 nmoles) wcrc activated endogenous fatty acids. Thus, of the me:tsured increase of 103 nmoles of activated acid with hexadecanedioic acid added as substrate, only 33 nmolcs (or about 1i3) was activated flexadecanedioic acid, and the rest was activated endogenous fatty acids. This corresponds to an activation of hexadecanedioic acid of about / .J /moles/g wet weight of liver per min, i.e. about t/lo ofthe activation capacity for palmitic acid”.““. Isolated m~tochon~~ria were used as enzyme source in a few ex~~r~l?l~nts. In some of these Triton X-loo was added (final concentration o.o~:‘;,), otherwise the incubation conditions were as described in legend to Fig. 4. Essentially the same amounts of activated hexadecanedioic acid was found with Triton X-100 added about 95(jo) as without this detergent present (100”:). This indicates that the activation of long-chain dicarboxylic acids takes place outside the inner mito~hondri~~1 compartment, as is also the case with the long-chain monocarboxylic acids”,23-3”.
HEXADECANEDlOlC TABLE
ACID
METABOLISM
II
IV
ACYLATION
OF
M~TOCHOND~IAL
COENZYME
A BY HEXAD~C~ANEDIOYLCA~NI-
TINE OR PALMITYL~ARNITINE Mitochondria (7 mg of protein) were preincubated for 3 min with 15 mM N-tris(hydroxymethyl)methyl-z-aminoethanesulfonic acid buffer (pH 7.4). about 0.1 M KCI, and 0.2 mM z,4-dinitrophenol in a total volume of 3.5 ml. Then 5 /4moles KCN and acylcarnitine were added. The temperature was 15 C. The mitochondria were precipitated by I M HCIO, (3.5 ml) after 3 min. .._ .._ _~ _ _.. ..-. Additior7 ijlid~~Sl
_____-. None Palmitylcarnitine, 0.2 Hexadecanedioylcarnitine, Hexadecanedioylcarnitine,
CoA puesmt in die tiClO,i~7s~i~~bt~~ prrcipifnte i fl~?~uies~,~~~ protein) . _______.. .__..__
0.2 1.0
0.39 1.67 0.62 0.84
Table TV shows the amounts of ~ClO~-insoluble CoA (long-chajn acyl-CoA) formed when mitochondria were incubated with hexadecanedioylcari~itine or paimitylcarnitine. Nearly 6 times as much long-chain acyl-CoA was obtained with palmitylcarnitine as substrate as with the same amounts of hexadecanedioylcarnitine. When larger amounts of hexadecanedioylcarnitine were used, more long-chain acylCoA was formed.
Fig. 5 shows the O2 uptake of rat liver mitochondria uncoupled with dinitrophenol, and with malate added to stimulate the tricarboxylic acid cycle. A but significant, increase in O1 uptake was seen when hexadecanedioylcarnitine added (Trace a). However, the 0, uptake decreased rapidly with time. When
2,4low, was pal-
Fig. 5. Oxygraph traces showing the 0, uptake of rat liver mitochondria with hexadecanedioylcarnitine (H) and/or palmitylcarnitine (P) as substrates. The incubation medium (2.5 ml) contained 0.1 M KCI, 6 mM MgC12. IO mM N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid buffer (pH 7.4), 0.5% (w/v) bovine serum albumin, 2 mM malate, 0.08 mM z,q-dinitrophenol, and mitochondria (3.5 mg of protein). The acylcarnitines were added in amounts corresponding to 0.04 mM of each. The temperature was 25 “C.
I2
J. E. Pt.TTEIISEN
mitylcarnitine then was added the 0, uptake reached immediately the same level as with palmitylcarnitine added as the sole substrate. Thus, no inhibition of the oxidation of palmitylcarnitine by hexadecanedioylcarnitine was observed. Neither did any decrease in 0, uptake occur when hexadecanedioylcarnitine was added to mitochondria oxidizing palmitylcarnitine (Trace b). With rat heart mitochondria similar results were obtained. When malonate was added, the 0, uptake was lower, but the results were otherwise similar to those in Fig. 5. DISCUSSION
The present results show that hexadecanedioic acid can be activated, but at a lower rate than palmitic acid. The activation mainly occurs outside the inner mitochondrial compartment, as is also the case with monocarboxylic long-chain fatty acids ‘7.24.30. We have shown that when dicarboxylic acids are added to the incubation medium, there is an increased activation of endogenous monocarboxylic acids in addition to the activation of dicarboxylic acids. This may be due to a release of endogenous fatty acids, bound for instance to proteins, in exchange of the added dicarboxylic acids. Therefore specific methods are necessary in studies on dicarboxylic acid activation to ascertain that the measured increase of activated acids really represents activated dicarboxylic acids. The carnitine acyltransferases in the inner mitochondrial membrane3’ catalyze the reversible transfer of acyl groups from acyl-CoA to t_-carnitine and thereby mediate the transport of activated fatty acids through that membrane32.33. Our results show that hexadecanedioic acid can use this transport mechanism. Hexadecanedioylcarnitine is substrate for the enzyme in the isotope exchange assay, and mitochondrial CoA may be acylated by hexadecanedioylcarnitine. The isotope exchange assays with hexadecanedioylcarnitine and palmitylcarnitine added together as substrates indicated that the two acylcarnitines used the same enzyme. Solberg et ~1.“~ have shown that clofibrate feeding increases significantly the activities of all carnitine acyltransferases (i.e. short-chain (EC 2.3. I .7), medium-chain, and long-chain (EC 2.3. I .-) carnitine acyltransferase) in rat liver, but not to the same extent. In the present studies we found that in fasted, diabetic, and clofbrate-fed animals the carnitine acyltransferase activity showed a parallel increase with hexadecanedioylcarnitine and with palmitylcarnitine as substrate (Table III). This further supports our suggestion that the same enzyme, pi:. hexadecanoyl-Co/i: carnitine O-hexadecanoyltransferase (EC 2.3. I .-), is responsible for the transport both of activated long-chain monocarboxylic and dicarboxylic acids across the inner mitochondrial membrane. An increased activity of this enzyme has been described previously in fat-fed”s.36, alloxan-diabetic3’, fasted3”,36, and clofibrate-fed’4.34 rats. An increased urinary excretion of short-chain dicarboxylic acids may be caused by increased amounts of precursors, 1li7. long-chain dicarboxylic acids (see Introduction), and/or by an increased activity of the enzymes participating in the degradation of long-chain dicarbocylic acids. The well-known increased liberation of fatty acids in ketosis, combined with the increased w-oxidation capacity which has been reported by Wada c’t al.‘.“, may yield increased amounts of long-chain dicarboxylic acids. Moreover, we have shown that the carnitine acyltransferase activity with hexadecanedioylcarnitine as substrate is increased in this metabolic condition. Thus, both factors,
HEXADECANEDIOlC
ACID
METABOLISM
‘3
viz. a formation of increased amounts of long-chain dicarboxylic acids and an augmented degradation of these acids, may be of importance for the urinary excretion of short-chain dicarboxylic acids in ketosis. We have shown that the short-chain dicarboxylic acids may be formed from long-chain monocarboxylic acids by a primary o-oxidation, followed by /i-oxidations of the long-chain dicarboxylic acids thus formed4. III viva experiments performed by various other groups3.37m39 also indicate that long-chain dicarboxylic acids are degraded by the P-oxidation mechanism. Following the ingestion of acids with oddnumbered carbon-chains, odd-numbered short-chain dicarboxylic acids are excreted in the urine; and following the ingestion of acids with an even-numbered carbonchain, even-numbered acids are excreted. However, the low 0, uptake of mitochondria with hexadecanedioylcarnitine as substrate necessitates further studies to clarify if other chain-shortening processes than ordinary fi-oxidation are of importance in the degradation of long-chain dicarboxylic acids. ACKNOWLEDGEMENTS
The author is indebted to Professor .J. Bremer, Dr M. Aas, and Dr S. Skrede for helpful suggestions and discussions. The technical assistance of Mrs Kari Frogner is gratefully acknowledged. Through the kind cooperation of the Chemistry Department (Mr K. Undheim and Mr G. Hvistendahl), University of Oslo, we had access to the high resolution mass spectrometer. REFERENCES I Pettersen,
J. E., Jellum, E. and Eldjarn, L. (1971) &and. J. C/in. Lab. Invest. 2.7, Suppl. I 18. 69 J. E., Jellum, E. and Eldjarn, L. (1972) C/in. C/rim. Acfa 38, I 7-24 3 Verkade, P. E.. Van der Lee, J. and Van der Alphen, A. J. S. (1937) Hoppe-.Se_r/ers Z. Phj,sio/. Chrm. 250, 47756 4 PetterSen. J. E. (1972) Clin. Chim. Acta, 41, 2-j-237 5 Wada. F.. Hirata, K., Shibata, H., Higashi, K. and Sakamoto, Y. (1967) J. Eiochem. Tokyo 62, 2 Pettersen,
134-136 6 Wada, F. (1966) J. Jap. Biochern. Sot. 38, 818-825 7 Stokke. 0. and Bremer, J. (1970) Biochim. Bioph_vs. Acta 218, 552-554 8 Brcmer. J. (1968) Biochem Prep. 12, 69973 9 Norum. K. R. (1964) Biochitn. Biophys. Acra 89, 95-108 IO Farstad, M., Bremer, J. and Norum, K. R. (1967) Biochim. Biaphys. Acra 132. 492-502 I I Christophersen, B. 0. and Bremer, J. (I 972) Biochim. Biophys. Acta 260, 5 15-526 12 Junod, A., Lambert, A. E., Orci, L., Pictet, R., Gonet, A. E. and Renold, A. E. (1967) Proc. Sac. Esp. Biol. Med. I 26, 201~205 I 3 Junod. A.. Lambert, A. E., Stauffacher, W. and Renold. A. E. (1969) J. C/in. rmest. 48, 2129-2139 14 Daae, L. N. W. and Aas, M. Atherusckrusis, in the press 15 Best. M. M. and Duncan, C. H. (1964) J. Lab. C/in. Med. 64, 634-642 16 Myers, D. K. and Slater, E. C. (1957) Biochrm. J. 67, 558-572 I 7 Aas, M. (1971) Biuchirn. BiophJx Acta 23 I, 32-47 18 Chappel, J. B. (1964) Bioc/rern. J. 90, 225-237 19 Lowry, 0. H., Rosebrough, N. J., Farr, A. 1.. and Randall, R. J. (195 I) J. Biol. Chem 193, 26520 2I 22 23
275 Bremer, Bremer, Aas. M. Bremer.
J. (1962) J. Biol. Chew. 237, 2228-2231 J. and Norum, K. R. (I 967) J. Biol. Chern. 242, I 74991755 and Bremer, J. (1968) Biochim. Biophvs. Acta 164, I 57-166 J. and Wojtczak, A. B. (1972) Biochim. Biophys. Acta 280, 515-530
J. E. PETTERSEN
is
S. and Bremer, J. (1970) Eur. J. Biuclre~~r. 14, 465-472 Hvistendahl. G., Undheim. K. and Bremer. J. (1970) Org. .&cfr~ss &wcf~~~u. 3. x433-1438 Solberg, H. E. and Eremer. J. (1970) Biocftiuz. Biopk_vs. Acfrr 222, 372-380 Holmes. J. L. and Jean, T. St. (1970) Oyr. Moss Specfrrtm. 3. 1y+r~r8 Hvistendahl, G. and Undheim, K. (1970) Org. Muss S~CITO~~I.3, 82i...824 Van Tot, A. and Hiilsmann, W. C. (1969) Biochi~u. BioplrJx. Acts I&, 342-353 Norum. K. R., Farstad, M. and Bremer, J. (1966) Biochr,r/. Bioph)~s. RPS. Comm~,/r. 24. 797-804 Norum, K. R. and Bremer. J. (1967) J. Bio/. Clwur. 242, 407-41 I l3remer. J. (196.3) J. Biul. Clxw. 238. 2774-2779 Fritz, 1. B. and Yue. K. T. N. (1963) J. Lipici’Res. 4, 279-288 Solberg. H. E., Aas, M. and Daae, L. N. W. ( 1972) Biocltiw. Biopfr.~,s.Actri z.80, 434-439 Norum, K. R. (1965) Biacizin~. Biuph_u. Acfrr 98. 652-654 Aas, M. and Daae, L. N. W. (1971) Biochim. Bioph,tx Artrr 239, zo%rr6 Bergstrclm, S., Borgstr@m, B.. Tryding, N. and Westnn, G. (1954) Biorheitr. j. 58, 604-608 Tryding. N. and Westetr, G. (1956) A&I Clwt~. Scwfd. IO, 1234-1242 Yamakawa, T. (1950) J. Biochr~u. 37, 343-353
24 Skrede, 25
26 27 28 29
30 31 32 33 34 35 36 37 38 39