Propionylcarnitine physiological variations in vivo

BIOCHIMICA

nB*

ET BIOPHYSICA

ACTA

559

55443

PROPIONYLCARNITINE PHYSIOLOGICAL

THOMAS

VARIATIONS

BOHMER

AND JON

January

I’WO

BREMER

University of Oslo, Hikshospitalet,

Institute of Clixical Biochemistry, (Received

IN

Oslo (Norway)

11th, 1968)

SUMMARY

I.

Propionylcarnitine

has been identified

as a relatively

important

derivative in liver and kidney in male rats. 2. In the liver of fed rats the ratio of propionylcarnitine/free nylcarnitine

was

22q~

Fasting

and in the kidney 49:.

carnitine

carnitine+propio-

reduced this ratio to 19/, in both

liver and kidney. In the other organs the ratio was ~“/dor less in both normally fed and fasted rats. 3. Feeding corn oil to rats previously given a diet rich in carbohydrate reduced the level of propionylcarnitine in the liver to fasting values within 4 h. 4. The changes in the propionylcarnitine level in the liver are exactly

opposite

to those in the long-chain acylcarnitines. 5. The results are discussed in relation

in mito-

chondrial

to the regulatory

mechanisms

metabolism.

INTRODUCTIOS

The relative has recently

distribution

of carnitine

derivatives

in different

tissues of the rat

been studied and it was found that the levels of long-chain

acylcarnitines

and of acetylcarnitines vary with the nutritional state of the animal. They increase in fasted, fat-fed and diabetic animals, while decreasing when carbohydrate is given to fasted animals1T2. During these studies two unknown carnitine derivatives were detected in aqueous extracts of liver and kidney. One of these unknown carnitine esters has now been identified as propionylcarnitine. A preliminary report on this observation has recently been publisheds. VF:e have now studied the physiological variations in the level of propionylcarnitine by the same technique as used for the study of the relative distribution of long-chain

acylcarnitine

MATERIALS

ASD

and acetylcarnitine*32.

METHODS

Chemicals: L3H]methyliodide (specific activity, 138 pC/pmole) was obtained from the Radiochemical Centre, Amersham, England. [MGHlButyrobetaine was Biochim. Biophvs.

.-lcta, 152 (1968) 559-567

TH. BGHMER, J. BREMER

$0 synthesized as previously describedl. Merck A.G., Darmstadt, trifluoroacetic was purchased

from Schuchardt,

of the Otsuka

Pharmaceutical

acetyltransferase

(EC 2.3.1.7)

Silicic acid (G after STAHL) was obtained from acid from Fluka, Buchs, Switzerland. Diketene

Miinchen, Company,

Germany. Osaka,

were obtained

L-Carnitine

Japan.

was the kind gift

CoA and carnitine+CoA

from Boehringer,

Mannheim,

Germany.

Synthesis of L-acylcarnih~es L-Acetylcarnitine hydrochloride was synthesized as previously described”. The L-propionylcarnitine-, L-butyrylcarnitine-, L-isovalerylcarnitineand L-crotonylhydrochlorides were synthesized by treating a solution of L-carnitine hydrochloride in trifluoroacetic

acid with the corresponding

acid chlorides at 40-50~.

Approx. 300 mg of carnitine was dissolved in 0.5 ml of trifluoroacetic acid and mixed with I ml of the acid chloride in a test tube and left in a glycerol bath at 40-45’ overnight.

Moisture

was kept out by a CaCl, tube. Subsequently

the reaction

vessel

was cooled to room temperature, 5 ml of acetone were added, and the tube was left on ice for a couple of hours. Any undissolved material was removed by centrifugation. Subsequently

ethyl

ether was added at room temperature

to incipient

cloudiness.

After crystallization had started, another IO ml of ethyl ether were added and the tube was cooled on ice. The crystallization products were dissolved in I ml of methanol or ethanol and 4-5 ml of acetone were added. Ethyl ether was then added to incipient cloudiness.

After

crystallization

was started

another

5 ml of ether

(Acetone was used to avoid formation of oils upon the addition 70-90;; were obtained by this procedure. When quantitative theoretical ester contents

Hestrin tests were obtained.

with CHCl,-CH,OH-conc.NH, one spot with iodine vapour. were also present. trace amounts

as previously on thin-layer

Yields

of

described3 silicic acid

(50:30:8, v/v/v) as eluting agent usually gave only In some preparations small amounts of free carnitine

Chromatography

of an impurity

were performed Chromatography

were added.

of ether.)

of the crotonylcarnitine

with a higher

RF value

showed the presence

of

than the main spot. Solutions

of the crotonylcarnitine discoloured KMnO, solutions, showing L-Acetoacetylcarnitine hydrochloride was prepared by L-carnitine hydrochloride (500 mg) in trifluoroacetic acid (0.5 mately equimolar amount of diketene (0.225 ml) at 0“. After a

a double bond. treating a solution of ml) with an approxicouple of hours on ice

the reaction mixture was left at room temperature overnight. 5 ml of acetone were added, and the reaction mixture was left on ice for some hours. Undissolved material was removed by centrifugation. Subsequently the acetoacetylcarnitine was precipitated as a sticky mass with a great volume of ether (about 50 ml). (It proved impossible to obtain crystalline material.) The sticky mass was precipitated twice from ethanol with ether and subsequently dried under reduced pressure. A crisp, foamy, extremely hygroscopic material was obtained. Micro-Kjeldahl analysis showed 5.08;/, of N. (The theoretical content of N is 4.98yb), and determination of acetoacetate content according to the method of WALKERS showed 9036 of the theoretical value. Thin-layer chromatography showed one main spot and traces of a material with a higher RF value. L-/!-Hydroxybutyrylcarnitine was prepared from acetoacetylcarnitine by treatment with sodium borohydride : 140 mg of acetoacetylcarnitine were dissolved in I ml of ethanol containing 0.3 ml of glacial acetic acid. After cooling the solutinn on ice, Riochim.

Rioph_vs. dcta,

152 (19681 559-567

PROPIONYLCARNITINE VARIATIONS ifi ViVO

561

about 75 mg of sodium borohydride

were added in small portions.

of ethyl ether were added. Insoluble

material

carnitine material

Subsequently

was removed by centrifugation

5 ml

and the

ester was precipitated as a solid material with about 15 ml of ethyl ether. The contained no Cl- (no precipitate was formed with silver nitrate). When

reacted with alkaline hydroxylamine, a stable hydroxamic acid was obtained. The quantitative Hestrin test3 showed that its ester bond content was 95:; of that expected. L-Succinylcarnitine was prepared by treating L-carnitine (zoo mg) in trifluoroacetic acid (0.5 ml) with succinyl chloride (I ml) at room temperature for 3 h. (When a temperature

of 40-50”

was used, the reaction

product

discoloured

KM,O,

showing

that crotonbetaine probably had been formed.) Acetone (IO ml) was added. -4fter standing overnight at o’, undissolved material was removed. The succinylcarnitine was precipitated as an oil with 50 ml of ethyl ether. I ml of water was added to the precipitated

oil. The solution

was repeatedly

shaken with ether to remove

cinic acid and most of the water. The remaining

free suc-

oil was dried under reduced pressure.

The slightly sticky residue contained Cl-. The ester bond content corresponded to about 700/6 of the theoretical value for succinylcarnitine hydrochloride. Thin-layer chromatography carnitine.

showed

that

the

product

contained

significant

amounts

A small amount (15 mg) was purified by column chromatography

50 (NH,+) (see below), for use in enzymatic

Animals Male rats of Wistar

(Mall) strain,

of free

on Dowex

tests.

with a 3H-labelled

carnitine

as before1*2. Two experiments were performed with a rabbit received an intraperitoneal injection of [Me-3H]butyrobetaine

pool, were used

and a guinea pig. They 12 h before they were

killed.

Methods For separation graphic 200-400

of carnitine

esters in tissue extracts,

the following

chromato-

systems were used: (I) Column chromatography on Dowex 50-X8, mesh; 50 cm x 1.2 cm) equilibrated with 0.2 M ammoniumformate

(pH 4.2) (modified according shows how different synthetic

(NH,+, buffer

to the technique of HIRS, MOORE AND STEINS). Fig IA carnitine esters are eluted by 0.2 M ammonium formate

(pH 4.2) from this column. (2) Paper chromatography (Whatman No. I) with propanol-conc.NH,-water (85:5: IO, v/v/v) as eluting agent. (3) Thin-layer silicic acid chromatography with (CHCl,CH,OH-conc.NH,, 50: 30: 8, v/v/v) as eluting agent. Fig. I shows the RF values obtained. The assay for the radioactive propionylcarnitine was performed as previously described with the thin-layer silicic acid chromatographic system2,3. The reactivity of the different acylcarnitines with carnitine CoA acetyltransferase was determined spectrophotometrically at 233 rnp in an Zeiss RPQ 20 AV spectrophotometer. Further details are given in the legends to the figures and tables.

Statistics The Wilcoxon

two-sample

test was used (twotailed) Biochim.

as before2.

Bioph_vs.

Acta,

152 (1968)

559-567

TH. BQHMER,

562 Presentation

J. BREMER

of results

The tissue level of propionyl carnitine carnitine+propionylcarnitine as previously

is given as the “/6 propionylcarnitine/free done for other carnitine ester9.

RESULTS

Identificatiola

of propionylcardine

Fig. IB shows that three radioactive peaks were obtained when an aqueous extract of liver was chromatographed on Dowex 50 (NH,+). The first peak was eluted exactly as acetylcamitine,also when co-chromatographed with carrier acetylcarnitine. The second peak was eluted at the position of carnitine/propionylcarnitine, and contained two compounds as shown by chromatography on paper (Fig. IC). The RF

A

(3)

n

X

Fig. IA. Chromatography of different acylcarnitines on a column of Dowes 50-X8 (zoo-400 mesh; 50 cm x 1.2 cm) with 0.2 M ammonium formate buffer (pH 4.2). Carnitine was eluted after approx. 35 fractions (280 ml) of this buffer. The figure is the result of a series of runs with different acylcarnitines and [aH]carnitine. The acylcarnitines were converted to the corresponding hydroxamic acids and measured spectrophotometrically at 540 m,n. The relative elution volumes of the different acylcarnitines in relation to carnitine were as follows: (I), /!-hydroxybutyrylcarnitine 0.57; (2), succinylcarnitine 0.6; (3), acetylcarnitine 0.7; (4), acetoacetylcarnitine o.S4; (5), propionylcarnitine 0.95; (6), butyrylcarnitine 1.5; (7). crotonylcarnitine 2.5. Fig. rB. Column chromatography of a liver extract from a rabbit with a [3H]-carnitine pool. Fig. IC. Chromatography of Peak II (from the column) on paper (Whatman No. I) with propanolconc.NH,-water (85: 5: IO, v/v/v) as eluting agent for 74 h with standards: (I), carnitine; (L), acetylcarnitine; (3), propionylcarnitine. Fig. ID. The peak corresponding to propionylcarnitine on the paper chromatogram ( IC) was eluted with methanol and cochromatographed with synthetic acylcarnitines on thin-layer silicic acid with CHCl,-CH,OH-cont. NH, (50:30:8, v/v/v) as eluting agent. The front was run 13-14 cm. The radioactivity was assayed by scraping fractions of 0.3 cm of the silicic acid into counting vials and counted in a Packard Tri-Carb liquid scintillation spectrometer after addition of scintillation solution. The acylcarnitines were localized by exposure to iodine \-apour. The RF values were as follows: (I) carmtine 0.08, (2) succinylcarnitine 0.08; (3) acetylcarnitine 0.12~0.1g; (4) propionylcarnitine 0.20~0.25; (5) acetoacetylcarnitine o.20--0.25; (6) butyrylcarnitine 0.35; (7) crotonylcarnitinc 0.35.

PROPIONYLCARNITINE

VARIATIONS in Vi80

values of these compounds of carnitine

563

in the paper system corresponded

and propionylcarnitine.

In the thin-layer

exactly

to the RF values

system too, the two compounds

were found at the positions of carnitine and propionylcarnitine (Fig. ID). The unknown carnitine ester was eluted from the paper chromatogram with methanol and reacted with alkaline hydroxylamine. The hydroxamic acid formed chromatographed as propionylhydroxamic acid in two different system+. After acid hydrolysis of the carnitine ester the radioactive compound chromatographed as carnitine in the thinlayer system used, and also in a system separating carnitine from butyrobetaine and metylcholine (System A in ref. I). In addition recrystallization of the isolated radioactive carnitine ester with carrier L-acetylcarnitine, L-propionylcarnitine, and L-butyrylcarnitine The identity

gave constant

specific activity

only with propionylcainitine3.

of the third peak in the column chromatography

unknown. However, by acid hydrolysis it was converted this compound also is most probably a carnitine ester. Pvopionylcavfzitine Ovgax

TABLE

(Fig. IB) remains

to carnitine,

showing that

in uivo

distribution. Table

I shows that the ratio propionylcarnitine/free

carrn-

I

PROPIONYLCARNITINE

IN

ORGANS

OF

NORMALLY

FED

AND

FASTED

RATS

Exflt. I. Liver. The rats were injected with 0.1 mC [3H]butyrobetaine. Mean values are given (in parentheses, S.D.). The results for acetylcarnitine and long-chain acylcarnitine are given for comparison. Number

Acylcavnitine

of’animals

Normally fed Fasted (24 h)

8 12

“/’ Aqilcarnitim+fvee

cavnitine

Pvopionylcavnitine

Acetylcav&ine

Long-chain acylcarwitimzs

II I.5

26 5’

20

(8.6) (0.7)

(6.4) (2.9)

2.8

(1.2)

(4.8)

Expt. II. Other organs. The rats were injected with 0.3 mC [3H]butyrobetaine. The livers of the respectively. The unfed rats were fasted fed rats contained 14, 24 and 38:; of propionylcarnitine, for 36 h. The organs mere dissected out in the following order: liver, kidney, heart, adipose tissue (fat pad), testis, epididymis, muscle, brain. o, Propionylcamitine ” Pvopiolz.vlcavnitirle_(~fvee camitiwe Organ

Fed

Kidney Heart Adipose tissue Testis Epididymis

3.3 1.3

0.2

Muscle

Brain

Fasted

I.0

4.0 0.7 0.3

0.7

1.0

0.4

0.1

0.3 0.8

0.2

0.1 I.0

I.0

I.0

I.0

4.0

I.0

I.0

0.8

0.4

0.1

2.0

0.2

0.3

0.8 0.5

0.1

0.2

0.5

tine+propionylcarnitine was highest in the liver and the kidney of the normally fed rat. In the other organs the propionylcarnitine level was much lower. However, the delay in the extraction of these organs may have caused changes in the acylcarnitine levels as postmortum changes have been shown to occur1T7. It may especially be noted Biochim. Bioph~ts. A4cta, 152 (1968) 559-567

TH. BBHMER, J. BREMER

564 that in the liver of fed rats the propionylcarnitine

level was as high as that of acetyl-

carnitine. Table I shows that the propionylcarnitine level was reduced in liver and kidney in fasting while the levels of long-chain acyl- and

EfSect offasting.

drastically

acetylcarnitine were increased1y2. In some of the other tissues there may be some effect of fasting upon the propionylcarnitine level, but the low content even in the TABLE

11

THE EFFECTOF CORN OIL FEEDINGON THE PROPIONYLCARi%ITINE IN

LEVEL

IS

RATS

PREVIOUSLY

GIVEN

A

DIET

RICH

CARBOHYDRATE

All rats had free access to stock diet, with IoO,, sucrose substituted for water on the last day. Mean consumption of sucrose solution was 50 ml. One group of rats was killed without handling. The other rats were given either 5 ml of corn oil or water with a gastric tube, and killed after 2 or 4 h. As there was no significant difference between the txvo control groups, the oil-fed groups were compared statistically with the pooled control groups killed at S and I L a.m. Mean values are given (in parentheses, S.D.). The changes in the level of long-chain acylcarnitines are given for comparison2. (Number of rats in each group are given in square brackets.) o

GlYIl@

Pvopionylcavnitine u FGfiG?qqz

Controls, killed at 8 a.m. 1.~1 Oil-fed at 8 a.m., killed at IO a.m. [6! Oil-fed at S a.m., killed at 11 a.m. [O] Sham-fed at 8 a.m., killed at 12 a.m. 13 1 * P = ** P =

o carn2tine

LI IL 1.6

(1-r) (b.3) + (o.+)**

18

(11.6)

Lotlg-chain acylcarnitines

” Long-chain acyl- ffvee cavnitine ~~~_ ~~___~ 2.7

11 23 3.6

Ii::;

(10) (1.0)

_

_. ._ ~~

0.13 when compared with pooled control groups. 0.00~ when compared with pooled control groups.

fed animals makes accurrate Effect

of COY~Z oil feeding

determinations

of the propionylcarnitine

to rats prezriody

level difficult.

giaen a diet rich iva carbohydrate.

Tube

feeding of corn oil to rats previously given a diet rich in carbohydrate rapidly changes the metabolic pattern towards fatty acid oxidation 2. Table II shows that the level was reduced within 2 h and had decreased to fasting values 4 h after corn oil had been given. Concomitantly the level of long-chain acylcarnitines changed in the opposite directions. Diurnal

puttem

in the nomzally

Fig. 2 shows that the level of propionyl-

fed rd.

.

: , long-chain cornitine

ocyl-

F?F s! 9 0 80.m.

aI 3p.m.

I 10 p.m.

0 4a.m.

s-

Time

Fig. L. Diurnal pattern of propionylcarnitine in the liver of the normally fed rat. The diurnal pattern of longchain acylcarnitines is given for comparison (only mean values)2. Biockim. Biophys.

Acta, 152 (1968) 559-567

PROPIONYLCARNITINE

VARIATIONS in VhO

565

carnitine was reduced in the middle of the day, and increased about the same level as in the morning. of propionylcarnitine

were exactly

again in the evening

to

It can be seen that these changes in the level

opposite

to those

of long-chain

acylcarnitinesz.

The decrease in the level of propionylcarnitine in the middle of the day most likely is caused by a short physiological fasting during the day, as rats eat mainly at nights. Substrate specificity of carnitine-Cod acetyltransferase. Propionyl-CoA is known to be reversibly converted to succinyl-CoA s- Il. It might be expected, therefore, that propionyl-CoA and succinyl-CoA vary in the same manner in viva. However, no succinylcarnitine was detected (Fig. I). We have therefore made some studies on the TABLE

III

SURSTRATESPECIFICITYOF CARNITINE-Cd

ACETYLTRANSFERASE

The cuvette contained zo+oo punits of carnitine acetyltransferase, 0.3 ml of 0.1 M TES-buffer (trishydroxymethylamino sulfonic acid, pH 7.5) and 5 pmoles of the respective carnitine esters in a total volume of 1.5 ml. The temperature was approx. 25’. The initial reaction rates measured at 233 rnp are given in relation to the velocity measured with L-acetylcarnitine. L-Acetylcarnitine L-Propionylcarnitine L-Butyrylcarnitine L-Acetoacetylcarnitine L-/j-Hydroxybutyrylcarnitine L-Isovalerylcarnitine L-Succinylcarnitine

substrate

specificity

I I.3 0.4 0.2

<0.05 <0.05 0

of carnitine-CoA

acetyltransferase.

Table

III

shows that L-pro-

pionylcamitine is the most active substrate for the carnitine-CoA acetyltransferase, with a higher reactivity than obtained with L-acetylcarnitine and L-butyrylcarnitine. It is likely therefore that the propionylcarnitine is formed by this enzyme. When the butyryl group was modified by introducing metyl, keto, or hydroxyl groups, the reactivity

was reduced

and succinylcarnitine

was found not to react at all.

DISCUSSION

Previously we have shown that the levels of long-chain acylcarnitine and acetylcarnitine change in the same direction as do the corresponding CoA esters in different physiological

conditions lt2. The relatively

high concentration

of propionylcarnitine

in the liver of fed animals therefore indicates a corresponding high level of propionylCoA in liver and kidney of fed animals. The experiments in Go show that the level of propionylcarnitine drops rapidly in fasting and fat-feeding. A corresponding drop in propionyl-CoA most likely takes place, as the reaction Propionyl-CoA + carnitine + propionylcarnitine + CoA is freely reversible with an equilibrium constant close to I. This reaction most likely is catalysed by carnitine-CoA acetyltransferase, since propionylcarnitine was found to be the most active substrate for this enzyme in vitro (Table III). Propionyl-CoA may also be in equilibrium in the mitochondria with methylmalonyl-CoA and succinyl-CoA9-11. It is possible, therefore, that all these esters of CoA in the mitochondria change in the same manner as does propionylcarnitine. When TUBBS AND GARLAND’~ determined the acid-soluble CoA derivatives in liver, a large fraction could not be accounted for as free CoA or acetyl-CoA. WILLIAMSON et a1.13 Biochim.

Biophys.

24cta, 152 (1968)

559-567

TH. BoHMER, J. BREMER

566 found that the sum of free CoA, acetyl-CoA from 412 to 708 mpmoles/g dry weight after likely that a large fraction

and long-chain 24 h of fasting.

of liver CoA in the normally

acyl-CoA increased It seems, therefore,

fed animal is bound to acids

other than acetic and long-chain fatty acids. Our results indicate that an appreciable fraction of CoA may be bound as propionyl-CoA. Appreciable amounts of CoA may also be bound to succinate the absence

and methylmalonate.

of succinylcarnitine

This possibility

itz zlivo, as succinyl-CoA

is not excluded

apparently

by

does not react

with carnitine. The acetyl-CoA

level in liver has been proposed

to be the main regulator

of

ketone body production and gluconeogenesis 12-14. The acetyl-CoA level increases only by a factor of 2-3 during fasting12>14, and gluconeogenesis has been induced by oleic acid perfusion of rat liver without any concomitant increase in the acetyl-CoA leve115. It therefore seems likely that increased gluconeogenesis and ketogenesis also depend on changes in the levels of CoA esters other than acetyl-CoA in the liver. Our results indicate that propionyl-CoA, vary more extensively than propionyl-CoA

inhibits

and possibly metylmalonyl-CoA and succinyl-CoA, does acetyl-CoA. Recently we have also found that

the formation

of acetoacetate

in mitochondrial

extracts.

Pyruvate carboxylase is stimulated by propionyl-CoA, while it is inhibited by methylmalonyland malonyl-CoA 16. At present therefore it is not possible to assess the combined effect of acetyl-, propionyl-, methylmalonyl-, and succinyl-CoA on the pyruvate

carboxylase

in e&lo.

However, a regulating effect of methylmalonate on glyconeogenesis is supported by a recent observation of OBERHOLZER et al.‘?. They have studied a patient who excretes great amounts of methylmalonate in the urine, most likely because of a lack of methylmalonyl-CoA racemase (EC 5.1.99.1) or methylmalonyl-Coil 5.4.99.2). When this patient was given a load of propionate, increased

mutase (EC excretion of

methylmalonate and hypoglycaemia were observed. Propionyl-CoA or its metabolites may also have other effects. Recently PEARSOS AND TUBBS~ have shown that perfusion of rat hearts with propionate induces a nearly complete

disappearance

of acetylcarnitine

from the heart.

Carnitine has been shown to stimulate gluconeogenesis from propionate in kidney slices’*. In fasting animals amino acids are rapidly converted to glucose in liver and kidneys. Since propionyl-CoA is an intermediate in the breakdown of several amino acids, it is also an intermediate in gluconeogenesis. It seems paradoxical then, that the level of propionyl-CoAdecreases in fasting. This indicates that the metabolism of propionyl-CoA is inhibited in animals fed with carbohydrate, but the mechanism for this inhibition is unknown. ACKNOWLEDGEMENTS One of the authors (T.B.) is a Fellow of the Norwegian Council for Cardiovascular Diseases. The authors are indebted to Mrs. KARI GAUPAS for skilled assistance. REFERENCES I T. L T. .3 T.

BOHMER,

BQHMER, BoHnlER

Ii. R. NORUM AND J. BREMIER, Biochim. BwphTs. rlcta, Biochim. Biophys. 24cta. I++ (1967) 259. AND J. BREMER, Biochirn. Biophys. Acta, 152 i1968) +$o.

B~ochirn. Biophys.

Acta,

152 (1968)

559-567

125 (1966) ‘14.

PROPIONYLCARNITINE

VARIATIONS

in ViVO

567

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.Icta. 152 (1968)559-567