253

Biochimica et Biophysica Acta, 583 (1979) 253--260 © Elsevier/North-Holland Biomedical Press

BBA 28836

I N C O R P O R A T I O N OF THE H Y D R O G E N ATOMS OF E T H A N O L INTO AMINO ACIDS IN R A T L I V E R IN VIVO

TOMAS CRONHOLM

Department of Chemistry, Karolinska Institutet, S-104 01 Stockholm (Sweden) (Received August 14th, 1978)

Key words: Ethanol incorporation; Amino acid synthesis; Deuterium; (Rat liver)

Summary Rats were injected intraperitoneally with [1-2H2]ethanol, (1R)-[1-2H] ethanol, (1S)-[1-2H]ethanol or [2-2H3]ethanol. After different times, the rats were anesthetized and the liver was freeze-clamped in situ. Amino acids were isolated, converted into n-butyl ester N-trifluoroacetates and the labelling of alanine, proline, aspartate and glutamate was analyzed by gas chromatographymass spectrometry. Aspartate became labelled during metabolism of [1-2H~] ethanol, the deuterium excess in one position being 13 atoms% in samples isolated after 20 min of ethanol oxidation. Taken together with previous results on the labelling of malate this indicates that at least 60% of the liver aspartate was derived from oxaloacetate. A b o u t 24% of the transferred hydrogen was derived from the 1-pro-S position of the ethanol, indicating that both alcohol dehydrogenase and aldehyde dehydrogenase contribute NADH to malate dehydrogenase. Alanine and proline were labelled with deuterium after 4 h of [1-2H2]ethanol oxidation, the excess in one position being a b o u t 2 and 3 atoms%, respectively. Labelling of alanine indicates its formation from pyruvate derived from malate, and labelling of proline is explained by its formation in NAD(P)H-dependent reductions. All amino acids studied incorporated deuterium from [2-2H3]ethanol. The highest incorporation was observed in glutamate, and it was calculated that a b o u t 26% of the liver free glutamate was formed from ethanol after 4 h of ethanol oxidation. The presence of monodeuterated glutamate and proline molecules indicated exchange of hydrogens at C-2 of ethanol during the incorporation.

254 Introduction Alcohol oxidation in the liver results in the formation of excessive amounts of acetate and NADH. This leads to changes in the metabolism of endogenous compounds. The interaction of ethanol metabolism with normal metabolism may be studied in vivo by following the incorporation of deuterium from ethanols labelled at different positions with deuterium (see e.g. refs. 1 and 2). The constant rate of ethanol oxidation in vivo should result in a constant degree of labelling of NADH and acetyl-CoA in the liver, making it possible to study kinetics of endogenous compounds and precursor-product relationships [2]. The hydrogens at C-1 in ethanol are incorporated via NADH into acids of the citric acid cycle [1], and the carbon atoms are known to be incorporated via acetyl-CoA into oxo acids which may become transaminated [3] and into protein [4]. A study of the incorporation of the hydrogen atoms of ethanol into amino acids in vivo was therefore expected to give information both on the metabolism of amino acids and on possible sites of interaction of ethanol metabolism with amino acid biosynthesis in the liver. Experimental procedure

Materials. [1-2H2]Ethanol having 94.6% deuterium at C-1 was obtained from Merck AG (Darmstadt, Germany), and [ 1-2H2]ethanol having 99.5% deuterium at C-1 was obtained from Merck, Sharp and Dohme, Canada Ltd. (Kirkland, Que., Canada}. (1R)-[1-2H]Ethanol and (1S)-[1-2H]ethanol were prepared, purified and analyzed as previously described [5]. One batch had a deuterium excess of 80.3% in the 1-pro-R position and 5.8% in the 1-pro-S position, and the other batch had a deuterium excess of 10.5% in the 1-pro-S position and 92.4% in the 1-pro-S position. [2-2H3]Ethanol having 99.5% deuterium at C-2 was a gift from Dr. A.L. Burlingame, Berkeley, CA, U.S.A. Animal experiments. Female Sprague-Dawley rats weighing about 200 g were used. Water and food pellets were given ad libitum. Labelled ethanol (1 g/kg body weight) was injected intraperitoneally as a 20% (w/v) solution in saline. In the 240-min experiments, a second dose of ethanol equal to the first was injected after 2 h. The abdomen was opened under light ether anesthesia and a portion of a liver lobe (0.1--0.3 g) was freeze-clamped with tongs precooled in liquid nitrogen [6] 5, 20 or 240 min after the ethanol administration. Isolation of amino acids. Frozen liver samples were pulverized in liquid nitrogen and 5 ml 0.6 M H 2 S O J m e t h a n o l (1 : 4, v/v) was added together with liquid nitrogen. After thorough grinding to a pulver, the mixture was allowed to thaw, and was centrifuged. The pellet was resuspended with 2 ml of 0.6 M H2SO4/methanol (1 : 4, v/v). After centrifugation the supernatants were pooled and passed through a column (2 × 1 cm) of Dowex 50W-X8 in the H* form in water. The column was rinsed with 5 ml 0.5 M acetic acid and 15 ml water. The amino acids were eluted with 30 ml 2 M triethylamine in acetone/water (1 : 4, v/v) [7], and the solvent was removed in vacuum. The residue was dissolved in 3 ml acetyl chloride/n-butanol (1 : 9, v/v), refluxed for 20 min in an oil bath and the reagents were removed in a stream of nitrogen. The residue was dissolved in 0.75 ml CH2C12 and 0.25 ml of trifluoroacetic anhydride, refluxed for

255 10 min in an oil bath and evaporated to dryness in a stream of nitrogen at room temperature [8]. The sample was immediately dissolved in 0.6 ml ethyl acetate, and 2.4 ml n-hexane was added. The solution was passed through a 200 mg column of silicic acid (Unisil, Clarkson Chemical Co., Williamsport, PA, U.S.A.) in ethyl acetate/hexane (1 : 4, v/v), and the column was washed with 3 ml of the same solvent. The pooled fractions were evaporated to dryness and the residue was dissolved in ethyl acetate. The stability of the hydrogen atoms in the amino acids to exchange with the medium was tested in the following way. Frozen liver samples from two rats, and a mixture of 1 mg each of glutamic acid, aspartic acid, proline, and alanine were treated as described above using 2H2SO4/CH302H. The samples were passed through a column of Dowex 50W-X8 washed with 2H20 that was then rinsed with acetic acid in 2H20 and with 2H20. The eluted and derivatized amino acids were analyzed by gas chromatography-mass spectrometry as described below. The deuterium c o n t e n t was below the detection limit (1%), indicating that there are no losses of deuterium during the work-up of the amino acids in the experiments. The overall recovery in the procedure was studied by addition of ~4C-labelled glutamate, alanine and proline (Radiochemical Centre, Amersham, England) to the liver extract. Radioactivity was measured in a liquid scintillation counter using quenching correction with an internal standard. The recoveries of these amino acids were 90--100%. Gas chromatography-mass spectrometry. An LKB 9000 gas chromatographymass spectrometry instrument (LKB-Produkter AB, Bromma, Sweden) was used with a 3% OV-17 column, temperature programmed from 90 to 220°C at a rate of 3°C per min. The electron energy was 22.5 eV and the ion source temperature 310°C. Spectra for identification purpose were recorded on magnetic tape by the incremental technique, and spectra for isotope excess measurements were recorded on magnetic tape by continuous (sampling rate 10 kHz) recording of partial spectra obtained by accelerating voltage scanning over six mass numbers in 2 s. In the latter case, the values were bunched, giving 12 values per mass number [9]. The isotope excess was calculated using the intensities of the fragment ion resulting from loss of COO(CH2)3CH3 from the molecular ions [8]. The fractional i n c o r p o r a t i o n of hydrogen from the 1-pro-R and the 1-pro-S position of ethanol was calculated as previously described [5]. Results

The amino acid derivatives were identified by comparison of mass spectra and retention times with those of standards. The purity of the gas chromatographic peaks was checked by mass spectrometry. Fig. 1 shows typical plots of fragment ion current chromatograms obtained in the gas chromatographic-mass spectrometric isotope analyses. Administration of [1-2H2]ethanol resulted in rapid deuterium incorporation into aspartate (Table I). Alanine and proline were labelled to a small extent after 4 h of ethanol metabolism, whereas glutamate was n o t significantly labelled. In all instances, only m o n o d e u t e r a t e d molecules were detected. In

256 100

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F i g . 1. F r a g m e n t i o n c u r r e n t c h r o m a t o g r a m s obtained in the analyses of reference glutamate (A) and proline (B) and of glutamate (C) and proline (D) from the liver of a rat given [2-2H3]ethanol 2 and 4 h prior t o k i l l i n g . T h e i o n s m e a s u r e d w e r e a t role 2 5 4 ( e ) , 2 5 5 ( o ) , 2 5 6 ( o ) a n d 2 5 7 0~) i n t h e a n a l y s i s o f g l u t a m a t e , a n d a t m]e 1 6 6 ( $ ) , 1 6 7 ( o ) , 1 6 8 ( o ) a n d 1 6 9 (t~) i n t h e a n a l y s i s o f p r o l i n e .

TABLE

I

ABUNDANCE METABOLISM

(%) O F M O N O D E U T E R A T E D OF [1-2H2]ETHANOL

T h e v a l u e s ( m e a n +_ S . D . ) a r e c o r r e c t e d Amino acid

Duration

for the protium

Alanine Proline Aspartate Glutamate

0.8 0.6 13.3 0.1

+_ 0 . 4 _+ 0 . 4 _+ 1 . 2 +_ 0 . 1

ACID

content

MOLECULES

of the ethanols.

of ethanol metabolisin

20 min %

AMINO

240 rain n

%

3 3 4 4

1.7 2.8 15.4 2.3

tl + 0.5 +_ 1 . 4 +_ 1 . 7 +_ 2 . 3

3 4 4 4

IN

LIVER

DURING

257 TABLE II DEUTERIUM CONTENT (2H ATOMS/100 MINISTRATION OF [2-2H3]ETHANOL

MOLECULES)

I N A M I N O A C I D S I S O L A T E D A F T E R AD-

R e s u l t s are t h e m e a n ± S.D. o f f o u r e x p e r i m e n t s a n d e x p r e s s e d as p e r c e n t a g e . A m i n o acid

Alanine Proline Aspartate Glutamate

Duration of ethanol metabolism 5 min

20 m i n

0.4 1.3 0.5 7.8

0.8 3.5 1,7 18.8

_+ 0.6 ± 1.3 ± 0.2 ± 0.7

± + + +

240 min 0.5 0.9 0.7 6.0

1.7 11.8 4.8 42.8

± 0.8 ± 2.5 _+ 0 . 5 ± 3.2

separate experiments, four rats were given (1R)-[1-2H]ethanol and four rats were given (1S)-[1-2H]ethanol. The aspartate obtained from liver samples removed after 20 min of ethanol metabolism, contained a deuterium excess of 6.0--9.3% in the former case, and 3.5--4.1% in the latter case. The calculated transfer of the 1-pro-R hydrogen was 10.1%, and that of the 1-pro-S hydrogen 3.2%. Admininistration of [2-2H3 ]ethanol resulted in the formation of amino acids labelled with deuterium. Alanine and aspartate only became monodeuterated, whereas both m o n o d e u t e r a t e d and dideuterated proline and glutamate molecules were present. In no case were molecules containing more than two deuterium atoms detected. The total deuterium c o n t e n t in the molecules is given in Table II. The presence of m o n o d e u t e r a t e d proline and glutamate molecules indicates exchange of deuterium for protium since biosynthesis of these amino acids from acetyl-CoA involves the incorporation of two carbon-bound hydrogens from C-2. The ratio between the abundances of di- and m o n o d e u t e r a t e d proline and glutamate molecules were 1.11 ± 0.65 (S.D.) and 2.42 ± 0.15 (S.D.), respectively, in the 240-min samples. The degree of exchange (e) can be calculated from this ratio (r), if it is assumed t h a t the exchange is random: e = (2r + I) -I This equation is derived from the equations given in ref. 2. The results from such calculations are given in Table Ill. If these values for the degree of exchange are applied to the total deuterium contents given in Table II, the fraction of the amino acid that is derived from ethanol can be calculated [2]. For

TABLE In CALCULATED FRACTION OF DEUTERIUM IN [2-2H3]ETHANOL THAT METHYL CARBON PRIOR TO INCORPORATION INTO AMINO ACIDS R e s u l t s a r e t h e m e a n ± S.D. o f f o u r e x p e r i m e n t s . A m i n o acid

5 min

20 m i n

240 rain

Proline Glutamate

0 . 7 7 +- 0 . 3 3 0.23 + 0.01

0.31 +_ 0 . 1 0 0 . 1 2 +_ 0 . 0 6

0 . 3 6 +- 0 . 1 5 0 , 1 7 -+ 0 . 0 1

IS L O S T F R O M

THE

258 proline the figures are 2.5 and 9.2% in the 20-min and the 240-min samples, respectively, and for glutamate the figures are 5.1, 10.7 and 25.8% in the 5-min, 20-min and 240-min samples, respectively. Discussion The m e t h o d used in the present study is based on the in vivo administration of ethanols labelled with deuterium. The efficient labelling of the amino acids indicates the usefulness of this technique in studies on humans, where the use of radioactive isotopes is to be avoided. One problem in the quantitative interpretation of the results is the presence of kinetic isotope effects. This has been discussed previously [1,2], and the major consequence is a possible underestimation of the incorporation of hydrogen either via NADH or bound to acetylCoA. Likewise the extent of hydrogen exchange might be underestimated, since the primary isotope effect would result in a preferential loss of protium during citrate formation from partially deuterated acetyl-CoA molecules.

Aspartate The incorporation of deuterium from C-1 of ethanol was lower in liver aspartate (13--15%) than in corresponding positions of malate and fumarate (about 21%, Ref. 1). This indicates that 60--70% of the liver aspartate was formed from malate. However, the value may be an underestimation since deuterium could be lost from oxaloacetate by enolization [10]. The relative incorporation of the 1-pro-S and the 1-pro-R hydrogens of ethanol into aspartate is independent of losses of deuterium from oxaloacetate and contributions from unlabelled compounds. The ratio between the incorporation from the 1-pro-S and 1-pro-R positions was about the same as previously found for hydrogen at C-3 in the glycerol moiety of phosphatidylcholines [11]. This supports the validity of this ratio as a characteristic of coenzyme pools, since in both cases the hydrogens are primarily incorporated in malate in the malate dehydrogenase reaction (EC 1.1.1.37). Aspartate was much less labelled than glutamate during metabolism of [2-2H3]ethanol. This cannot be due to slow reactions in the sequence fumarate ~ malate-~ oxaloacetate-~ aspartate, since metabolism of [1-2H2]ethanol rapidly resulted in extensive labelling of these compounds. Thus, it is likely that the conversion of labelled 2-oxoglutarate to fumarate is slow compared to enolization and loss of deuterium in oxaloacetate.

Alanine Hydrogens at C-1 and C-2 of ethanol may appear in alanine via malate, oxaloacetate and pyruvate. Alanine was much less labelled than aspartate after administration of [1-2H2]ethanol. This can only partly be explained by losses during transamination (about 25%, ref. 12). Thus, the results indicate that liver alanine was derived from other sources, e.g. lactate, glucose, and extrahepatic alanine. As expected from the low labelling of aspartate, alanine had a low deuterium excess when [2-2H3]ethanol was administered.

Glutamate No deuterium was incorporated in glutamate from [1-2H2]ethanol. However,

259

this does not permit any conclusions to be drawn regarding the labelling of the intramitochondrial NADH pool since deuterium exchange would occur in the reactions catalyzed by aminotransferases [ 13]. The [2-2H3]ethanol experiments showed that at least 26% of the acetyl-CoA precursor of glutamate was derived from ethanol. Some deuterium was lost (12--23%) due to exchange, probably at the acetaldehyde or acetyl-CoA stage. This loss has previously been observed in other acetyl-CoA metabolites [14]. Proline Glutamate is a precursor of proline, both in vivo [15,16] and in rat liver slices [17], although the biosynthetic route is incompletely known. Proline may also be formed from arginine and ornithine [18]. The incorporation of deuterium in proline during metabolism of [2-:H3]ethanol is most likely due to formation from glutamate, via glutamic 7-semialdehyde. A larger fraction of the deuterium had been exchanged for protium in the formation of proline than in the formation of glutamate. This may be due to reversible enolization of glutamic ~-semialdehyde prior to cyclization. After 4 h of [2-2H3]ethanol metabolism, 9% of the proline and 26% of the glutamate had incorporated C-2 of ethanol. Thus, at least 36% of the free liver proline had been synthesized from glutamate. This value is comparable to that calculated from the ratio between the specific activities of proline and glutamate in guinea pig skin collagen after injection of [ 14C] glutamate [ 16]. It has been suggested that alcoholic liver cirrhosis may be due to elevated concentrations of proline [19]. Thus, addition of ethanol to normal rat liver slices results in increased formation of proline from 2-oxoglutarate [19], and high proline concentrations may result in increased collagen synthesis [ 17], and thus possibly in the development of liver cirrhosis [ 18,20]. Increased formation of proline during ethanol oxidation might be due to the increased concentrations of reduced pyridine nucleotides [ 19]. If formation of NADH used in proline synthesis were closely coupled to the oxidation of ethanol a high degree of hydrogen transfer from C-1 of ethanol might be expected. However, this was n o t observed and only 3% of the hydrogen in proline was derived from C-1 of ethanol when more than one third of the proline was newly synthesized. Thus, the present study does not support the hypothesis that alcoholic liver cirrhosis is due to an increased proline production caused by the increased NADH concentration. On the other hand, the efficient formation of amino acids from acetate derived from ethanol suggests that there may be other mechanisms by which ethanol interferes with amino acid metabolism.

Acknowledgements This work was supported by grants from the Swedish Medical Research Council (project 25X-2189) and from Karolinska Institutet. References 1 Cronholm, T., Matern, H., Matern, S. and SjSvaU, J. (1974) Eur. J. Biochem. 48, 71--80 2 Cronholm, T., Burlingame, A.L. and SjSvaU, J. (1974) Eur. J. Biochern. 49, 497--510

260 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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