Effect of the presence of labile methyl groups in the diet on labile methyl neogenesis

88

VOL. 12

I31OCH1MICA F.T BIOPHYSI(A ACTA

EFFECT

OF THE PRESENCE

(I953)

OF LABILE METHYL GROUPS IN THE

D I E T ON L A B I L E M E T H Y L N E O G E N E S I S by VINCENT DU VIGNEAUD,

J O H N M. K I N N E Y ,

AND J U L I A N

JOHN

E. W I L S O N

R. R A C H E L E

Department o/Biochemistry, Cofnell University Medical College, New York (U.S..~.)

Tissue slice experiments employing 14C-labelled precursors 1-3 and experiments with germ-free animals receiving deuterium-labelled drinking water4, s have demonstrated that "biologically labile" methyl groups are synthesized in the tissues of the rat. In the latter experiments the deuterium concentration of the body water of both germ-free and nonsterile animals was maintained at about 3 atom per cent. After a period of approximately 3 weeks the choline was isolated from the tissues of these rats and the deuterium concentration of the trimethylamine chloroplatinate prepared from the isolated choline was determined. The level of deuterium in the methyl groups of the choline of these animals was between 6. 4 and 9.6% of that in the body water. These values agreed closely with the levels obtained in a much earlier experiment in which ordinary animals were given deuterium-labelled drinking water G. In interpreting these results it was reasoned that if synthesis of the labile methyl group did occur from some precursor in the presence of D20, then the methyl group so formed would have deuterium introduced into it during the synthetic process. From our previous experience that deuterium attached to the carbon of the labile methyl group did not exchange appreciably with the hydrogens of the body water 7 9, it appeared highly unlikely that in such experiments a direct exchange between the hydrogens of the methyl group and the deuterium of the D20 in the body water had taken place to bring about the appearance of deuterium in the methyl group. Thus, the finding of deuterium in the methyl groups of choline under these conditions justified the conclusion that synthesis of the methyl group must have occurred. In all of this work on the uptake of deuterium, the diet of the animals contained labile methyl groups, so that synthesis was apparently occurring to the extent observed, even in the presence of a dietary source of labile methyl groups. If the interpretation were correct that the appearance of deuterium in the labile methyl groups of the body is a reflection of the degree of neogenesis of methyl groups, then the appearance of a larger amount of deuterium in tile methyl groups of choline should result under dietary conditions in which the animal was forced to synthesize its entire supply of labile methyl groups. The present paper reports a set of experiments designed to test whether this was the case. The amount of incorporation of deuterium from body water into the labile methyl group in animals receiving dietary methyl groups was compared with t h a t in Re/erences p. 9 ±.

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LABILE METHYL NEOGENESIS

89

a n i m a l s on a m e t h y l - f r e e diet. B o t h groups of a n i mal s r e c e i v e d B ~ an d folic acid in sufficient a m o u n t s to ensure synthesis of labile m e t h y l groups a n d p r o m o t e g r o w t h w h e n t h e labile m e t h y l groups were n o t p r e s e n t in t he diet. T h e results fitted in w i t h w h a t w o u ld be p r e d i c t e d on t h e basis of t h e c o n c e p t outlined. I n t h e case of th e a n i m a l s on t h e m e t h y l - c o n t a i n i n g diets, f r o m 5.5 to lO.4% of t he choline m e t h y l was d e r i v e d f r o m t h e b o d y water, whereas in t h e case of t h e a nima l s on t h e m e t h y l - f r e e diet, the p e r c e n ta g e s r a n g e d f r o m 24.4 to 34.3. T h u s t h e i n c o r p o r a t i o n of d e u t e r i u m into t h e m e t h y l g r o u p of choline in t h e an i m al s on t h e m e t h y l - f r e e diet was a l m o s t four times as g r e a t as t h a t in t h e an i m al s r ecei v i n g diets c o n t a i n i n g labile m e t h y l c o m p o u n d s .

EXPERIMENTAL The eight rats used for these e x p e r i m e n t s were k e p t in raised cages e q u i p p e d for urine collection. D r i n k i n g w a t e r was m a d e c o n t i n u o u s l y available. Th e animals were placed on an a m i n o acid diet c o n t a i n i n g h o m o c y s t i n e a n d choline, b u t no m e t h i o n i n e . T he c o m p o s i t i o n of this diet, which was fed a d l i b i t u m , is shown in Tab l e I. Th e weights TABLE I PERCENTAGE

C O M P O S I T I O N OF I N I T I A L D I E T

Sucrose Salt mixture 1° Amino acid mixture* Water soluble vitaminst§ Corn oil Vitamins E and K in corn oil** Vitamins A and D conc.*** DL-Homocystine Choline chloride

73 4 17.2 I 2 I 0.5 drop I 0.8

* Glycine, o.I; L-hydroxyproline, o.I; L-proline, o.2; nL-serine, o.2; L-aspartic acid, o.2; nLalanine, 0. 4; L-tryptophan, 0. 4; L-arginine. HC1, o.6; L-histidine. IlC1. H20, o.7; L-tyrosine, I.O; DL-tbreonine, 1.4; nL-phenylalanine, 1.5 ; DL-isoleucine, 1.8 ; nL-valine, 2.o ; L-glutamic acid, 2.o; L-leucine, 1.3; L-lysine-HC1, 1.9; sodium bicarbonate, 1.4. * Biotin, o.oi nag; folic acid, o.i mg (increased to 0. 4 mg in the case of Rats 3o, 31); thiamin chloride, riboflavin, pyridoxine hydrochloride, nicotinic acid, and p-aminobenzoic acid, i mg each; calcium d-pantothenate, 5 mg; inositol, IO rag; Vitamin B12, 20 #g (except for Rats 3° and 31, which received 15/~g); sucrose to make i g. § In the case of Rats 3° and 31, the vitamins were removed from the diet on the 2oth day and the following vitamin solution was given per os in two 0. 5 ml portions daily (rag per ml of solution) : thiamin chloride, riboflavin, nicotinic acid, pyridoxine hydrochloride, p-anlinobenzoic acid, o.8 each; calciuni d-pantotbenate, 0.40; inositol, o.8o; folic acid, o.oo8; biotin, 0.0008, sucrose, ioo rag. ** Containing 4 mg of a-tocopherol acetate and o.i mg of 2-methyi-I,4-naphthoquinone. *** Containing 75o I.U. of vitamin A and 125 I.U. of vitamin D. of the an i m al s at v ar i o u s stages of the e x p e r i m e n t are s u m m a r i z e d in Tab l e I I , along w i t h t he days on wh i ch t h e d i e t a r y r e g i m e n s were changed. F o u r of the rats (3 ° , 31, 4o an d 41) were m a i n t a i n e d on a labile m e t h y l - f r e e diet t h r o u g h o u t the e x p e r i m e n t a l period. Tw o of t h e an i m al s (42 a n d 43) r e c e i v e d t h e diet gi v en in Tab l e I c o n t a i n i n g choline a nd t he o t h e r t w o (44 a n d 45) r e c e i v e d a diet in w h ich the choline was r e p l a c e d b y a 1% level of m et h i o n i n e. A f t e r the a n i m a l s h a d g r o w n on these regimens for t h e t i m e s Re/erences p. 9 L

v. I)U VmNEAUD et al.

9°

VOL. 12 (1953)

indicated in Table II, deuterimn oxide was administered in the drinking water at a level of Io atom per cent for 4 days and then at a level of 4 atom per cent. for the remainder of the 3-week experimental period. The animals were then sacrificed with chloroform and immediately frozen in dry ice. TABLE

II

x,VEIGHTS OF ANIMALS IN GRAMS 28

38

57

78

Rat No.

Sex

Wearied

o

Initial

Choline* removed

D20

begun

Sacrificed

3or 317 4° 41 42 43 44 45

~ d c~ ~ 9 9 ~ 9

4t 39 38 32 34 26 36 33

41 39 136 lO 4 92 93 112 98

I21 I2I i48 122 t22 12o 123 119

214 208 188 192 172 164 168 162

Day of Experiment

Diet

238 234 23I 226 187 17o 202 t76

* R a t s 42 a n d 43 w e r e c o n t i n u e d on t h e initial c h o l i n e - c o n t a i n i n g diet; rats 44 a n d 45 w e r e p l a c e d on a diet in w h i c h t h e c h o l i n e w a s r e p l a c e d b y m e t h i o n i n e . ? R a t s 3 ° a n d 31 w e r e b e g u n on t h e a m i n o acid diet at w e a n i n g , c h o l i n e w a s r e m o v e d 3 ° d a y s later, D~O w a s b e g u n 79 d a y s l a t e r a n d t h e a n i m a l s w e r e sacrificed on t h e i o o t h d a y .

The choline was isolated as the chloroplatinate and degraded to trimethylamine according to procedures described previously 5. The average deuterium content of the body water during the last 3 weeks of the experiment was determined by measuring by the mass spectrometer methodU, 12 the deuterium content of water from the pooled urine of each animal for this period. The deuterium contents of the trimethylamine chloroplatinates were likewise determined. These values were then used to calculate the extent to which the deuterium of the body water had been incorporated into choline methyl groups. The experimental results are summarized in Table III. TABLE

III

ISOTOPIG CONTENTS OF BODY WATER AND CHOLINE METHYL GROUP A verage deuterium Rat No.

3 °* 31 ~ 4° 41. 42 43 44 45

Deuterium content

in body water (A)

TMA chloroplatinate

Methyl group of choline (B)

atom per cent. excess

atom per cent. excess

atom per cent. excess

2.71 2'63 2.95 3.3 ° 3.26 4.04 3.04 3.44

0.84 o.75 o.65 0.84 o.31 0.34 0.24 o.17

0.93 0.83 0.72 0.93 0.34 0.38 0.27 o.19

* T h e s e rats on t h e labile m e t h y l - f r e e diet. R e / e r e n c e s p. 9~.

Per cent. of methyl hydrogen derived from body water

(B/A X Ioo)

34-3 31.6 24.4 28.3 lO.4 9.4 8.9 5.5

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LABILE METHYL NEOGENESIS

91

SUMMARY T h e i n c o r p o r a t i o n of d e u t e r i u m from isotopically labelled b o d y w a t e r into t h e m e t h y l g r o u p s of b o d y choline h a s been used as a n i n d i c a t i o n of m e t h y l s y n t h e s i s in t h e r a t in vivo. As a t e s t of t h i s c o n c e p t t h e a m o u n t of isotope a p p e a r i n g in t h e b o d y choline of r a t s on a m e t h y l - f r e e diet h a s b e e n c o m p a r e d w i t h t h a t in r a t s receiving choline or m e t h i o n i n e in t h e diet. T h e a n i m a l s g r o w i n g well w i t h o u t a source of m e t h y l g r o u p s in t h e diet were f o u n d to i n c o r p o r a t e a p p r o x i m a t e l y four t i m e s as m u c h isotope into b o d y choline as a n i m a l s g i v e n choline or m e t h i o n i n e . Rt~SUM£ L ' i n c o r p o r a t i o n de d e u t e r i u m 5. p a r t i r de l ' e a u m a r q u 6 e d a n s les g r o u p e s m 6 t h y l i q u e s de la choline de l ' o r g a n i s m e a 6t6 eonsid6r6e c o m m e u n signe de la s y n t h ~ s e m 6 t h y l i q u e chez le r a t in vivo. P o u r vdrifier la validit6 de c e t t e h y p o t h ~ s e , les a u t e u r s o n t c o m p a r 6 la q u a n t i t 6 d ' i s o t o p e d6celable d a n s la choline de l ' o r g a n i s m e chez des r a t s carenc6s en g r o u p e m e n t s m 6 t h y l i q u e s et chez des r a t s r e c e v a n t de la choline ou de la m 6 t h i o n i n e d a n s leur r6gime. Les a n i m a u x q u i croissent n o r m a l e m e n t a v e c u n r6gime s a n s g r o u p e m e n t s m 6 t h y l i q u e s i n c o r p o r e n t e n v i r o n q u a t r e fois p l u s d ' i s o t o p e d a n s la choline de leur o r g a n i s m e q u e c e u x q u i re~oivent de la choline ou de la m 6 t h i o n i n e . ZUSAMMENFASSUNG D e r E i n b a u v o n D e u t e r i u m a u s m i t I s o t o p e n m a r k i e r t e m K 6 r p e r w a s s e r in die M e t h y l g r u p p e n des K 6 r p e r c h o l i n s w u r d e b e n u t z t als ein A n z e i c h e n der M e t h y l s y n t h e s e in der R a t t e in vivo. Z u r P r f i f u n g dieser V o r s t e l l u n g w u r d e die M e n g e Isotop, die i m K 6 r p e r c h o l i n der R a t t e n bei m e t h y l f r e i e r Di/it erscheint, v e r g l i c h e n m i t d e r j e n i g e n y o n Cholin oder M e t h i o n i n in der Di~t e m p f a n g e n d e n R a t t e n . E s w u r d e g e f u n d e n , class o h n e eine Quelle f/Jr M e t h y l g r u p p e n in der Di~t g u t w a c h s e n d e Tiere u n g e fiihr 4 real so viel I s o t o p e in d a s K 6 r p e r c h o l i n e i n b a u e n , als Tiere, die Cholin oder M e t h i o n i n erhielten. REFERENCES 1 w . SAKAMI AND A. D. WELCH, J. Biol. Chem., 187 (195 o) 379. 2 W . SAKAMI, Federation Proc., 9 (195 o) 222. P. BERG, J. Biol. Chem., 19 ° (1951) 31. 4 V. DU VIGNEAUD, C. RESSLER AND J. R. RACHELE, Science, 112 (195o) 267. 5 V. DU VIGNEAUD, C. RESSLER, J, R. RACHELE, J. A. REYNIERS AND T. D. LUCKEY, J. Nutrition, 45 (1951 ) 361. 6 V. DU VIGNEAUD, S. SIMMONDS, J. P. CHANDLER AND M. COHN, J. Biol. Chem., 159 (1945) 795. 7 V. DU VIGNEAUD, M. COHN, J. P. CHANDLER, J. R. SCHENCK AND S. SIMMONDS, J. Biol. Chem., 14o (1941 ) 625. s E. B. KELLER, J. R. RACHELE AND V. DU VIGNEAUD, J. Biol. Chem., 177 (1949) 733. 9 V. DU VIGNEAUD, W . G. VERLY, J. E. WILSON, J. R. RACHELE, C. RESSLER AND J. M. KINNEY, J. Am. Chem. Soc., 73 (1951) 2782. 10 j . H. JoNEs AND C. FOSTER, J. Nutrition, 24 (1942) 245. n j . GRAFF AND D. RITTENBERG, Anal. Chem., 24 (1952) 878. 12 H. W. WASHBURN, s u p p l e m e n t to U. S. N a v a l Medical Bulletin, B u r e a u of Medicine a n d Surgery, U. S. N a v y , W a s h i n g t o n , D.C., p. 60 (1948). Received

March 23rd, 1953