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High concentration of free trimethyllsine in red blood cells
Biochimica et Biophysica Acta. 1033 (1990) 119-123 Elsevier
119
BBAGEN 23234
High concentration of free trimethyllysine in red blood cells Mutsuhiko Mizobuchi 1, M a s a h a r u M i y a k e 2, Akira Sano i and Yasuo Kakimoto 1 J The Department of Neuropsyehiatry and ? Central Research Laboratory, Ehime University, School of Medicine, Ehime (Japan)
(Received 10 August 1989)
Key words: Trimethyllysine;Methylatedamino acid; Red blood cell A high concentration of a basic unidentified amino compound was found in the blood of rats. It was isolated and identified as N~,N',N'-trimethyllysine by paper chromatography, thin-layer chromatography, high-performance liquid chromatography and amino acid analyzer. It was localized exclusively in red blood cells in the blood of rats. Free trimethyllysine was also determined in the liver, kidney, spleen, brain, muscle, heart and testis of rat. The concentration of free trimethyilysine in red blood cells was more than lO-times as high as that in the other tissues. This compound in red blood cells was found in different species of animals. The relationship between this free trimethyllysine and carnitine was discussed.
Introduction Protein N-methylation is one of the major posttranslational modifications principally involving histidine, lysine and arginine residues. Methylated amino acid residues such as 3-methylhistidine, N'-monomethyllysine, N~,N'-dimethyllysine, N~,N~,N~-trimethyllysine, NG-monomethylarginine, N ~ , N G - d i m e t h y l arginine and N , N -dlmethylargmlne occur in various proteins [1-4]. They are released as free amino acids when methylated proteins are subjected to metabolic breakdown and are not reutilized for protein synthesis. Most of these free methylated amino acids are transported in blood plasma and excreted into urine without being reabsorbed by the kidney [3,5], although some methylated amino acids are metabolized in the body [6,7]. It was demonstrated that high concentrations of free methylated amino acids were present only in urine [1,3,8]. We found an unknown compound in the blood of rats, which was ninhydrin positive and was fractionated into a basic amino acid fraction during purification. We identified this compound as free trimethyllysine. It was localized in red blood cells. Trimethyllysine enters the carnitine biosynthetic pathway by the action of mitochondrial hydroxylase [3,9]. The essential role of carnitine in the mitochondrial B-oxidation of fatty acids is now well established [10,11]. The origin of this trimethyllysine is, however, unclear. (3
tG
•
-
•
Correspondence: M. Mizobuchi, Department of Neuropsychiatryand Central Research Laboratory, Ehime University, School of Medicine, Shigenobu-cho, Onsen-gun, Ehime, Japan.
The present study describes the presence of free trimethyllysine in red blood cells and a species difference in the amounts of this amino acid. This may provide useful information for carnitine biosynthesis. Materials and Methods Animals and diets We used Wistar rats (220-365 g), C 5 7 / B L mice (13-26 g), Beagle dogs (7-12 kg), chickens (line 412, 1.2-1.8 kg), Japanese monkeys (6-13 kg), rabbits (3-4.5 kg) and adult human blood. The care and use of laboratory animals in these experiments followed the guidelines of the National Research Council. Preparation of blood and tissue samples Blood was collected in a tube containing heparin usually from the cervical artery of animals and was immediately placed on the ice box. Human blood was obtained from the brachial vein. The blood was centrifuged at 1400 x g for 3 min. The supernatant fraction, which contained plasma and platelets, was separated into pure plasma by centrifugation at 2250 x g for 15 min. Red blood cells were obtained by full aspiration of buffy coat, washed with cold saline solution and centrifuged at 1400 x g for 3 min. Unless otherwise specified, red blood cells in all experiments were washed twice with saline solution. Hematocrits were determined using an International Micro-capillary Centrifuge Model MB and an International Microcapillary Reader. Rats were decapitated and perfused with cold saline solution, via the blood vessel, to remove blood. Liver,
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
120 kidney, spleen, brain, muscle, heart and testes were immediately removed. To obtain reticulocytes, rats were made anemic by intraperitoneal injections with 0.5 ml of HCl-neutralized solution of phenylhydrazine (20 m g / k g body weight) once a day for six days [12]. The animals were killed on the third day after cessation of injections and blood was collected and washed as described above. Blood and tissue samples were homogenized in 9 vol. of 10% trichloroacetic acid, sonicated for 1 min and centrifuged at 10000 × g for 15 min. The precipitate was suspended in the same volume of 10% trichloroacetic acid and centrifuged at 10000 × g for 15 min. After repeated suspension and centrifugation, these supernatants were passed through a 1 × 4 cm column of Amberlite IR-120 (100-200 mesh, H + form) and the resin was washed with 20 ml of water. Amino acids were eluted with 20 ml of 3 M ammonium hydroxide, evaporated to dryness and applied to a 1 × 4 cm column of Amberlite CG-50 (100-200 mesh, N H J form) to remove histidine and its derivatives. The column was washed with 20 ml of water. Basic amino acids were eluted with 20 ml of 3 M ammonium hydroxide and evaporated. These samples were subjected to analysis of the basic amino acids.
Analysis of methylated derivatives of lysine Paper chromatography was performed in two solvent systems. The solvent systems were p y r i d i n e / a c e t o n e / 3
B
A
e-J ¢:o.
62
e. o e-
0
ew
m u~
% 7.
o
Ik~jJ
M ammonium hydroxide ( 1 0 : 6 : 5 ) and isopropyl alc o h o l / f o r m i c a c i d / w a t e r (4:1 : 1). Chromatographic paper was Toyoroshi No 51A which is similar to Whatman No. 1 paper. The thin-layer chromatographies were carried out in two solvent systems of methanol/3 M ammonium hydroxide (3 : 1 : 2). The chromatographic sheet was DCPlastikfolien KJeselgel 60 (Merk). Basic amino acids were applied in the high-performance liquid chromatography on a column (15 cm × 4 mm internal diameter) of Shimazu ISC-07/S 1504 resin and eluted with 0.8 M lithium citrate buffer, pH 5.5 at 40 ° C at a flow rate of 0.5 ml/min. The usual analytical pattern in the whole blood is shown in Fig. lB. The concentration of lysine and methylated derivatives of lysine was determined routinely by the automatic amino acid analyzer according to the method of Kakimoto and Akazawa [8]. Methylated lysine derivatives were synthesized according to procedures described in the literature [3]. Results
Isolation and identification of free trimethyllysine in the blood Blood (180 g) was collected from 20 male rats and homogenized in 9 vol. of 10% trichloroacetic acid. The supernatant solution was successively applied to Amberlite IR-120 and Amberlite CG-50 column chromatography as described in Materials and Methods. Basic amino acids were fractionated on a column (0.9 x 30 cm) of cation exchange resin (Aminex 21-23) with 0.51 M NaC1 in 0.2 M sodium citrate buffer by an automatic amino acid analyzer. An unidentified compound was eluted from 112 to 128 min. This fraction was collected and passed through a 2.2 x 10 cm column of Amberlite IR-120 (H + form) to desalt and the column was washed with 200 ml of water. This compound was eluted with 200 ml of 3 M ammonium hydroxide. The eluate was evaporated to dryness under reduced pressure (yield 1.2 rag). The compound was analyzed by paper chromatography, thin-layer chromatography, amino acid analyzer and high-performance liquid chromatography. R r values and retention times are summarized in Table I. The R F values and retention time of this compound matched completely those of synthesized trimethyllysine.
Retention time (min)
Methylated derivatives of lysine in red blood cells and plasma of rats
Fig. 1. The chromatographic pattern of lysine and methylated lysine by high-performance liquid chromatography. (A) Chromatogram of synthesized lysine and methylated lysine (1 nmol). Orn, ornithine; Lys, lysine; MML, monomethyllysine; DML, dimethyllysine; TML, trimethyllysine (B) Chromatogram of the free basic amino acids fraction of rat whole blood. The retention time of the unknown compound, indicated by an arrow, corresponded to trimethyllysine.
Red blood cells and plasma were obtained as described in Materials and Methods. Red blood cells were pelleted by centrifugation and washed five times with cold saline. More than 95% of the trimethyllysine of non-washed cells was recovered in the well-washed cells. Therefore, red blood cells can be washed with
121 TABLE I
TABLE III
R F values and retention time of isolated compound and synthesized methyl derivatives of lysine by thin-layer chromatography, paper chromatography, amino acid analyzer and high.performance liquid chromatography Thin-layer chromatography (TLC) and paper chromatography (PCG) were both performed in two different solvent systems (A and B, C and D~ respectively).About 5 nmol of isolated compound and synthesized methyl derivativesof lysine were applied and visualizedwith ninhydrin. These compounds were separated by an amino acid analyzer and high-performance liquid chromatography (HPLC), in which [hey reacted with ninhydrin and orthophthalaldehyde, respectively. Compound
Isolated compound Trimethyllysine Dimethyllysine Monomethyllysine Lysine
R v value TLC
PCG
A
C
B
Retention time (min) D
amino acid analyzer
HPLC
0.86 0 . 2 2 0 . 1 6 0.64 0 . 8 6 0 . 2 2 0 . 1 6 0.64 0.46 0 . 7 0 0.48 0.66
120 120 155
58.6 58.6 52.3
0.38 0.32
168 170
45.9 35.9
0 . 4 6 0.33 0.62 0 . 5 6 0 . 3 7 0.52
A, phenol/water/3 M ammonium hydroxide (3 : 1 : 2); B, methanol/3 M ammonium hydroxide (1:2); C, pyridine/acetone/3 M ammonium hydroxide (10 : 6 : 5); D, isopropyl alcohol/formic acid/water (4:1:1).
saline solution to remove a n y p l a s m a t r a p p e d in red b l o o d cells w i t h o u t a n y loss. Unless otherwise specified, red blood cells in all experiments were washed twice with saline solution. The c o n c e n t r a t i o n of m e t h y l a t e d derivatives of lysine in whole blood, red b l o o d cells a n d p l a s m a are presented in T a b l e II. The c o n c e n t r a t i o n s of dimethyllysine a n d m o n o m e t h y l l y s i n e in all samples were less t h a n 1 n mol per g wet weight. O n calculation from hematocrit, approx. 97% of trimethyllysine in the b l o o d was calculated to be present in red b l o o d cells,
TABLE II The concentrations of methylated lysine and lysine in whole blood, red blood cells and plasma of rats The preparation of whole blood, red blood cells and blood plasma are described in Materials and Methods. Data represent mean _+S.D. for seven rats. Rat
Whole blood Red blood cells Blood plasma
nmol/g wet weight trimethyllysine
dimethyllysine
monomethyllysine
lysine
32 + 5
<1
<1
428 __+18
69 + 5 <1
<1 <1
<1 <1
480 + 24 317 + 32
The concentration of free trimethyllysine and lysine of various organs in rat
Blood and tissues were homogenized in 9 vol. of 10% trichloroacetic acid, sonicated for 1 min and centrifuged. These supernatants were passed through the column described in the Materials and Methods. These samples were applied to the amino acid analyzer. Data represent mean_+S.D. Values in parentheses represent the numbers of animals used. Rat organs Red blood cell Liver Kidney Spleen Brain Muscle Heart Testis
Number of
nmol/g wet weight
samples
trimethyllysine
lysine
7 5 5 5 5 5 5 5
69 _+5 3_+1 6 _+1 5 _+1 3_+1 4_+0 3_+0 2_+0
480_+ 24 566_+ 51 743 _+l l 1 456_+ 37 290_+ 36 362_+ 75 513_+ 8 396_+ 56
a n d the rest was calculated to be present in b l o o d plasma. T h e trimethyllysine c o n c e n t r a t i o n s of the platelet fraction a n d buffy coat which c o n t a i n e d white b l o o d cells were u n d e r 1 n mol per g wet weight. W a s h e d red b l o o d cells were b r o k e n by the a d d i t i o n of a n equal v o l u m e of deionized water a n d by freezing a n d t h a w i n g three times. After centrifugation, trimethyllysine was recovered in the s u p e r n a t a n t fraction, which was free from the p l a s m a m e m b r a n e of red blood cells. This indicates that free trimethyllysine in red blood cells is w i t h i n the cytoplasm. Concentration of free trimethyllysine in various organs of rat T h e c o n c e n t r a t i o n s of trimethyllysine a n d lysine of the liver, kidney, spleen, brain, muscle, heart a n d testis in rats are s h o w n in T a b l e III. The c o n c e n t r a t i o n s of trimethyllysine in these organs were between 6.3 a n d 1.8 n mol per g wet weight. The c o n c e n t r a t i o n of trimethyllysine in red b l o o d cells was more than 10-times as high as i n these organs, while the c o n c e n t r a t i o n of lysine was similar to that of other organs. Concentration of free trimethyllysine in red blood cells from various blood vessels Blood was o b t a i n e d from a b d o m i n a l artery, portal vein a n d inferior a b d o m i n a l vein. The c o n c e n t r a t i o n s of free red b l o o d cell trimethyllysine in a b d o m i n a l artery a n d inferior a b d o m i n a l vein were the same as that in cervical artery. The c o n c e n t r a t i o n s of free trimethyllysine a n d lysine of red b l o o d cells in portal vein were 165 _+ 5 a n d 1371 _+ 162 n m o l / g , respectively. O n l y in the portal vein, they were higher t h a n in the other vessels.
122 TABLE IV
The concentration of free trimethyllysine in reticulocytes and erythrocytes of rat and in red blood cells of various animals Reticulocytes were obtained from phenylhydrazine induced anemic rats as described in Materials and Methods. Data represent mean _+ S.D. Values in parentheses represent the numbers of experimental animals. Species
Rat erythrocyte reticulocyte Mouse Rabbit Dog Monkey Chicken Man
Number of
nmol/g wet weight
samples
trimethyllysine
lysine
7 5 6 2 5 5 5 4
69+_ 5 54+ 4 47+ 6 79_+ 26 36_+ 12 27+_ 7 8+ 2 2+_ 1
480+_ 24 815_+158 218_+ 19 220_+ 43 319_+ 56 338+_ 50 194_+ 93 76+_ 29
Concentrations of free trimethyllysine in reticulocytes and erythrocytes of rat and in red blood cells of different animal species Reticulocytes were obtained from phenylhydrazineinduced anemic rats as described in Materials and Methods. More than 90% of red blood cells consisted of reticulocytes. As seen in Table IV, the concentrations of trimethyllysine and lysine in reticulocytes were 54 and 815 n mol per g wet weight, respectively. The concentration of trimethyllysine in reticulocytes was somewhat lower than that in mature erythrocytes. The concentrations of blood trimethyllysine and lysine were determined in some animal species including mice, rabbits, dogs, monkeys, chickens and Man (Table IV). The concentrations of plasma trimethyllysine were less than 1 n mol per g wet weight in any species of animals, while those of red blood cells varied extremely among species, ranging from 2 to 79 n mol per g wet weight. The reason why the trimethyllysine concentration of human red blood cells was very low may be related to the preparation being from blood in the brachial vein. Discussion
Free trimethyllysine, dimethyllysine, Ne-monomethyllysine, NG, N°-dimethylarginine and N ° , N ° - d i methylarginine have been isolated from human urine, while in plasma they have been determined in very low concentrations [8,13]. This report describes, for the first time, a high concentration of free methylated amino acid found in mammalian red blood cells. Methylation of free lysine has been demonstrated in the mold Neurospora crassa [14,15], but not in mammalian organs. We incubated either [2,3-3H]lysine or S-adenosyl[methyl-3H]methionine with erythrocytes or
reticulocytes of rats, but no radioactivity was found on the spot of trimethyllysine in these experiments (unpublished observation). Free methylated amino acids are partially derived from dietary proteins and endogenous proteins upon degradation. The origin of the high concentration of free trimethyllysine in red blood cells is unclear. One explanation is that they move actively from plasma. Another explanation is that methylated proteins in red blood cells are degraded and free trimethyllysine is retained without being released through the red blood cell membrane. The concentration of free red blood cell trimethyllysine in the portal vein was about 2.4-times higher than in any other vessel. This suggests that trimethyllysine in red blood cells does not originate from the breakdown of its own methylated proteins. Rather trimethyllysine-containing proteins are digested in the gastrointestinal tract, absorbed, accumulated in the portal vein, transported and retained in red blood cells. It is necessary to show that only trimethyllysine is selectively transported into red blood cells among various methylated amino acids. The concentrations of carnitine in red blood cells and plasma have been reported by many investigators [16-18]. In adult rats, approx. 70-80% of whole blood carnitine was found in plasma, while 20-30% was in red blood cells. Trimethyllysine exists almost exclusively in red blood cells. Three possibilities have been suggested concerning the origin of trimethyllysine for carnitine biosynthesis in the rat. Zaspel et al. [19] have postulated that, since liver is the only tissue (in rat) which can hydroxylate "f-butyrobetaine to form carnitine, the liver proteins could provide all of the trimethyllysine required for conversion into carnitine. Carter and Frenkel [20] have suggested that free trimethyllysine is generated in various tissues from protein degradation, transported to the kidney, and converted to "y-butyrobetaine. This compound is then transported to the liver, where it is hydroxylated to form carnitine. In both experiments, transport and metabolism of trimethyllysine were examined only under conditions of malnutrition such as starvation, and only by the intravenous injection of labeled trimethyllysine. Rebouche et al. [17] have proposed that the availability of trimethyllysine is ratelimiting for the synthesis of carnitine in the growing rat and considered the diet proteins as a source of the precursor for carnitine biosynthesis. In this experiment, rats were raised with standard assorted feed, which had 0.3 /~mol of trimethyllysine residues per g. They were fed about 20 g of the diet per day, which contained 6/~mol of trimethyllysine residue. Calculating from the turnover rate of body protein and the content of trimethyllysine in the body, it is considered that about 14/~mol of trimethyllysine per 300 g rat is formed from whole body protein in a day [21]. Cederblad and Lindstedt [22] showed that about 3/~mol
123 of carnitine per day is formed in a 200 g rat. Both dietary a n d e n d o g e n o u s m e t h y l a t e d proteins provide e n o u g h trimethyllysine to meet the needs of c a r n i t i n e biosynthesis. It is n o t clear which is a m a j o r source for the synthesis of carnitine, a n d the q u e s t i o n r e m a i n s of whether red b l o o d cells f u n c t i o n as carrier of trimethyllysine a n d some other a m i n o acids between tissues. T h e issue that r e m a i n s to be resolved is whether trimethyllysine is i n c o r p o r a t e d actively i n t o red b l o o d cells from p l a s m a of the portal vein a n d is t r a n s p o r t e d from red blood cells to m a n y tissues, especially the liver, for carnitine synthesis.
Acknowledgement The authors are greatly i n d e b t e d to Dr. M a s a y u k i Matsuoka, H i r o s h i m a Daiichi Hospital, for his interest a n d suggestions.
References 1 Paik, K. and Kim, S. (1975) Adv. Enzymol. 42, 227-286. 2 Miyake, M. and Kakimoto, Y. (1976) Metabolism 25, 885-896. 3 Paik, K. and Kim, S. (1980) Protein Methylation (Mister, A., ed.), Vol 1, Biochemistry: A Series of Monographs, Wiley-Interscience, New York. 4 Kakimoto, Y., Matsuoka, Y., Miyake, M. and Konishi, H. (1975) J. Neurochem. 24, 893-902.
5 Young, V. and Munro, H. (1978) Fed. Proc. 37, 2291-2300. 6 McDermott, J. (1976) Biochem. J. 154, 179-184. 7 Young, V., Alexis, S. Baliga, S., Munro, H. and Muecke, W. (1972) J. Biol. Chem. 247, 3592-3600. 8 Kakimoto, Y. and Akazawa, S. (1970) J. Biol. Chem. 245, 5751-5758. 9 Henderson, L., Nelson, P. and Henderson, L. (1982) Fed. Proc. 41, 2843-2847. 10 Fritz, B. and Marquis, R. (1965) Proc. Natl. Acad. Sci. USA. 54, 1226-1233. 11 Hoppel, C. (1982) Fed. Proc. 41, 2853-2857. 12 Luque, J., Roncales, P., Tejero, C. and Pinilla, M. (1977) Acta Biol. Med. Germ. 36, 631-638. 13 Lange, H., Loewer, R. and Hempel, K. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 117-118. 14 Rebouche, C. and Broquist, H. (1976) J. Bacteriol. 126, 1207-1214. 15 Borum, P. and Broquist, H. (1977) J. Biol. Chem. 252, 5651-5655. 16 Borum, P., York, C. and Bennett, S. (1985) Am. J. Clin. Nutr. 41, 653-656. 17 Rebouche, C., Lehman, L. and Olson, L. (1986) J. Nutr. 116, 751-759. 18 Perlman, J., Farrell, P. and Gutcher, G. (1986) Am. J. Clin. Nutr. 43, 77-84. 19 Zaspel, B., Sheridan, K. and Henderson, L. (1980) Biochim. Biophys. Acta 631, 192-202. 20 Carter, A. and Frenkel, R. (1979) J. Biol. Chem. 254, 10670-10674. 21 Rebouche, C. (1982) Fed. Proc. 41, 2848-2852. 22 Cederblad, G. and Lindstedt, S. (1976) Arch. Biochem. Biophys. 175, 173-180.
119
BBAGEN 23234
High concentration of free trimethyllysine in red blood cells Mutsuhiko Mizobuchi 1, M a s a h a r u M i y a k e 2, Akira Sano i and Yasuo Kakimoto 1 J The Department of Neuropsyehiatry and ? Central Research Laboratory, Ehime University, School of Medicine, Ehime (Japan)
(Received 10 August 1989)
Key words: Trimethyllysine;Methylatedamino acid; Red blood cell A high concentration of a basic unidentified amino compound was found in the blood of rats. It was isolated and identified as N~,N',N'-trimethyllysine by paper chromatography, thin-layer chromatography, high-performance liquid chromatography and amino acid analyzer. It was localized exclusively in red blood cells in the blood of rats. Free trimethyllysine was also determined in the liver, kidney, spleen, brain, muscle, heart and testis of rat. The concentration of free trimethyilysine in red blood cells was more than lO-times as high as that in the other tissues. This compound in red blood cells was found in different species of animals. The relationship between this free trimethyllysine and carnitine was discussed.
Introduction Protein N-methylation is one of the major posttranslational modifications principally involving histidine, lysine and arginine residues. Methylated amino acid residues such as 3-methylhistidine, N'-monomethyllysine, N~,N'-dimethyllysine, N~,N~,N~-trimethyllysine, NG-monomethylarginine, N ~ , N G - d i m e t h y l arginine and N , N -dlmethylargmlne occur in various proteins [1-4]. They are released as free amino acids when methylated proteins are subjected to metabolic breakdown and are not reutilized for protein synthesis. Most of these free methylated amino acids are transported in blood plasma and excreted into urine without being reabsorbed by the kidney [3,5], although some methylated amino acids are metabolized in the body [6,7]. It was demonstrated that high concentrations of free methylated amino acids were present only in urine [1,3,8]. We found an unknown compound in the blood of rats, which was ninhydrin positive and was fractionated into a basic amino acid fraction during purification. We identified this compound as free trimethyllysine. It was localized in red blood cells. Trimethyllysine enters the carnitine biosynthetic pathway by the action of mitochondrial hydroxylase [3,9]. The essential role of carnitine in the mitochondrial B-oxidation of fatty acids is now well established [10,11]. The origin of this trimethyllysine is, however, unclear. (3
tG
•
-
•
Correspondence: M. Mizobuchi, Department of Neuropsychiatryand Central Research Laboratory, Ehime University, School of Medicine, Shigenobu-cho, Onsen-gun, Ehime, Japan.
The present study describes the presence of free trimethyllysine in red blood cells and a species difference in the amounts of this amino acid. This may provide useful information for carnitine biosynthesis. Materials and Methods Animals and diets We used Wistar rats (220-365 g), C 5 7 / B L mice (13-26 g), Beagle dogs (7-12 kg), chickens (line 412, 1.2-1.8 kg), Japanese monkeys (6-13 kg), rabbits (3-4.5 kg) and adult human blood. The care and use of laboratory animals in these experiments followed the guidelines of the National Research Council. Preparation of blood and tissue samples Blood was collected in a tube containing heparin usually from the cervical artery of animals and was immediately placed on the ice box. Human blood was obtained from the brachial vein. The blood was centrifuged at 1400 x g for 3 min. The supernatant fraction, which contained plasma and platelets, was separated into pure plasma by centrifugation at 2250 x g for 15 min. Red blood cells were obtained by full aspiration of buffy coat, washed with cold saline solution and centrifuged at 1400 x g for 3 min. Unless otherwise specified, red blood cells in all experiments were washed twice with saline solution. Hematocrits were determined using an International Micro-capillary Centrifuge Model MB and an International Microcapillary Reader. Rats were decapitated and perfused with cold saline solution, via the blood vessel, to remove blood. Liver,
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
120 kidney, spleen, brain, muscle, heart and testes were immediately removed. To obtain reticulocytes, rats were made anemic by intraperitoneal injections with 0.5 ml of HCl-neutralized solution of phenylhydrazine (20 m g / k g body weight) once a day for six days [12]. The animals were killed on the third day after cessation of injections and blood was collected and washed as described above. Blood and tissue samples were homogenized in 9 vol. of 10% trichloroacetic acid, sonicated for 1 min and centrifuged at 10000 × g for 15 min. The precipitate was suspended in the same volume of 10% trichloroacetic acid and centrifuged at 10000 × g for 15 min. After repeated suspension and centrifugation, these supernatants were passed through a 1 × 4 cm column of Amberlite IR-120 (100-200 mesh, H + form) and the resin was washed with 20 ml of water. Amino acids were eluted with 20 ml of 3 M ammonium hydroxide, evaporated to dryness and applied to a 1 × 4 cm column of Amberlite CG-50 (100-200 mesh, N H J form) to remove histidine and its derivatives. The column was washed with 20 ml of water. Basic amino acids were eluted with 20 ml of 3 M ammonium hydroxide and evaporated. These samples were subjected to analysis of the basic amino acids.
Analysis of methylated derivatives of lysine Paper chromatography was performed in two solvent systems. The solvent systems were p y r i d i n e / a c e t o n e / 3
B
A
e-J ¢:o.
62
e. o e-
0
ew
m u~
% 7.
o
Ik~jJ
M ammonium hydroxide ( 1 0 : 6 : 5 ) and isopropyl alc o h o l / f o r m i c a c i d / w a t e r (4:1 : 1). Chromatographic paper was Toyoroshi No 51A which is similar to Whatman No. 1 paper. The thin-layer chromatographies were carried out in two solvent systems of methanol/3 M ammonium hydroxide (3 : 1 : 2). The chromatographic sheet was DCPlastikfolien KJeselgel 60 (Merk). Basic amino acids were applied in the high-performance liquid chromatography on a column (15 cm × 4 mm internal diameter) of Shimazu ISC-07/S 1504 resin and eluted with 0.8 M lithium citrate buffer, pH 5.5 at 40 ° C at a flow rate of 0.5 ml/min. The usual analytical pattern in the whole blood is shown in Fig. lB. The concentration of lysine and methylated derivatives of lysine was determined routinely by the automatic amino acid analyzer according to the method of Kakimoto and Akazawa [8]. Methylated lysine derivatives were synthesized according to procedures described in the literature [3]. Results
Isolation and identification of free trimethyllysine in the blood Blood (180 g) was collected from 20 male rats and homogenized in 9 vol. of 10% trichloroacetic acid. The supernatant solution was successively applied to Amberlite IR-120 and Amberlite CG-50 column chromatography as described in Materials and Methods. Basic amino acids were fractionated on a column (0.9 x 30 cm) of cation exchange resin (Aminex 21-23) with 0.51 M NaC1 in 0.2 M sodium citrate buffer by an automatic amino acid analyzer. An unidentified compound was eluted from 112 to 128 min. This fraction was collected and passed through a 2.2 x 10 cm column of Amberlite IR-120 (H + form) to desalt and the column was washed with 200 ml of water. This compound was eluted with 200 ml of 3 M ammonium hydroxide. The eluate was evaporated to dryness under reduced pressure (yield 1.2 rag). The compound was analyzed by paper chromatography, thin-layer chromatography, amino acid analyzer and high-performance liquid chromatography. R r values and retention times are summarized in Table I. The R F values and retention time of this compound matched completely those of synthesized trimethyllysine.
Retention time (min)
Methylated derivatives of lysine in red blood cells and plasma of rats
Fig. 1. The chromatographic pattern of lysine and methylated lysine by high-performance liquid chromatography. (A) Chromatogram of synthesized lysine and methylated lysine (1 nmol). Orn, ornithine; Lys, lysine; MML, monomethyllysine; DML, dimethyllysine; TML, trimethyllysine (B) Chromatogram of the free basic amino acids fraction of rat whole blood. The retention time of the unknown compound, indicated by an arrow, corresponded to trimethyllysine.
Red blood cells and plasma were obtained as described in Materials and Methods. Red blood cells were pelleted by centrifugation and washed five times with cold saline. More than 95% of the trimethyllysine of non-washed cells was recovered in the well-washed cells. Therefore, red blood cells can be washed with
121 TABLE I
TABLE III
R F values and retention time of isolated compound and synthesized methyl derivatives of lysine by thin-layer chromatography, paper chromatography, amino acid analyzer and high.performance liquid chromatography Thin-layer chromatography (TLC) and paper chromatography (PCG) were both performed in two different solvent systems (A and B, C and D~ respectively).About 5 nmol of isolated compound and synthesized methyl derivativesof lysine were applied and visualizedwith ninhydrin. These compounds were separated by an amino acid analyzer and high-performance liquid chromatography (HPLC), in which [hey reacted with ninhydrin and orthophthalaldehyde, respectively. Compound
Isolated compound Trimethyllysine Dimethyllysine Monomethyllysine Lysine
R v value TLC
PCG
A
C
B
Retention time (min) D
amino acid analyzer
HPLC
0.86 0 . 2 2 0 . 1 6 0.64 0 . 8 6 0 . 2 2 0 . 1 6 0.64 0.46 0 . 7 0 0.48 0.66
120 120 155
58.6 58.6 52.3
0.38 0.32
168 170
45.9 35.9
0 . 4 6 0.33 0.62 0 . 5 6 0 . 3 7 0.52
A, phenol/water/3 M ammonium hydroxide (3 : 1 : 2); B, methanol/3 M ammonium hydroxide (1:2); C, pyridine/acetone/3 M ammonium hydroxide (10 : 6 : 5); D, isopropyl alcohol/formic acid/water (4:1:1).
saline solution to remove a n y p l a s m a t r a p p e d in red b l o o d cells w i t h o u t a n y loss. Unless otherwise specified, red blood cells in all experiments were washed twice with saline solution. The c o n c e n t r a t i o n of m e t h y l a t e d derivatives of lysine in whole blood, red b l o o d cells a n d p l a s m a are presented in T a b l e II. The c o n c e n t r a t i o n s of dimethyllysine a n d m o n o m e t h y l l y s i n e in all samples were less t h a n 1 n mol per g wet weight. O n calculation from hematocrit, approx. 97% of trimethyllysine in the b l o o d was calculated to be present in red b l o o d cells,
TABLE II The concentrations of methylated lysine and lysine in whole blood, red blood cells and plasma of rats The preparation of whole blood, red blood cells and blood plasma are described in Materials and Methods. Data represent mean _+S.D. for seven rats. Rat
Whole blood Red blood cells Blood plasma
nmol/g wet weight trimethyllysine
dimethyllysine
monomethyllysine
lysine
32 + 5
<1
<1
428 __+18
69 + 5 <1
<1 <1
<1 <1
480 + 24 317 + 32
The concentration of free trimethyllysine and lysine of various organs in rat
Blood and tissues were homogenized in 9 vol. of 10% trichloroacetic acid, sonicated for 1 min and centrifuged. These supernatants were passed through the column described in the Materials and Methods. These samples were applied to the amino acid analyzer. Data represent mean_+S.D. Values in parentheses represent the numbers of animals used. Rat organs Red blood cell Liver Kidney Spleen Brain Muscle Heart Testis
Number of
nmol/g wet weight
samples
trimethyllysine
lysine
7 5 5 5 5 5 5 5
69 _+5 3_+1 6 _+1 5 _+1 3_+1 4_+0 3_+0 2_+0
480_+ 24 566_+ 51 743 _+l l 1 456_+ 37 290_+ 36 362_+ 75 513_+ 8 396_+ 56
a n d the rest was calculated to be present in b l o o d plasma. T h e trimethyllysine c o n c e n t r a t i o n s of the platelet fraction a n d buffy coat which c o n t a i n e d white b l o o d cells were u n d e r 1 n mol per g wet weight. W a s h e d red b l o o d cells were b r o k e n by the a d d i t i o n of a n equal v o l u m e of deionized water a n d by freezing a n d t h a w i n g three times. After centrifugation, trimethyllysine was recovered in the s u p e r n a t a n t fraction, which was free from the p l a s m a m e m b r a n e of red blood cells. This indicates that free trimethyllysine in red blood cells is w i t h i n the cytoplasm. Concentration of free trimethyllysine in various organs of rat T h e c o n c e n t r a t i o n s of trimethyllysine a n d lysine of the liver, kidney, spleen, brain, muscle, heart a n d testis in rats are s h o w n in T a b l e III. The c o n c e n t r a t i o n s of trimethyllysine in these organs were between 6.3 a n d 1.8 n mol per g wet weight. The c o n c e n t r a t i o n of trimethyllysine in red b l o o d cells was more than 10-times as high as i n these organs, while the c o n c e n t r a t i o n of lysine was similar to that of other organs. Concentration of free trimethyllysine in red blood cells from various blood vessels Blood was o b t a i n e d from a b d o m i n a l artery, portal vein a n d inferior a b d o m i n a l vein. The c o n c e n t r a t i o n s of free red b l o o d cell trimethyllysine in a b d o m i n a l artery a n d inferior a b d o m i n a l vein were the same as that in cervical artery. The c o n c e n t r a t i o n s of free trimethyllysine a n d lysine of red b l o o d cells in portal vein were 165 _+ 5 a n d 1371 _+ 162 n m o l / g , respectively. O n l y in the portal vein, they were higher t h a n in the other vessels.
122 TABLE IV
The concentration of free trimethyllysine in reticulocytes and erythrocytes of rat and in red blood cells of various animals Reticulocytes were obtained from phenylhydrazine induced anemic rats as described in Materials and Methods. Data represent mean _+ S.D. Values in parentheses represent the numbers of experimental animals. Species
Rat erythrocyte reticulocyte Mouse Rabbit Dog Monkey Chicken Man
Number of
nmol/g wet weight
samples
trimethyllysine
lysine
7 5 6 2 5 5 5 4
69+_ 5 54+ 4 47+ 6 79_+ 26 36_+ 12 27+_ 7 8+ 2 2+_ 1
480+_ 24 815_+158 218_+ 19 220_+ 43 319_+ 56 338+_ 50 194_+ 93 76+_ 29
Concentrations of free trimethyllysine in reticulocytes and erythrocytes of rat and in red blood cells of different animal species Reticulocytes were obtained from phenylhydrazineinduced anemic rats as described in Materials and Methods. More than 90% of red blood cells consisted of reticulocytes. As seen in Table IV, the concentrations of trimethyllysine and lysine in reticulocytes were 54 and 815 n mol per g wet weight, respectively. The concentration of trimethyllysine in reticulocytes was somewhat lower than that in mature erythrocytes. The concentrations of blood trimethyllysine and lysine were determined in some animal species including mice, rabbits, dogs, monkeys, chickens and Man (Table IV). The concentrations of plasma trimethyllysine were less than 1 n mol per g wet weight in any species of animals, while those of red blood cells varied extremely among species, ranging from 2 to 79 n mol per g wet weight. The reason why the trimethyllysine concentration of human red blood cells was very low may be related to the preparation being from blood in the brachial vein. Discussion
Free trimethyllysine, dimethyllysine, Ne-monomethyllysine, NG, N°-dimethylarginine and N ° , N ° - d i methylarginine have been isolated from human urine, while in plasma they have been determined in very low concentrations [8,13]. This report describes, for the first time, a high concentration of free methylated amino acid found in mammalian red blood cells. Methylation of free lysine has been demonstrated in the mold Neurospora crassa [14,15], but not in mammalian organs. We incubated either [2,3-3H]lysine or S-adenosyl[methyl-3H]methionine with erythrocytes or
reticulocytes of rats, but no radioactivity was found on the spot of trimethyllysine in these experiments (unpublished observation). Free methylated amino acids are partially derived from dietary proteins and endogenous proteins upon degradation. The origin of the high concentration of free trimethyllysine in red blood cells is unclear. One explanation is that they move actively from plasma. Another explanation is that methylated proteins in red blood cells are degraded and free trimethyllysine is retained without being released through the red blood cell membrane. The concentration of free red blood cell trimethyllysine in the portal vein was about 2.4-times higher than in any other vessel. This suggests that trimethyllysine in red blood cells does not originate from the breakdown of its own methylated proteins. Rather trimethyllysine-containing proteins are digested in the gastrointestinal tract, absorbed, accumulated in the portal vein, transported and retained in red blood cells. It is necessary to show that only trimethyllysine is selectively transported into red blood cells among various methylated amino acids. The concentrations of carnitine in red blood cells and plasma have been reported by many investigators [16-18]. In adult rats, approx. 70-80% of whole blood carnitine was found in plasma, while 20-30% was in red blood cells. Trimethyllysine exists almost exclusively in red blood cells. Three possibilities have been suggested concerning the origin of trimethyllysine for carnitine biosynthesis in the rat. Zaspel et al. [19] have postulated that, since liver is the only tissue (in rat) which can hydroxylate "f-butyrobetaine to form carnitine, the liver proteins could provide all of the trimethyllysine required for conversion into carnitine. Carter and Frenkel [20] have suggested that free trimethyllysine is generated in various tissues from protein degradation, transported to the kidney, and converted to "y-butyrobetaine. This compound is then transported to the liver, where it is hydroxylated to form carnitine. In both experiments, transport and metabolism of trimethyllysine were examined only under conditions of malnutrition such as starvation, and only by the intravenous injection of labeled trimethyllysine. Rebouche et al. [17] have proposed that the availability of trimethyllysine is ratelimiting for the synthesis of carnitine in the growing rat and considered the diet proteins as a source of the precursor for carnitine biosynthesis. In this experiment, rats were raised with standard assorted feed, which had 0.3 /~mol of trimethyllysine residues per g. They were fed about 20 g of the diet per day, which contained 6/~mol of trimethyllysine residue. Calculating from the turnover rate of body protein and the content of trimethyllysine in the body, it is considered that about 14/~mol of trimethyllysine per 300 g rat is formed from whole body protein in a day [21]. Cederblad and Lindstedt [22] showed that about 3/~mol
123 of carnitine per day is formed in a 200 g rat. Both dietary a n d e n d o g e n o u s m e t h y l a t e d proteins provide e n o u g h trimethyllysine to meet the needs of c a r n i t i n e biosynthesis. It is n o t clear which is a m a j o r source for the synthesis of carnitine, a n d the q u e s t i o n r e m a i n s of whether red b l o o d cells f u n c t i o n as carrier of trimethyllysine a n d some other a m i n o acids between tissues. T h e issue that r e m a i n s to be resolved is whether trimethyllysine is i n c o r p o r a t e d actively i n t o red b l o o d cells from p l a s m a of the portal vein a n d is t r a n s p o r t e d from red blood cells to m a n y tissues, especially the liver, for carnitine synthesis.
Acknowledgement The authors are greatly i n d e b t e d to Dr. M a s a y u k i Matsuoka, H i r o s h i m a Daiichi Hospital, for his interest a n d suggestions.
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