Urinary excretion of NG-dimethylarginines in multiple sclerosis patients: preliminary observations

JOURNAL

OF THE

NEUROLOGICAL SCIENCES

EL-SEVIER

Journal

of the Neurological

Sciences

129 (1995)

186-191

Urinary excretion of iVG-dimethylarginines in multiple sclerosis patients: preliminary observations Nenoo Rawal a, Yong-Ju Lee a, John N. Whitaker b, Jong Ok Park ‘, Woon Ki Paik a, Sangduk Kim a,* a Fels Institute

for Cancer

b Department

of Neurology,

Research

and Molecular

University

Biology,

of Alabama ’ Department

Received

Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140, USA at Birmingham, and Neurology and Research Services, Birmingham Veterans Medical Center, Birmingham, AL 35294, USA of Chemistry, Kyung Sung University, Pusan, South Korea

4 March

1994; revised

8 November

1994; accepted

18 November

1994

Abstract The concentrations mined

in the urine

of NG,WG-dimethylarginine samples from multiple

[Me,(sym)Arg]

sclerosis (MS)

and NG,NG-dimethylarginine

and control

subjects,

[Me,(asym)Arg] using a highly sensitive HPLC

were deterpost-column

o-phthaldialdehydederivatization method. The presenceof approximately equal amountsof both dimethylarginine isomers,of Arg concentration nearly half of Me,Arg, and of the undetectable amount of NG-monomethylargininewere the characteristic urinary excretion pattern in all human samplesstudied. The urinary excretion of Me,(asym)Arg and Me,(sym)Arg from MS (n = 9) and control (n = 7) were analyzed: the mean values from the samples were approximately 20% (for all MS) and 33% (for chronic-progressive MS) lower than those from the control for both dimethylarginine-derivatives when compared to the respective compounds. Although there were contrasting trends between controls and MS patients in the relationship of urinary NG-dimethylarginines and myelin basic protein like material (MBPLM), the correlations were not significant. Differences in the

ratios of the concentrations of the two dimethyl derivatives, Me,(sym)Arg/Me,(asym)Arg,

were not significantly different

between MS and control groups. These findings warrant further investigation of possible links between urinary excretion of NG-dimethylarginine and MBPLM in MS. The possible significance of myelin metabolism in relation to urinary NG-dimethylarginines in MS is discussed. Keywords:

material;

NG,NG-Dimethylarginine; HPLC

NG,WG-Dimethylarginine;

1. Introduction Myelin basic protein (MBP) ’ is the major myelin protein, constituting approximately 30-40% of the total myelin protein (Campagnoni, 1988). The presence of posttranslationally formed NG-methylarginines in MBP (bovine/human MBP), catalyzed by MBP-specific

* Corresponding author. Tel.: (215) 707-4327; Fax: (215) 707-4318. 1 Abbreviations: MS, multiple sclerosis; CSF, cerebrospinal fluid; MBP, myelin basic protein; MBPLM, myelin basic protein like material; PMI, protein methylase I; OPA, o-phthaldialdehyde; HPLC, high performance liquid chromatography; MeArg, NC-monomethylarginine; Mez(sym)Arg, NG,NfGdimethylarginine; Me,(asym)Arg, NG,NG-dimethylarginine; 0022-510X/95/$09.50 0 1995 Elsevier SSDI 0022-510X(94)00277-0

Science

B.V.

All rights

reserved

Urinary

excretion;

Multiple

sclerosis; Myelin basic protein

like

protein methylase I (PMI; S-adenosylmethione:protein-arginine N-methyltransferase, EC 2.1.1.231, has been reported earlier (Baldwin and Carnegie, 1971; Brostoff and Eylar, 1971; Paik and Kim, 1980; Ghosh et al., 1988). Further, exclusive nature of MBP-methylation was also demonstrated in vitro studies in which MBP was the only protein methylated in an isolated intact myelin by highly purified MBP-specific protein methylase I (Ghosh et al., 1991). Temporal correlation between the level of MBP-PM1 activity and brain development/myelination has been observed by several workers (Miyake, 1975; Crang and Jacobson, 1981; Amur et al., 1984; Chanderkar et al., 1986). Particularly, interesting observation was that MBP-PM1 activity was greatly reduced in the brains of dysmyelinating

N. Rawal et al. /Journal

of the Neurological

mutant mice in comparison with those from the normal (Rawal et al., 1991), and the enzyme activity in cerebrospinal fluid (CSF) obtained from multiple sclerosis (MS) patients was found to be slightly elevated than the normal (Park et al., 1989). MBP-PM1 methylates residue-107 arginine among 19 arginine residues in MBP (bovine or human), yielding mainly NG-monomethylarginine (MeArg) and NG,N’G-dimethylarginine [Me,(sym)Arg] (Baldwin and Carnegie, 1971; Brostoff and Eylar, 1971; Deibler and Martenson, 1973). However, our recent observation (Rawal et al., 1992) showed that MBP isolated from early myelinating and mutant brain contained not only MeArg and Me,(sym)Arg, but also NG,NG-dimethylarginine [Me,(asym)Arg]. Under demyelinating conditions, MBP is dissociated from the membrane, and MBP-fragments formed by intracellular proteolysis (Whitaker and Heinemann, 1983; Bever and Whitaker, 1985; Whitaker, 1987) as well as free amino acids find their way into the body fluids (Kakimoto and Akazawa, 1970; Carnegie et al., 1977; Lou, 1979; Yudkoff et al., 1984). Since methylated arginines are not reutilized for protein biosynthesis, it seems to be quite possible to assessmyelin-associated abnormalities by analyzing the amino acid derivatives excreted in the urine. In light of our recent improvement in resolving methylated arginine derivatives by HPLC analysis, coupled with highly sensitive post-column o-phthaldialdehyde (OPA) derivatization method (Rawal et al., 1992), we have investigated the urinary excretion of methylated arginines in healthy and MS individuals.

Sciences 129 (1995) 186-191

187

equilibrated in 0.2 M pyridine. The column was washed with about 100 ml of 0.2 M pyridine to remove unadsorbed acidic and neutral amino acids. The retained basic amino acids in the column were then eluted with 60 ml of 3 M NH,OH solution, and the eluate was evaporated to dryness under reduced pressure. The dried sample was then dissolved in double distilled water and was lyophilized again to completely remove any trace amount of NH,OH. Finally, the washed sample was microfuged in an eppendorf tube for 5 min and stored at - 20” C until HPLC analysis. Amino acid analysis by HPLC

Methylated arginine derivatives were quantified with a strong cation Amino Acid Analysis Column (0.4 X 25 cm; Waters Assoc.) on HPLC, applying an isocratic elution with 0.3 M sodium citrate (pH 3.8) at 60°C (Rawal et al., 1992). The amino acids eluted were detected by post-column OPA derivative formation. Routinely, urine samples (corresponding to 0.05 mg of creatinine) pre-cleaned as above were used. Immunoassay

Radioimmunoassay for the detection of myelin basic protein-like material (MBPLM) was carried out using a previously described method (Whitaker, 1987), but with a standard of MBP peptide 83-89 rather than MBP peptide 80-89 (Whitaker et al., 1994). Urinary MBPLM was expressed as ng/mg creatinine (Whitaker, 1987). 3. Results

2. Materials

and methods

Materials

MeArg, Me,(sym)Arg, OPA and Dowex 50(Hf) were purchased from Sigma Chemical Co. (St. Louis, MO). Me,(asym)Arg was from Calbiochem (San Diego, CA). All other reagents were from various commercial sources and of the highest grade available. Specimens of human urine were obtained from 9 MS patients following the informed consents from each patient with prior approval by the review committee at the University of Alabama School of Medicine, Birmingham, Alabama. In this group, there were 5 males and 4 females. One patient had a relapsing-remitting pattern of disease, 3 were relapsing-progressive, and 5 were chronic-progressive (Table 2). Urine was also collected from 7 normal controls. Preparation of urine for HPLC analysis

Frozen urine sample was thawed and was centrifuged in a clinical centrifuge for 10 min to remove any precipitate. Five ml of the clear sample was loaded on a Dowex 5O(H+) column (1 X 5 cm) which was

Separation of arginine and its methylated derivatives on HPLC

The HPLC post-column OPA method used in the present studies was found to be extremely sensitive, thus, enabling us to detect sub-nanomole quantity of methylarginine derivatives with less than 0.5 ml of urine (or corresponding to 0.05 mg creatinine). The elution pattern for the standard methylated arginine derivatives are illustrated in Fig. lA, showing clear separation of these amino acids: the retention times for Me,(asym)Arg, Me&m&g, MeArg and Arg are 236 min, 268 min, 345 min and 376 min, respectively. The analysis of a control urine sample (Fig. 1C) indicates the presence of Me,(asym)Arg, Me,(sym)Arg and Arg whose retention times are well corresponding to those of the standard compounds (Fig. 1A). No MeArg peak is observed, however, a small peak eluting at 328 min and close to the region of MeArg (345 min; Fig. 1A) was observed. In order to identity a possible relationship of this unknown peak with MeArg, a small amount of the urine sample was cochromatographed with standard MeArg (Fig. 1B): MeArg eluting at 345 min did not overlap with the unknown peak (shown as an arrow

N. Rawal et al. /Journal

250

Retention

300

350

of the Neurological

400

time ( min )

Fig. 1. Separation of NG-methylarginine derivatives by HPLC. A strong cation amino acid analysis column was used to separate basic amino acids on a HPLC apparatus (Waters Assoc.) and the amino acids eluted were detected by OPA derivatization as described in Materials and methods. Panel A, standard amino acids (2 nmol each); B, cochromatogram of control urine sample corresponding to 0.005 mg creatinnine and 2 nmol of standard MeArg; C, control urine (0.016 mg creatinine) and D, MS urine.

in Fig. 10, thus, confirming the absence of MeArg in the human urine. This has been previously observed also by other investigators (Kakimoto and Akazawa, 1970; Carnegie et al., 1977; Lou, 1979). Further identification of the unknown peak was not attempted. Concentrations of methylated argimne deriwtives in urine from MS and normal subjects

Urine samples from 7 normal subjects (laboratory personnel) and 9 MS patients were analyzed by HPLC (Table 1). First of all, an absence of MeArg and relatively low amount of Arg compared to two isomers of Me,Arg are seen in all cases. The amount of asymmetric isomer is almost equal or slightly higher than that of symmetric isomer in both control and MS urine, with the mean value of 24.45 nmol/mg creatinine for the former and 22.48 for the latter. The value for Arg (13.34 nmol/mg creatinine) is about half of the Me,Arg. These values are in close agreement with

Sciences 129 (1995) 186-191

those reported by others (Kakimoto and Akazawa, 1970; Lou, 1979). As noted in previous studies (Whitaker et al., 19941, the level of urinary MBPLM varied broadly, 16.7-300.0 ng/mg creatinine, in normals. Linear regression of urinary dimethylarginines and MBPLM resulted in I = -0.645 with a p value of 0.118 (Table 2). In urine samples from MS patients, individual variations of dimethylarginines were somewhat greater than that of the controls. The mean values for both dimethylarginines in the MS patient specimens are approximately 17-20% lower than the control (24.45 vs. 19.45 for Me,(asym)Arg and 22.48 vs. 18.63 for Me,(sym)Arg). However, when comparing the mean values only from five chronic progressive MS (patients no. 2, 3, 6, 8 and 9) with the control, greater differences in both isomers are seen; the MS urine being approximately 33% lower than the control. Linear regression of urinary dimethylarginines and MBPLM in all MS patients showed a different and positive correlation (r = 0.626) from controls but it was insignificant (p = 0.071) (Table 2). The ratio of the urinary concentrations of dimethylarginines [Me,(sym)Arg/Me,(asym)Arg] has often been implicated to represent a meaningful index in the evaluation of muscular dystrophy where urinary excretion of dimethylarginine was found to be about 10 times that of the control urine (Lou, 1979). We have herewith examined a single sample of muscular dystrophic urine in order to verify our analytical method Table 1 Amounts of methylarginine (mmol/mg creatinine) derivatives in human urine. Amino acids were determined on HPLC as described in Methods and materials with urine samples corresponding to 0.05 mg creatinine. Sample

Me,(asym)Arg

Mez(sym)Arg

Arg

Control subject 1 28.05 2 18.10 3 27.13 4 26.74 5 28.89 6 24.22 7 18.02 Mean f SD 24.45 f 4.60

28.05 18.56 26.26 24.68 23.56 20.05 16.18 22.48 + 4.33

12.00 6.35 14.88 21.94 12.91 15.04 10.29 13.34 + 4.82

Multiple sclerosis 1 23.14 2 17.86 3 10.46 4 24.15 5 24.11 6 17.12 7 21.43 8 14.29 9 22.50 Mean + SD 19.45 f 4.82

25.73 19.67 13.33 24.15 22.03 13.76 20.92 10.59 17.50 18.63k5.2

13.48 7.76 6.96 10.25 10.12 8.45 6.89 10.59 10.00 9.39k2.11

Muscular dystrophy 1 166.6

105.3

N. Rawal et al. /Journal Table 2 Ratio of amounts of dimethylarginines Data are based on Table 1.

excreted

of the Neurological

in human urine.

Ratio Mez(sym)Arg/ Me,(asym)Arg

MBPLM (ng/mg creatinine)

Control subject 1

1.00

2 3 4 5 6 7

1.03 0.97 0.92 0.82 0.83 0.90

49.0 37.1 16.7 300.0 245.0 123.8 205.3

Range Mean + SD

0.82-1.03 0.911+0.08

Multiple 1

sclerosis

2 3 4 5 6 7 8 9

Range Mean + SD

1.11 (RP) a 1.10 (CP) 1.27 (CP) 1.00 (RP) 0.91 (RP) 0.80 (CP) 0.98 (RR) 0.74 (CP) 0.78 (CP) 0.74-1.27 0.943 +0.17

139.3 222.5 294.5

155.1 227.0

124.6 287.9

145.2 121.5

Muscular dystrophy 0.63

a Clinical conditions for MS patients are indicated in parentheses: RP for relapsing-progressive; CP, chronic-progressive; and RR, relapsing-remitting (Whitaker et al., 1993).

and found that the amounts of both dimethyl isomers were indeed greatly elevated (Table 1). However, the ratio of two isomers (0.63) was not significantly different from those found with MS or normal urine samples i;f,“bES?) (0.82-1.03 for the normal and 0.74-1.27 for

4. Discussion

Because of the variation in severity of progression of MS, a number of laboratory methods have been applied to body fluids of MS patients in an effort to provide a surrogate marker of disease activity (Whitaker and Snyder, 1982), of response to treatment (Whitaker et al., 1993) or of change from relapsing-remitting to progressive phases (Whitaker et al., 1994). The purpose of the present, preliminary investigation was to determine if urinary NG-dimethylarginines were altered in MS patients. In the present study, we have observed about 20% lower level of NG-dimethylarginine derivatives in urine samples from all MS and 33% decrease from the chronic-progressive condition, thus indicating that the lower urinary concentration of NG-dimethylarginines to be intimately correlated with the degree of myelin abnormality associated with MS. Although the control

Sciences 129 (1995) 186-191

189

and the MS samples contained MBPLM, neither the control nor the MS groups had the urinary levels of dimethylarginines and MBPLM significantly correlated. Of possible importance, however, was the observation that the correlation was negative in the control group while it was positive in the MS group. Larger numbers and different types of control patients, including other neurological diseased and MS patients with renal, cardiovascular and muscle diseases, will be required to confirm this observation. Biochemical significance of the observed decrease of the urinary levels of NG-dimethylarginines in MS patients could be found in the following discussion. NGDimethylarginines are found exclusively at residue 107 of MBP (human/bovine), catalyzed by MBP-specific protein methylase I. This highly specific methyl modification of the basic protein has been implicated to increase hydrophobicity of the protein molecule, thereby stabilizing the structurally important hair-pin structure and helping the conformation of myelin double membrane structure (Brostoff and Eylar, 1971). In support of this hypothesis, protein methylase I activity in dysmyelinating jimpy mutant mice brain was shown to be greatly reduced compared to that of the normal brain (Kim et al., 1984; Rawal et al., 1991). In addition, the enzyme activity temporally correlated with the myelin formation during early development in mice brain (Chanderkar et al., 1986). It was, therefore, expected that the methylation level in vivo would also be reduced under demyelinating condition such as MS in human and that the affected myelin membrane would loose its compactness. Consequently, the dissociated MBP would be readily cleaved by intracellular proteases, generating free amino acids including NG-dimethylarginines. Since the dimethylarginines are not reutilized for the protein biosynthesis, the reduced urinary concentration of NG-dimethylarginine is an indicative of reduced level of MBP methylation. Several laboratories in the past attempted to establish the urinary concentration of NG-methylarginines as a marker to correlate disease state, however, no significant difference has been observed with samples from cerebrovascular accident, cancer or systemic lupus erythematosus (Yudkoff et al., 1984). On the other hand, the ratio of Me,(sym)Arg/Me,(asym)Arg in the case of liver disease and muscular dystrophy was lower than unity, indicating relatively larger amount of the asymmetric than the symmetric derivative excreted in these urine (Lou, 1979). In our present study, however, it was found that the analysis of such a ratio was not useful. In conclusion, the present study, indicating a lower level of NG-dimethylarginine excretion in the urine from MS patients, could be implicated as supporting evidence to evaluate the severity of the demyelinating conditions, although further studies are required with larger numbers of samples.

190

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of the Neurological

Although NG-methylated arginines are posttranslationally formed and the methylated amino acids in the body fluids are intracellular proteolytic products, renal reabsorption and/or its metabolism will be a major contributing factor to the eventual concentrations of these compounds in the urine. Relatively low level of Arg and an unmeasurable amount of MeArg in the urine clearly indicated the presence of an efficient reabsorption mechanism and/or of active metabolism of these compounds. Significance of the presence of nearly equal amounts of the dimethylarginine isomers in urine is difficult to assessat present, since in rodents these compounds are metabolically quite different. For example, Me,(asym)Arg but not Me,(sym)Arg is metabolized in vitro by an enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, in rat kidney (Ogawa et al., 1987; Ogawa et al., 1989). In addition, in vivo metabolic studies also revealed a different turnover rate: Only 13% of the asymmetric isomer but 75% of the symmetric isomer was excreted in rat urine (Ogawa et al., 1987). Furthermore, McDermott observed that the concentration of Me,(sym)Arg in rabbit urine is 30-times greater than that of either MeArg or Me,(asym)Arg (McDermott, 1976). In the present study, however, approximately equal amounts of Me,Args in both diseased or control normal human urine samples were observed (Table 1). This observation suggests that both dimethyl derivatives are metabolized equally in the human subject, quite unlike in rat and rabbit. It is of interest to note here that recent studies indicated that Me,(asym)Arg can function as an inhibitor for nitric oxide synthetase to a degree equal to that of MeArg, a well known inhibitor of this synthetase (Hibbs et al., 1987; Oken and Marletta, 1993). Me,(sym)Arg, however, does not function as an inhibitor (Valiance et al., 1992). Finally, the present studies also suggest that human urine should be an excellent and easily available natural source for obtaining both Me,Args. Since the difference in the retention time between the symmetric and asymmetric isomers is 30 min apart, the currently described HPLC procedure, without the OPA reaction, permits excellent purification of Me,(asym)Arg and Me,(sym)Arg in the range of 6.4-6.9 mg/24 h urine (taken as 1.4 g creatinine/day). Thus, this procedure could be used as an alternative to a tedious chemical synthesis involving elaborate subsequent purification of the compounds (Kakimoto and Akazawa, 1970). Acknowledgements

This work was supported in part by Grants from the National Cancer Institute (5-P30-CA 122270), the National Institute of Health, (AMO9602, NS23240, and NS29719), the Research Program of the Veterans Ad-

Sciences 129 (1995) 186-191

ministration, the Korean Science and Engineering Foundation (KOSEF 911-0301-036-2) and the Kil Chung-Hee Fellowship Fund. References Amur, S.G. Shanker, G. and Pieringer, R.A. (1984) Regulation of myelin basic protein (arginine) methyltransferase by thyroid hormone in myelinogenic cultures of cells disassociated from embryonic mouse brain, J. Neurochem., 43: 494-498. Baldwin, G.S. and Carnegie, P.R. (1971) Specific enzymatic methylation of an arginine in the experimental allergic encephalomyelitis protein from human myelin, Science, 171: 579-581. Bever, C.T. and Whitaker, J.N. (1985) Proteinases in inflammatory demyelinating disease, Springer Semin. Immunopathol., 8: 235250. Brostoff, S. and Eylar, E.H. (1971) Localization of methylated arginine in the Al protein from myelin, Proc. Natl. Acad. Sci. USA, 68: 765-769. Campagnoni, A.T. (1988) Molecular biology of myelin proteins from the CNS, J. Neurochem., 51: 1-14. Carnegie, P.R., Fellows, F.C. and Symington, G.R. (1977) Urinary excretion of methylarginine in human disease, Metabolism, 26: 531-537. Chanderkar, L.P., Paik, W.K. and Kim, S. (1986) Studies on myelin basic protein methylation during mouse brain development, Biochem. J., 240: 471-479. Crang, A.J. and Jacobson, W. (1981) The Relationship of myelin basic protein (arginine) methyltransferase to myelination in mouse spinal cord., J. Neurochem., 39: 244-247. Deibler, G.E. and Martenson, R.E. (1973) Determination of methylated basic amino acids with the amino acid analyzer, J. Biol. Chem., 248: 2387-2391. Ghosh, S., Paik, W.K. and Kim S. (1988) Purification and molecular identification of two protein methylases I from calf brain: myelin basic protein- and h&one-specific enzyme, J. Biol. Chem., 263: 19024-19033. Ghosh, G.K., Rawal, N., Syed, S.K., Paik, W.K. and Kim, S. (1991) Enzymatic methylation of myelin basic protein in myelin, Biochem. J., 275: 381-387. Hibbs, J.B., Taintor, R.R. and Varrin, Z. (1987) Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite, Science 235: 473-476. Kakimoto, Y. and Akazawa, S. (1970) Isolation and identification of NG,NG-and NG,NfG-dimethylarginine, N’-mono-, di-, and trimethyllysine, and glucosylgalactosyl- and galactosyl-&hydroxylysine from human urine, J. Biol. Chem., 245: 5751-5758. Kim, S., Tuck, M., Kim, M., Campagnoni, A.T. and Paik, W.K. (1984) Studies on myelin basic protein-specific protein methylase I in various dysmyelinating mutant mice. Biochem. Biophys. Res. Commun., 123: 468-474. Lou, M.F. (1979) Human muscular dystrophy: elevation of urinary dimethylarginines, Science, 203: 668-670. McDermott, J.R. (1976) Studies on the catabolism of NG-methylarginine, NG,NfG-dimethylarginine and NG,NG-dimethylarginine in the rabbit, Biochem. J., 154: 179-184. Miyake, M. (1975) Methylases of myelin basic protein and histone in rat brain, J. Neurochem., 24: 909-915. Ogawa, T., Kimoto, M., Watanabae, H. and Sasaoka, K. (19871 Metabolism of NG,NG- and NG,NfG-dimethylarginine in rats, Arch. Biochem. Biophys., 252: 526-537. Ogawa, T., Kimoto, M. and Sasaoka, K. (1989) Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylamino hydrolase, from rat kidney, J. Biol. Chem., 264: 1020510209.

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Whitaker, J.N. and Heinemann, M.A. (1983) The degradation of human myelin basic protein peptide 43-88 by human renal neutral proteinase, Neurology, 33: 744-749. Whitaker, J.N. and D.S. Snyder (1982) Myelin components in the cerebrospinal fluid in diseases affecting central nervous system myelin. Clin. Immunol. Allergy, 2: 469-482. Whitaker, J.N., Layton, B.A., Herman, P.K., Kachelhofer, R.D., Burgard, S. and Bartolucci, A.A. (1993) Correlation of myelin basic protein-like material in cerebrospinal fluid of multiple sclerosis patients with their response to glucocorticoid treatment, Ann. Neurol., 33: 10-17. Whitaker, J.N., Williams, P.H., Layton, B.A., McFarland, H.F., Stone, L.A., Smith, M.E., Kachelhofer, R.D., Bradley, E.L., Burgard, S., Zhao, G., Paty, D.W., (1994) Correlation of clinical features and findings on cranial magnetic resonance imaging with urinary myelin basic protein-like material in patients with multiple sclerosis. Ann. Neural., 35: 577-585. Yudkoff, M., Nissim, I., Pereira, G. and Segal, S. (1984) Urinary excretion of dimethyl-arginines in premature infants, Biochem. Med., 32: 242-251.