Mass spectrometric approaches in structural identification of the reaction products arising from the interaction between glucose and lysine

0039-9140/91$3.00+ 0.00 Copyright0 1991PergamonFYessplc

Tolanfa, Vol. 38, No. 4, pp. 4OS4-412,1991

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MASS SPECTROMETRIC APPROACHES IN STRUCTURAL IDENTIFICATION OF THE REACTION PRODUCTS ARISING FROM THE. INTERACTION BETWEEN GLUCOSE AND LYSINE A. LAPOLLA,C. GERHARDINGER, G. CREPALDIand D. FEDELE Instituto di Medicina Interna, Patologia Medica I, Policlinico Universita di Padova, Italy M. PALUMBOand D. DALZOPP~ Dipartimento di Chimica Organica, Universita di Padova, Italy C. J. PORTER VG Analytical, Altrincham, England E. GHEZZO,R. SERAGLIAand P. TRALDI Consiglio Nazionale delle Ricerche, Area di Ricerca, Padova, Italy (Received

9 October

1990. Accepted

15 November

1990)

Summary-The products arising from the reaction of a-protected lysine with glucose have been studied by different techniques, viz. high-performance liquid chromatography (HPLC) with UV detection, fast atom bombardment (FAB) mass spectrometry (MS), and HPLC/MS. Most of the analytical data were obtained by the last approach and allowed identification of many molecular species for a thorough knowledge of possible reaction pathways or structural data already available in the literature.

Accumulation of brown advanced glycation products in proteins has been proposed as relevant to the development of long-term complications of diabetes mellitus.‘** In the past, the structure of these products has been studied, starting from glycated proteins, by means of chemical and enzymatic hydrolysis and other separative and analytical techniques.3-s New information has been added to that already known by Njoroge et al., who demonstrated that chemical hydrolysis can lead to artifacts.6 In a recent study’ we compared data from chemical and enzymatic hydrolysis and were able to confirm these earlier6 findings. However, since the structure of the brown products in the complex hydrolysis mixture is difficult to determine by those techniques, we decided to take a different approach, namely to study the products of the chemical reaction of glucose and protected lysine. This approach is expected to provide useful information about the mechanism of this reaction and form a promising starting point for future studies in vivo.

EXPERIMENTAL

Synthesis

of N-cr-acetyl-L-lysine

methyl ester

N-u -Acetyl-L-lysine methyl ester was prepared and purified according to Irving and Gutmann.’ It was a dense yellow oil. The protected lysine (100 mg) and o-glucose (5 g) were dissolved in 5 ml of sodium phosphate buffer (pH 7.5, 0.05M Na) and the solution was incubated for 28 days at 37”, and then was lyophilized. HPLC

separation

A Perkin-Elmer series 3B liquid chromatograph connected to an LC-75 spectrophotometric detector was used. The detector wavelength was set at 320 nm. A p-Bondapak C-18 reverse phase column was used for the separation. Each sample was diluted tenfold with distilled water before injection. Gradient elution at a flow-rate of 2 ml/min was used, with a mixture of acetonitrile and water in ratio progressively changed from 1: 99 to 20 : 80 in 30 min. 405

A. LAP~LLA~~ al.

406

Mass spectrometric

measurements

Fast atom bombardment (FAB) measurements were made with a VGZAB 2F mass spectrometer.’ Glycerol solutions of the sample were bombarded with 8 keV Xe atoms. GC/MS was performed on a Finnigan ITD 800 system operating in electron ionization mode (100 eV, 100 PA), with a fused-silica capillary column (25 m long, i.d. 0.32 mm) coated with a 0.4~pm layer of SE54. The temperature was programmed to rise from 70 to 250” at 7”/min. HPLC/MS measurements were performed with a VG ZAB-VE instrument operating under plasma-spray conditions with a probe temperature of 250” and a source temperature of 240”. The HPLC conditions were as described above. RESULTS AND DISCUSSION

The HPLC chromatogram of the reaction of protected lysine and glucose is shown in Fig. 1. There are three main peaks, with retention times of 6, 11 and 12 min respectively. GC/MS analysis of each peak led to unsatisfactory results, possibly owing to the high polarity of the compounds investigated. However, FAB MS of the whole reaction mixture, shown in Fig. 2a, gave a spectrum which differed from that of the pure protected lysine (Fig. 2b). For the mixture, ions at m/z 285 and 263 were detectable (with a

TIME (min)

2b

IO

Fig. 1. HPLC chromatogram obtained by detection 320 nm.

263285 203 L 0

100

IO

.L.:: 200 250

MASS 300

350

400

2 3

100 90 SO 70

b

60 SO

0

50

100

,1,. 150

200

A

250

MASS

300

350

400

Fig. 2. FAB mass spectra of (a) whole reaction mixture; (b) a-protected lysine.

at

Interaction between glucose and lysine

407

’

100

266

285

go80-

a

706050403020 10

224 130

0

MASS 100

120

140

160

160

100. 90

200

220

240

260

260

171

3

263

I

244

80

I

I

20

10 0

1t4 I 100

I 120

140

I

t60

I 180

200

220

I

240

MASS

260

Fig. 3. Daughter ion spectra: (a) ion at m/z 285; (b) ion at m/z 263.

signal/chemical background ratio of about 20). Daughter ion spectra (Fig. 3) suggested that these species had structures 1 and 2 respectively (Scheme 1). The very high chemical noise observed under the FAB conditions suggests the presence of many other molecular species. For this reason, HPLC/MS measurement was the method of choice. The reconstructed total ion chromato-

gram obtained under the same HPLC conditions is shown in Fig. 4. Practically no chromatographic resolution was obtained. However, mass spectra obtained at different elution times led to an effective description of the molecular species present in the complex mixture. Figure 5 shows the spectra obtained for scans l&25 and 37. In the first, many different ionic species are easily detected in the range m/z

c CH zOH

CH,OH

I

I

I

CHOH

/O

I

0

\

CHOH 171 r+

NH

CHOH

I

+H+

KHi)4

164

5H2

-CH=NH

178

NHz 224 1 mlt

mir 263 Scheme t

285

+H+

A.

408 100

LAPOLLA~~

al. rZtl6 'XIOZ

x10.00

h

-2310 80 -1732

-1155


140

do

260

160

210

210

SCM

Fig. 4. Reconstructed ion chromatogram for HPLC/MS analysis of reaction mixture under plasma-spray conditions.

150-500, and in the third new molecular species becomes detectable at m/z 324. Neither GC/MS analysis of every component obtained by preparative HPLC runs, nor mass spectrometric analysis of the whole mixture by FAB was satisfactory. No compound related to the Amadori reaction could be detected by CC/MS, possibly because of the high polarity of such molecules. The FAB mass spectrum of the whole reaction mixture differed from that of protected lysine (Fig. 2b), mainly by the presence of ions at m/z 285 and 263 (Fig. 2), but it must be stressed that this spectrum is quite unsatisfactory. Ionic species are present at every m/z

value. Also the ions of interest have a signal/ chemical background ratio of about 20, which suggests many other molecular species may be present but are concealed by the chemical background. For the ions at m/z 285 and 263, the daughter ion spectra (Fig. 3) suggest the structures 1 and 2 (in Scheme 1) which could originate from lysine and glucose (SchifYs base) with subsequent Amadori rearrangement and decarboxylation. Both compounds 1 and 2 show the formal loss of H, 0’ (possibly from sequential losses of H’ and H,O) in the daughter spectra, leading to ions at m Jz 244 and 266 respectively. 67631 x10

a

64232 Xl0

b 32111

50

324

-25662

Fig. 5. Mass spectra obtained at different elution times by HPLC/MS analysis: (a) mass spectrum of scan No. 18, (b) mass spectrum of scan No. 25, (c) mass spectrum of scan No. 37.

Interaction between glucose and lysine

To obtain more satisfactory results, we performed a series of HPLC/MS measurements. Under the HPLC conditions described we

409

obtained the total ion chromatogram reported in Fig. 4. The practically complete loss of resolution in comparison with UV detection

Table 1. Possible structures detected by plasma-spray HPLC/MS

I

+ NH,+

aluaose

198

I

7 f= 182

Structures

Im/zl

~H-c-c~,-~H~~-cH~H-cH,~~ 1::

j

b

v 0

HO

I

n 1** cool+CH*-CM*-O‘- H-CM, E? i

;’ L!”

OH +

iw,’

?

I

I

1+

i;fl,O@l

”

e

1+*

cn*or

‘fV4

_

iSi,-q-NH-Cti-COOCH, 0

242

u 0

HC

MHz-fN-COOH fp*

OH p%

i

MS@,+

COOH

k 249

f + @IN,+

I

880

m +

\

NH:

1” LO” _b”OH C%OH

A. LAPOLLAet al.

410

shows that the latter is artificially selective, in that it can detect those components containing the chromophoric moiety but not describe the real composition of the mixture. In contrast, the mass spectra obtained at different elution times

can give an effective description of the molecular species in the complex mixture (e.g., Fig. 5). The spectrum for scan 18 corresponding to the maximum of the reconstructed total ion current (Fig. 4) shows many different ionic species in the Glucose +Lysine

-Hz0

11 HO

NHR

I CH3

CH2

I c-o

I c-o

I I HCOH

I

c-o

HCOH

I

-Lysine

I

I

HCOH

HCOH

I

H 2bOH

H$OH

//’

0 OH

HO

OH ti

0

g,

FW 162

-Lysine

HCOH

a

11

CH3

OH HO

NH2-

OH

CH -

CH3 C’

I

II

CH2

I COOH

k, FW 204

OH CH3

h, FW 144

P”

ii COOH-

CH2CH2-

OC-C-_-H3

I H i ,FW

162 Scheme

2, Part

1.

COOH

I Wb)4 I NH

OH

0

COOH

I

HCOH

I

HCOH

+

I

CH,OH

Interaction between glucose and lysine

411

HOCH&j-CH

HC-0

d . FW 252

H,COH b

HOCH 2

,

e FW 162

CH~H

CH,OH

CH,OH

CHOH

CHOH

CHOH

CHOH

CH~H

CHOH

I I

I

I

I

I

+ HC

CH

CH

j , FW 252 Scheme 2, Part 2. Scheme 2. Possible pathways leading to structures compatible with the molecular weight found in HPLC/MS analysis, where R = -(CH,),--CH(NH)--COOH.

range m/z 150-500. Though some of them are easily attributable to molecules present in the reaction mixture (as for example m/z 180 and 198 corresponding to M+’ and [M + NH,]+ for glucose, respectively), others are rather difficult to account for, since their structure is inferred only from their molecular weight. For these reasons we propose for them isobaric structures already suggested and/or described in the

literature, as intermediate and final glycation products. At longer retention times most of the ionic species shown in scan No. 18 disappear, but new molecular species at m/z 324 become detectable. In recent years many efforts have been made to obtain a general view of the possible patterns related to the Maillard reaction. Some useful results have been obtained either from theoreti-

412

A.

LAPOLLA~~

cal or expe~mental approaches. The startles deduced from HPLC/MS data were proposed on the basis of studies of the Maillard reaction under physiolo~cal conditions. Our own opinion is that structure assi~ment based solely on molecular weight can sometimes give misleading results, but at this stage of the present research, reliable information on the molecular weights of the Maillard products is important. Possible pathways leading to structures compatible with the molecular weights found by ~PLC~MS are reported in Scheme 2. Loss of lysine from the Schiff’s base arising from the reaction of glucose and lysine (Scheme 2, structure a proposed by Paulsen and P~ughaupt3) can lead at first to two di%rent structu~s (b and c in Scheme 2). Such behaviour was already proposed by Anet,“’ Kate” and Beck et al.,jz and, considering the reaction conditions seems more than reasonable. Both structures b and c undergo further rearrangements with losses of neutral moieties such as furan~2~aldehyde and analogous products. By this pathway structures &, e and .f are obtained; they have been well described by Led1 and co-workers4~1~~i4and SengLJs It is significant that several ions, at m/z 252 frd), 222 (f) and 162 (e) are present in the mass spectrum reported in Fig, 5a. The further rearrangement of compound c to c’, favoured from a thermodynamic point of view, accounts for the formation of both compounds g and h (molecular weights 162 and 144 respectively). The latter, in aqueous solution, readily adds water to yield the compound with molecuiar weight 162. The pathways described above, leading to compounds c’, g, h and i, have already been described by Led1 and Severin,’ SengS5 and Mills et &.I6 The right-hand part of Scheme 2 shows the reaction of compound b with a through condensation and further cleavage of the polyol chain, followed by oxidation. This process leads to compound j, the molecular ion of which is still present in the spectrum in Fig, 5a at m/z 252, The formation of such a compound has already been proposed by KrBnig.” Also the fo~ation of ~arboxymethyl-lysine (compound k), described by Ahmed et al.,” cannot be excluded. In fact, the ion at m/z 222, shown in Fig. Sa, could be due to the ~ationi~atio~ of compound k with NH:, as often observed under plasmaspray conditions. All these findings are shown in Table 1, where the possible structures related to the most abundant ionic species in the spectra shown in Fig. 5 are proposed.

al.

In addition to the ionic species which can be attributed to the structures described above and already reported in the literature, we detected some other molecular ions of unknown structure. Thus, the ions at m/z 342 and 360 can be attributed to structures m and m + NH: respectively (Table 1). These structures easily originate from selective oxidation of the side alcohol chain of molecule I (Scheme 2),L9-2i The ions at m/z 324are not so easily identified. Structure n in Table 1 can be tentatively proposed. In conclusion, by ~mparing HPLC, GC/MS, FAB and NPLC/MS data for the products arising from the interaction of glucose and proton lysine we were abie to identify many different molecular species, most of which have the same molecular weight as the products described in the literature. Other compounds, not reported in the literature, were assigned B structure on a mechanistic basis.

1. M. Brownlee, W. Vlassara and A. Cerami, Ann. liti. A4&., 1984, Ml, 527. 2. V. M. Monnier and A. Cerami, C&n. Endocrfnol. Met&., 1982, 11, 431. 3. H. Paulsen and K. W. ~ughaupt, in C~rboh~dr~t~.~, Vol. 13, W. Pigman and D. Horton feds.), pp. 883-927. Academic Press, New York, 1980. 4. F. Led1 and T. Severin, Z. Lebensm. Unters. Borsch., 1982, 175, 262. 5. f&n, ibid., 1979, 169, 173. 6. F. G. Njoroge, A. A. Fernandes and V. M. Monnier, J. Biai. C&m., 1988, 263, 10646. 7. C. Gerbardinger, A. Lapolla, E. Gbezzo, P. Trafdi, G. Crepaldi and D. Fedele, submitted to C&m. C&n. Acfa. 8. C. C. Irving and H. R. Gutmann, J. Org. Chem., 1959, 24 1979. 9. M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, Nature, 1981, 293, 270. 10. E. F. L. f. Anet, Ausf. J. Chem., 1960, 13, 396. 11. H. Kate, Bt& Agr. Chem. Sot. Jupun, 1960, 24, 1. 12. J. Beck, F. Ledl and T. Severin, Carbohyd. Rrs., f988, 178, 240. 13. F. Ledl, J. Hiebl and T. Severin, Z. Lebensm. Unter-s. ForscpI., 1983, xn, 353. I4. J. Beck, F, Ledi and T. Severin, ibid,, in the press. ‘15. M. Sengl, ~~sertut~~n, University of Munich, 1988. 16. F. D. Mills, D. Weisieder and J. E. Hodge, Ter~u~e~ro~ Lett., 1970, 1243. 17. U. Kranig, ~~ssertutj~~, University of Munich, 1974. 18. M. U. Ahmed, S. R. Thorpe and J. Baynes, Fed. Proc., 1985,44, 1621. 19. J. G. Farmar, P, C. Ulrich and A. Cerami, J. Org. chtlm.. 1988, 53, 2346. 20. F. G. Njorogc, L. M. Sayre and V. M. Monnier, Carbohyd. Res,, 1987, 167, 211. 21. T. Severin and Cr. Krijnig, Z. Le&tsm. Unters. Forsck., 1973, IS%,42,