Stereoselective Determination of Amino Acids in β-Amyloid Peptides

Anal. Chem. 2001, 73, 2625-2631

Stereoselective Determination of Amino Acids in β-Amyloid Peptides and Senile Plaques Gunnar Thorse´n,*,† Jonas Bergquist,‡,§ Anita Westlind-Danielsson,| and Bjo 1 rn Josefsson†

Department of Analytical Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden, Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, Go¨teborg University, Sahlgrenska University Hospital, Mo¨lndal, Sweden, Institute of Chemistry, Department of Analytical Chemistry, Uppsala University, SE-751 21 Uppsala, Sweden, and Bioscience, Discovery RA, CNS & Pain Control, AstraZeneca, SE-151 85 So¨derta¨lje, Sweden

A novel method for the determination of the enantiomeric composition of peptides is presented. In this paper, the focus has been on β-amyloid peptides from deceased Alzheimer’s disease patients. The peptides are hydrolyzed using mineral acid. The free amino acids are derivatized with the chiral reagent (+)- or (-)-1-(9-anthryl)-2-propyl chloroformate and subsequently separated using micellar electrokinetic chromatography (MEKC) and detected using laser-induced fluorescence (LIF) detection. The high separation efficiency of the MEKC-LIF system, yielding ∼1 million theoretical plates/m for most amino acids, facilitates the simultaneous chiral determination of nine amino acids. The samples that have been analyzed were standard 1-40 β-amyloid peptides, in vitro precipitated β-amyloid fibrils, and human senile plaque samples. The proteins and peptides present in eucaryotes are synthesized from L-amino acids exclusively.1 D-Amino acids may be present in the proteins and peptides of eucaryotes due to the aging of long-lived proteins2-4 or as a result of posttranslational modifications.5,6 The presence of D-amino acids is more common in the proteins of bacteria and archaea than in eucaryotes.7-8 In humans, D-amino acids have been found in proteins from dentine,2 enamel,3 and eye lens,4 as well as in cerebral white and gray matter,9 myelin basic protein,10 and core proteins of senile plaques.11,12 The * Corresponding author: (e-mail) [email protected]; (fax) +46 8 15 63 91. † Stockholm University. ‡ Go ¨teborg University. § Uppsala University. | AstraZeneca. (1) Hardy, P. M. In Chemistry and Biochemistry of the Amino Acids; Barrett, G. C., Ed.; Chapman and Hall Ltd.: London, 1985; pp 10-11. (2) Helfman, P. M.; Bada, J. L. Nature 1976, 262, 279-281. (3) Helfman, P. M.; Bada, J. L. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 28912894. (4) Masters, P. M.; Bada, J. L.; Zeigler, J. S., Jr. Nature 1977, 268, 71-73. (5) Mor, A.; Amiche, M.; Nicolas, P. Trends Biochem. Sci. 1992, 17, 481-485. (6) Kreil, G. Science 1994, 266, 996-997. (7) Matsumoto, M.; Homma, H.; Long, Z.; Imai, K.; Iida, T.; Maruyama, T.; Aikawa, Y.; Endo, I.; Yohda, M. J. Bacteriol. 1999, 181, 6560-6563. (8) Nagata, Y.; Fujiwara, T.; Kawaguchi-Nagata, K.; Fukumori, Y.; Yamanaka, T. Biochim. Biophys. Acta 1998, 1379, 76-82. (9) Man, E. H.; Fisher, G. H.; Payan, I. L.; Cadilla-Perezrios, R.; Garcia, N. M.; Chemburkar, R.; Arends, G.; Frey, W. H., II. J. Neurochem. 1987, 48, 510515. (10) Shapira, R.; Chou, C. H. J. Biochem. Biophys. Res. Commun. 1987, 146, 1342-1349. 10.1021/ac000861q CCC: $20.00 Published on Web 04/26/2001

© 2001 American Chemical Society

determination of D-amino acids in biological fluids and proteinaceous samples have been reviewed by Imai et al.13,14 A correlation has been found between the presence of Daspartic acid and age in proteins that can be considered stable and stationary. An accumulation of 0.1% D-aspartic acid/year has been found for the proteins studied in dentine,2 enamel,3 and eye lens.4 In myelin basic protein, between 7 and 10%10 of the aspartic acid was found to be racemized. In the cerebral white matter, D/L ratios ranging from 0.013 to 0.045 was reported with lesser ratios in the cerebral gray matter, from 0.011 to 0.018.9 Shapira et al.11 found ∼5% D-aspartic acid and ∼2% D-serine in core proteins of senile plaques as compared to ∼2% D-aspartic acid in other proteins analyzed by the same method. Roher et al. did an extensive study on the racemization of aspartic acid in core proteins of senile plaques.12 The study provided information not only of the D-amino acid content, found to be 11% in the core peptides analyzed, but also on the location of the racemized aspartyl residues. A synthetic peptide, analyzed by the same method as the human samples, contained only 2% D-aspartic acid. The racemization rate of protein or peptide amino acids is dependent upon the amino acid sequence therein. The rate of racemization for aspartyl residues depends, for example, on the accessibility of solvent molecules to the peptide backbone15 and the rate of formation of succinimidyl residues.16 It has been suggested that the conformational changes of aspartyl residues in peptides is a time-dependent process involved in the aging and degradation of proteins and peptides.16,17 It has also been shown that conversion of L-aspartyl to D-aspartyl18 or isoaspartyl19 affects the aggregation rate of synthetic β-amyloid peptides (Aβ). To what extent the aggregation of peptides is affected depends on the position of the racemized aspartic acid.20 (11) Shapira, R.; Austin, G. E.; Mirra, S. S. J. Neurochem. 1988, 50, 69-74. (12) Roher, A. E.; Lowenson, J. D.; Clarke, S.; Wolkow, C.; Wang, R.; Cotter, R. J.; Reardon, I. M.; Zu ¨ rcher-Neely, H. A.; Heinrikson, R. L.; Ball, M. J.; Greenberg, B. D. J. Biol. Chem. 1993, 268, 3072-3083. (13) Imai, K.; Fukushima, T.; Santa, T.; Homma, H.; Hamase, K.; Sakai, K.; Kato, M. Biomed. Chromatogr. 1996, 10, 303. (14) Imai, K.; Fukushima, T.; Santa, T.; Homma, H.; Huang, Y.; Sakai, K.; Kato, M. Enantiomer 1997, 2, 143-145. (15) Lins, R. D.; Soares, T. A.; Ferreira, R.; Longo, R. L. Z. Naturforsch. 1999, 54c, 264-270. (16) Geiger, T.; Clarke, S. J. Biol. Chem. 1987, 262, 785-794. (17) Stephenson, R. C.; Clarke, S. J. Biol. Chem. 1989, 264, 6164-6170. (18) Zagorski, M. G.; Hang, J.; Shao, H.; Ma, K.; Zeng, H.; Hong, A. Methods Enzymol. 1999, 309, 189-204. (19) Fukuda, H.; Shimizu, T.; Nakajima, M.; Mori, H.; Shirasawa, T. Bioorg. Med. Chem. Lett. 1999, 9, 953-956.

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The enhanced generation of the amyloid precursor protein (APP) fragment Aβ1-40 and Aβ1-42 in brains of patients afflicted with Alzheimer’s disease (AD) is thought to play an important role in AD pathogenesis. These peptides contain three aspartic acid residues at positions 1, 7, and 23. Aβ1-40 and Aβ1-42 make up the bulk of the amyloid plaques that characterizes AD brains. The plaques are extremely resistant to proteolysis and are only soluble in strong acid. Although plaque formation may theoretically be dynamic and reversible, it is likely that the aging AD brain offers an environment that strongly shifts the reaction toward plaque formation. Since the disease debut is likely to precede apparent signs of clinical onset by decades, there is a possibility that the amino acids in Aβ1-40 and Aβ1-42 of plaques, especially aspartic acid residues, have a turnover coupled with a racemization rate that could give vital information concerning etiology, initiation, and progression of the disease. This, of course, provided that conformational changes of the amino acid are not inherent to the fibril formation process. We have taken a novel methodological approach to attempt to measure the racemization of amino acids in senile plaques from AD brains, analyzing of D-amino acid content by acid hydrolysis followed by derivatization of the amino acids by the chiral chloroformate reagents (+)- or (-)-1-(9anthryl)-2-propyl chloroformate (APOC).21 The APOC reagents form stable carbamate reaction products with primary and secondary amines. Diastereoisomers are formed after reaction with chiral compounds. The diastereoisomers can be resolved by chromatographic methods without the need for chiral stationary phases or chiral selectors. Separation of the derivatized amino acids is performed by micellar electrokinetic chromatography (MEKC), and the reaction products are detected by laser-induced fluorescence (LIF). In a single chromatographic run, the chiral separation and detection of nine different amino acids could be performed. The APOC reagent and the separation system employed for the analysis in this article were previously used for the enantiomeric determination of amino acids in urine and cerebrospinal fluid.22 The enantiomeric composition of amino acids in nonaggregated synthetic β-amyloid peptides, in vitro aggregated β-amyloid peptide fibrils, and human β-amyloid enriched samples was studied in order to investigate the origin of the D-amino acids and to explore the possibility of using the D-amino acids for age determination of the β-amyloid peptides. EXPERIMENTAL SECTION Chemicals. Boric acid, sodium tetraborate, and methanol were from Merck (Darmstadt, Germany). Sodium dodecyl sulfate (SDS) was from ICN (Aurora, OH). All amino acids, β-amyloid peptide (1-40, lot 47H49541), and the sodium salt of 7-deoxycholic acid were from Sigma (St. Louis, MO). β-Amyloid peptide (1-40, lot 519599) was also obtained from Bachem (Bubendorf, Switzerland). Sodium hydroxide pellets were from Eka Nobel AB (Bohus, Sweden). High-purity hexane for residue analysis was obtained from Fisher Scientific Ltd. (Loughborough, Leicestershire, U.K.). All buffer solutions were prepared using water from an Elgastat UHQII (Elga Ltd., High Wycombe, U.K.). (20) Tomiyama, T.; Asano, S.; Furiya, Y.; Shirasawa, T.; Endo, N.; Mori, H. J. Biol. Chem. 1994, 269, 10205-10208. (21) Thorse´n, G.; Engstro ¨m, A.; Josefsson, B. J. Chromatogr.. A 1997, 786, 347354. (22) Thorse´n, G.; Bergquist, J. J. Chromatogr., B 2000, 745, 389.

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In Vitro Aggregated Peptides. β-Amyloid 1-40 peptide (lyophilized powder) was dissolved in double-distilled H2O (ddH2O) and diluted to a concentration of 175 µM with 50 mM phosphatebuffered (pH 7.4) saline (0.1 M, PBS) containing 0.02% sodium azide. Samples were incubated in presilanized Eppendorf tubes in a 150-µL volume, in the dark, at 30 °C and without agitation. After 3 and 6 weeks, samples were centrifuged at 17900g for 5 min at 16 °C. Pellets from two tubes were immediately hydrolyzed for the racemization analysis. Transmission Electron Microscopy. The pellet from a third tube was used to verify fibrillization using transmission electron microscopy. To this pellet, 25 µL of ddH2O was added. The sample was vortexed, and 8-µL aliquots were added to carbon-stabilized Formvar grids (Ted Pella, Inc., Redding, CA). Samples were stained with 1% uranyl acetate (E. Merck, Darmstadt, Germany) and examined using a Philips CM10 transmission electron microscope. Size Exclusion Chromatography. To estimate the quantity of fibrils formed during the incubation period, the supernatants from the centrifuged samples were subjected to analysis using size exclusion chromatography (SEC). A Merck Hitachi D-7000 HPLC system, having a diode array detector model L-7455 and a model L-7100 pump, coupled to a Superdex 75 PC3.2/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden), was used for the chromatographic analysis. Samples were eluted at a flow rate of 0.08 mL/min (22 °C) using 50 mM Na2HPO4/NaH2PO4 (pH 7.4), 0.15 M NaCl. Chromatograms were obtained by measuring UV absorbance at 214 nm. Peak areas for soluble Aβ were integrated using Merck-Hitachi model D-7000 Chromatography Data Station software. A standard curve was produced by correlating integrated peak areas with peptide concentrations, as determined by quantitative amino acid analysis. The concentration of soluble peptide remaining in solution after centrifugation was calculated from the standard curve. Human Aβ Enriched Samples. At autopsy the left hippocampal formation and frontal cortex (Brodmann area 9) was dissected out, homogenized in liquid nitrogen, and stored at -80 °C until biochemical analyses. The two AD patients, age 66 and 84 years, fulfilled the clinical NINCDS criteria for probable AD.23 They had a histopathological score24 of five or above. The control patient, age 87 years, died from cardiac disease. The medical records revealed no history of dementia or psychiatric or neurological diseases. The control patient had a histopathological score24 lower than four. Samples subjected for CE analysis were dissolved in sample buffer (PBS containing 2% SDS) 10-20 mg of tissue in 1 mL, ultrasonicated for 15 min, and centrifuged for 10 min at 35000g. The supernatants (∼1 mg of total protein) were lyophilized and kept at -80 °C until biochemical analyses. Protein Hydrolysis. Aβ peptide (1-40) or plaque from brain tissue was dissolved in 150 µL of trifluoroacetic acid and transferred to a pyrolyzed borosilicate tube flame sealed at one end. The trifluoroacetic acid was evaporated by purging of nitrogen. Hydrochloric acid (300 µL of 6 N HCl) was added to the tube and the open end flame-sealed under reduced pressure. (23) McKhann, G.; Drachman, D.; Folstein, M.; Katzman, R.; Price, D.; Stadian, E. M. Neurology 1984, 34, 939. (24) Alafuzoff, I.; Iqbal, K.; Fride´n, H.; Adolfsson, R.; Winblad, B. Acta Neuropathol. (Berlin) 1987, 74, 209.

Figure 1. MEKC-LIF chiral analysis of amino acids showing reversal of elution order when optical form of the reagent is interchanged. Upper trace, (+)-APOC derivatized human senile plaque hydrolysate; lower trace, (-)-APOC derivatized human senile plaque hydrolysate. The samples were diluted 20- and 10-fold, respectively, prior to injection. Injection volumes are 0.3 nL. The separation voltage applied was 30 kV, resulting in a current of 9 µA. The symbol * denotes (-)-APO-D-glutamic acid and the symbol # denotes (-)-APO-D-aspartic acid.

The sealed ampule was incubated at 110 °C for a given time interval. The ampule was allowed to cool, opened, and the hydrochloric acid evaporated under nitrogen. Derivatization Procedure. The dry residue from the hydrolysis was dissolved in 200 µL of 0.2 M boric acid buffer adjusted to pH 8.2 with 6 M sodium hydroxide solution. The buffered solution was transferred to an Eppendorf tube. Reagent solution (100 µL of 10 mM APOC in acetonitrile) was then added to the sample solution and allowed to react for 5 min at room temperature. The derivatization reaction was terminated by the extraction of excess reagent with 200 µL of hexane. The sample solution was diluted 10-fold with deionized water prior to injection on the MEKC-LIF system to ensure a lower conductivity in the sample plug as compared with the separation electrolyte. Peak assessment is enhanced by dividing the sample into two equal portions and derivatizing each portion with a different optical isomer of the reagent. When the optical form of the reagent is interchanged, the elution order of the amino acid enantiomers is reversed.25 If the reagent used for derivatization is not optically pure, the impurity of the reagent must be determined and corrected for. The optical impurity of the (+)- and (-)-APOC reagents has been established according to the procedure presented in previous work.21,26 The (-)-APOC reagent has an optical impurity of 0.15% (+)-APOC while the (+)-APOC has an optical impurity of 0.1%. Racemization during the derivatization is further minimized since the derivatization reaction is performed at ambient reaction conditions, i.e., room temperature and pH 8.2. Identical derivatization conditions were used when the enantiomeric impurity of APOC was elucidated.21 (25) Allenmark, S. Chromatographic enantioseparation; Ellis Horwood: Chichester, U.K., 1988. (26) Engstro ¨m, A.; Wan, H.; Andersson, P. E.; Josefsson, B. J. Chromatogr., A 1995, 715, 151.

Separation Conditions. Chiral separation of the amino acids is performed using a mixed MEKC system. The separation electrolyte consisted of 20 mM borate buffer adjusted to pH 9.8 with 0.1 M sodium hydroxide solution. The micellar system consisted of 20 mM sodium dodecyl sulfate and 7.5 mM sodium deoxycholate. The optimization of the chromatographic separation, exhibiting separation efficiencies in the range of 8 × 105-1.4 × 106 theoretical plates/m for APOC-derivatized amino acids, has been described elsewhere.22 An example of a chromatographic separation, showing the reversal of elution order depending on the form of reagent used, is shown in Figure 1. The amino acids seen in the chromatograms are predominately L-amino acids. In the lower trace, showing (-)-APOC derivatized amino acids, the D-enantiomers of glutamic acid (*) and aspartic acid (#) can be clearly seen underneath their L-counterparts in the upper trace. The D-amino acids are readily quantifiable in injections of less diluted samples. Apparatus. The capillary electrophoresis (CE) equipment used was an in-house-built system consisting of a high-voltage power supply (Brandenburg, Thornton Heath, U.K.) and an untreated fused-silica capillary with an internal diameter of 25 µm and an external diameter of 150 µm (Polymicro Technologies, Phoenix, AZ). The length of the capillary was 92 cm with the polyimide coating removed at 84 cm from the injection end to enable LIF detection. The laser used for LIF detection was a fullframe argon ion laser, Innova argon 304 (Coherent, Palo Alto, CA), in an optical arrangement similar to that described by Yeung et al..27 The 351.0- and 351.5-nm emission lines from the laser were used for the detection of APOC-derivatized amino acids. An UG11 short-pass filter was employed to filter away light from other light sources in the room. Emitted light was collected at a 90° angle and focused onto a R212-UH photomultiplier tube (Hamamatsu, (27) Yeung, E. S.; Wang, P.; Li, W.; Giese, R. W. J. Chromatogr. 1992, 608, 73.

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Figure 2. Regression curves for Asx, Glx, alanine, and phenylalanine hydrolyzed 12, 24, 48, and 72 h. Analysis performed by derivatization with (+)-APOC.

Hamamatsu City, Japan) using a microscope objective. Scattered light was filtered away using a KV389 long-pass filter, and Raman emission from the buffer was filtered away using a 20-nm-wide band-pass filter centered around 412 nm. All filters used were from Schott (Mainz, Germany). The high-voltage electrode was placed in a Plexiglas box at the injection end of the capillary. A separation voltage of 30 kV was used in all of the experiments, resulting in a current of 9 µA. All instrumentation was placed on a springmounted optical bench. RESULTS AND DISCUSSION Hydrolysis Procedure. Proteins and peptides must be hydrolyzed before performing a chromatographic determination of the enantiomeric composition of the individual amino acids. The hydrolysis of peptides and proteins can be performed either chemically or enzymatically.28 Subjecting the protein or peptide to mineral acid or base at elevated temperatures is the most frequently used method for complete hydrolysis. While acidic hydrolysis induces racemization of the amino acids to some extent, basic hydrolysis will racemize the amino acids completely at the elevated temperatures used.28 Enzymatic hydrolysis was not used in this work as the aggregated Aβ peptides are not susceptible to proteolysis. The hydrolysis procedure must often be modified, depending on the sample type and the analysis being performed. The hydrolysis time has been kept short in most studies focused on the chiral determination of amino acid residues in peptides or proteins9-11 in order to avoid racemization artifacts. Only in the study performed by Roher et al.12 was a reference peptide with the same sequence cohydrolyzed along with the samples for measurement of the racemization induced by the sample preparation procedure. If a standard peptide can be obtained, containing only L-amino acids, then the racemization induced by hydrolysis can be measured and corrected for.29 It has been shown, however, that the method used for peptide synthesis affects the optical purity (28) Fountoulakis, M.; Lahm, H. W. J. Chromatogr., A 1998, 826, 109-134.

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of the peptides.18 The risk for misinterpretations due to optical impurity in the standard peptide can be significantly reduced if several amino acids can be analyzed simultaneously. The ratio of D-amino acids present before hydrolysis can also be elucidated by plotting the ratio of racemized amino acids against the hydrolysis time and interpolating to a value for the amino acid ratio prior to hydrolysis.8,30 Special consideration needs to be taken for asparagine, aspartic acid, glutamine, and glutamic acid. Asparagine and glutamine are deaminated during hydrolysis and are converted into aspartic acid and glutamic acid. Comparative studies between samples can be performed if the deamination of asparagine and glutamine is much faster than the conversion of L-amino acids to D-amino acids. If the deamination of, for example, asparagine is complete before the first sampling time for the hydrolysis regression, the regression curve will only be biased to a value different from that of the value corresponding to just aspartic acid. If the deamination of asparagine is slower or comparable with the conversion of L-aspartic acid to D-aspartic acid, the two competing reactions would cause nonlinearity in the regression curve of Asx (a combined value for asparagine and aspartic acid). The regression curves for Asx, Glx, phenylalanine, and alanine are presented in Figure 2. As the regression curves are linear, the conclusion can be drawn that the racemization of Asx and Glx occurs predominately as aspartic acid and glutamic acid. To determine the time needed for the hydrolysis of Aβ peptides, as well as the racemization induced by the hydrolysis, a synthetic Aβ peptide consisting of amino acid residues 1-40 was purchased. Aliquots of 2-4 µg of peptide were hydrolyzed in each experiment. The hydrolysis time varied between 4 and 72 h. Complete hydrolysis of the nonaggregated standard peptide was achieved by 12-h hydrolysis. A hydrolysis time of 48 h was, however, needed for the complete hydrolysis of the aggregated senile plaque peptides. To establish the reproducibility of the (29) D’Aniello, A.; Petrucelli, L.; Gardner, C.; Fisher, G. Anal. Biochem. 1993, 213, 290-295. (30) Frank, H.; Woiwode, W.; Nicholson, G.; Bayer, E. Liebigs Ann. Chem. 1981, 354-365.

Figure 3. Relative abundance of D-amino acids present in samples hydrolyzed 48 h. Samples: three aggregated standard β-amyloid peptide fibril samples, three reference (nonaggregated) β-amyloid peptide samples, and three human senile plaque core samples. Table 1. Repeatability of Peptide Hydrolysis (% D-Amino Acid Content)a average

std dev

reagent impurity

corrected value

Glx Asx Ser Phe

3.54 7.62 3.76 3.48

(-)-APOC Derivatized 0.30 1.5 0.63 1.5 0.33 1.5 0.54 1.5

2.04 6.12 2.26 1.98

Glx Asx Ser Ala

1.92 5.15 1.07 1.04

(+)-APOC Derivatized 0.41 0.1 0.49 0.1 0.27 0.1 0.11 0.1

1.82 5.05 0.97 0.94

a The values presented are averages of five replicate hydrolyses of standard β-amyloid peptide samples. Hydrolysis condidtions: 12 h at 110 °C in 6 N HCl. The values for the average D-amino acid content, the standard deviations, and the corrected values are presented as the percent D-amino acid content. The reagent impurity is presented as the percent enantiomeric impurity of the reagent.

sample preparation procedure, replicate hydrolyses of the standard peptide were performed. Five standard peptide samples were hydrolyzed for 12 h and then analyzed. These results are presented in Table 1. A comparative study was performed at 48 h of hydrolysis. The samples studied consisted of reference peptide (Aβ1-40 peptide), in vitro precipitated β-amyloid fibril samples, and human Aβ enriched samples. The enantiomeric ratios, in percent D-amino acid, for aspartic acid, glutamic acid, and serine are presented in Figure 3. Values were also obtained for valine, alanine, phenylalanine, leucine, and isoleucine. The great concentration range between the least abundant D-enantiomer to the most abundant L-enantiomer limited the number of amino acids that were measured in this study. Only the amino acids that were reliably determined in all of the samples are presented here. In Vitro Aggregated Peptides. The in vitro aggregation and precipitation of β-amyloid peptides was monitored using SEC

Figure 4. Transmission electron micrograph (117000×) of a negatively stained sample of Αβ1-40 fibrils formed after 2.6 weeks of incubation. Fibril diameter is estimated to be ∼10 nm. Scale bar, 100 nm.

analysis. More than 99% of all soluble Aβ1-40 initially present in the supernatant could be recovered in the sedimented pellet after both the 2.6- and 6.7-week incubation periods. From previous kinetic studies, it was clear that a 2-day incubation period under similar conditions was sufficient for fibril formation leaving ∼15% of the soluble Aβ1-40 unfibrillized in the supernatant.31 Fibrils were identified in the 2.6-week sample and conformed to dimensions and appearance of Aβ1-40 fibrils found in senile plaques of AD patients having a diameter of ∼10 nm with undeterminable length, as shown in Figure 4. (31) Nilsberth, C.; Westlind-Danielsson, A.; Eckman, C. B.; Axelman, K.; Forsell, C.; Luthman, J.; Younkin, S. G.; Na¨slund, J.; Lannfelt, L. Submitted to Science.

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Figure 5. Chromatographic separations of (a) human senile plaque core hydrolysate and (b) hydrolysate of aggregated β-amyloid peptide fibers. Both samples were diluted 100-fold prior to analysis. The injection volume for (a) was 0.7 nL and for (b) 1.4 nL. The separation voltage applied was 30 kV, resulting in a current of 9 µA.

The results gained for the standard peptide and the in vitro aggregated Aβ peptide imply that racemization is not a major contributing factor to the aggregation process. No significant increase in D-amino acid content could be seen in the transition from dissolved monomeric peptides to aggregated peptides in fibrils. Nor did the time the samples were incubated for fibril formation influence the D-amino acid content in the fibrils. These results do not exclude the possibility that racemization of individual amino acid residues may be involved in the initial nucleation process of the fibrils. However, it seems unlikely that the racemization of amino acids would be part of the aggregation process in the later stages of fibril formation. Human Aβ Enriched Samples, No significant difference in D-amino acid content could be observed between the nonaggregated standard peptide and the human Aβ enriched samples. There are some plausible explanations for this result. The reference peptide could have an optical impurity equal to the degree of racemization in the biological peptides. However, this seems unlikely as several amino acids were analyzed and in no case could any such trend be established. One interpretation of the results could be that the biological material contains no measurable amount of D-amino acids and that all D-amino acids found are artifacts from the sample preparation procedure. This would contradict the results published by Roher et al.12 and implies that no racemization occurs in the peptides when aggregated in senile plaque cores. It could also indicate that the human samples contain proteins or peptides that are not stationary. Figure 5 shows chromatographic separations of a human senile plaque core hydrolysate and a hydrolysate of aggregated Aβ standard peptide fibrils. The presence of threonine and proline in the human samples indicates that the samples analyzed do not entirely consist of Aβ peptide but rather a mixture of peptides. Earlier studies on the amino acid composition of purified senile plaque cores have shown large differences in the relative abun2630 Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

Table 2. Racemization Rate Constants (k × 10-8, s) of Selected Amino Acids amino acid

this worka Frank et al.30

glutamic acid

aspartic acid

alanine

phenylalanine

38b 23.1

81c 47.2

26b 15.5

30c 16.7

a The values presented from these experiments represent a combined value of glutamic acid and glutamine as well as aspartic acid and asparagine. The racemization rate constants are calculated as described by Frank et al.30 b Calculated as an average of six experiments. c Calculated as an average of five experiments.

dance of amino acids.11 If other proteins that are associated with the plaque cores are not stationary, then the changes in amino acid ratios expected in aged proteins could be suppressed by the presence of newly transcribed proteins. The senile plaque cores must therefore be more extensively purified by, for instance, reversed-phase high-performance liquid chromatography prior to an analysis focused on the conformational changes of the amino acids. In light of the results presented by Roher et al.,12 this would seem the most reasonable conclusion. The racemization rate constants of four of the amino acids were calculated from the regression curves of the human senile plaque samples and are presented in Table 2. ANOVA calculations showed F significance for the regression curves between 0.002 and 0.08, indicating a significant regression. The rate constants are in all cases higher than the rate constants earlier published in the literature.30 This indicates that the rate constants generated in this work are combinations of the rate constants of free amino acids and the rate constants of peptide bound amino acids. CONCLUSIONS The applicability of MEKC-LIF for the determination of the enantiomeric composition of peptides has been demonstrated by

the analysis of standard Aβ peptides, in vitro aggregated peptides, and human Aβ enriched samples. Two methods have been applied to elucidate the contribution of D-amino acids from the hydrolysis procedure: cohydrolysis of a standard peptide and interpolation of the D-amino acids produced during hydrolysis to a time prior to the start of the hydrolysis. Several amino acids could be studied in a single chromatographic separation due to the selectivity and resolving power of the MEKC separation system. Separation efficiencies surpassing 8 × 105 theoretical plates/m are achieved for APOC derivatized amino acids. Analysis of synthetic peptide aged into amyloid fibrils did not reveal any significant D-aspartic acid levels. This finding would preclude that Asp racemization is a driving force in the generation of amyloid fibrils and reveals that there is no significant racemization in vitro within the time span studied (6 weeks). Although a similar picture was seen for the human Aβ enriched samples, the samples revealed impurities that may overshadow or dilute racemization events inherent to the Aβ peptides. Thus, it seems necessary to couple even more rigorous peptide purification protocols to the described methodology in order for firm conclusions to be drawn.

ACKNOWLEDGMENT We thank Gunnel Arnerup for help with the electron microscopy. Drs. Rudolf Kaiser and Sven Petre´n are acknowledged for all their support with the SEC analysis. The authors thank Dr. Kaj Blennow, (Inst. Clin. Neuroscience, Department Psychiatry and Neurochemistry, Go¨teborg University, Sahlgrenska University Hospital, Mo¨lndal, Sweden) for providing the human brain samples. We also thank professor Sven Jacobsson for initializing the cooperation concerning the in vitro samples. This work was supported by the Fredrik and Ingrid Thuring Foundation, the Wilhelm and Martina Lundgren Foundation, the Magnus Bergvall Foundation, the Swedish Alzheimer Foundation, the Syskonen Svensson Foundation, the Gamla Trotja¨narinnor Foundation, the Swedish Lundbeck Foundation, the Swedish Society for Medical Research, the Swedish Natural Science Research Council (Grants K-AA/KU 12003-300 and 1084901), and the Swedish Medical Research Council (Grant 13123). Received for review July 27, 2000. Accepted January 4, 2001. AC000861Q

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