Current Clinical Pharmacology, 2008, 3, 144-152
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Serum Amyloid Beta Peptides in Patients with Dementia and Age-Matched Non-Demented Controls as Detected by Surface-Enhanced Laser Desorption Ionisation-Time of Flight Mass Spectrometry (SELDI-TOF MS) Suzanne V. Frankfort1,2, Jos P.C.M. van Campen1, Linda R. Tulner1 and Jos H. Beijnen2,3,* 1
Department of Geriatric Medicine, 2Department of Pharmacy & Pharmacology, Slotervaart Hospital, Louwesweg 6, 1066 EC Amsterdam, The Netherlands; 3Faculty of Pharmaceutical Sciences, Department of Biomedical Analysis, Division of Drug Toxicology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands Abstract: Background: By using surface enhanced laser desorption/ionisation- time of flight mass spectrometry (SELDITOF MS) an amyloid ß (Aß) profile was shown in cerebrospinal fluid (CSF) of patients with dementia. Objective: To investigate the A-profile in serum with SELDI-TOF MS, to evaluate if this profile resembles CSF profiles and to investigate the correlation between intensity of A-peptide-peaks in serum and clinical, demographical and genetic variables. Methods: Duplicate profiling of Aß by an SELDI-TOF MS immunocapture assay was performed in 106 patients, suffering from Alzheimer´s Disease or Vascular Dementia and age-matched non-demented control patients. Linear regression analyses were performed to investigate the intensities of four selected A peaks as dependent variables in relation to the independent clinical, demographic or genetic variables. Results: Aß37, Aß38 and Aß40 were found among additional unidentified Aß peptides, with the most pronounced Aß peak at a molecular mass of 7752. This profile partly resembled the CSF profile. The clinical diagnosis was not a predictive independent variable, however ABCB1 genotypes C1236T, G2677T/A, age and creatinine level showed to be related to Aß peak intensities in multivariate analyses. Conclusions: We found an Aß profile in serum that partly resembled the CSF profile in demented patients. Age, creatinine levels, presence of the APOE 4 allele and ABCB1 genotypes (C1236T and G2677T/A) were correlated with the A serum profile. The role of P-gp as an Aß transporter and the role of ABCB1 genotypes deserves further research. The investigated serum A profile is probably not useful in the diagnosis of dementia.
INTRODUCTION Dementia is a rapidly growing health and socioeconomic problem worldwide, as it is estimated that 24 million people have dementia nowadays and that this will increase to 42 million by 2020 and 81 million by 2040 [1]. Alzheimer’s Disease (AD) is the most common form of dementia [2] and Vascular Dementia (VaD) is the second in the developed world [3]. Amyloid precursor protein (APP) is degraded by secretases and -secretases into amyloid beta (A) peptides that play a key role in the pathogenesis of AD [2] and are also related to VaD [4]. A40 and A42 [5,6] have been frequently investigated and more recently also A38 [7,8] was found in cerebrospinal fluid (CSF) by using Enzyme Linked Immunosorbent Assay (ELISA). A38, A40 and A42 are candidate biomarkers for AD and A42 for its potential to differentiate from VaD [9-12]. A38, A40 and A42 are also found in plasma of patients with AD [13-15] and A40 and A42 in patients with VaD [15] as investigated with ELISA. Recently, van Ooijen et al. [15] showed in a longitu*Address correspondence to this author at the Department of Pharmacy & Pharmacology, Slotervaart Hospital, Louwesweg 6, 1066 EC Amsterdam, The Netherlands; Tel: +31-20-5124737; Fax: +31-20-5124753; E-mail:
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dinal follow-up study that elevated baseline A40 plasma levels, especially when combined with low baseline plasma levels of A42, indicate an increased risk for AD. Results from new proteomic techniques in dementia research may add to current knowledge regarding biomarkers obtained with ELISA. One of these techniques is the Protein Chip® array based Surface Enhanced Laser Desorption/ Ionisation in combination with Time of Flight Mass Spectrometry (SELDI-TOF MS). Arrays with different chromatographic surfaces enhance on chip purification and retained proteins are analysed on the same chip resulting in a profile of proteins characterised by mass and charge [16]. SELDITOF MS has been applied in CSF of AD [17,18] and in VaD (unpublished results Frankfort et al.) and revealed novel Apeptides besides A38, A40 and A42 resulting in an Afingerprint, consisting of A37, A38, A39, A40 and A42, in CSF. To our best knowledge, only Lewczuk et al. [19] described an A-fingerprint in plasma of AD patients, using gel electrophoresis. SELDI-TOF MS analysis in plasma or serum of patients suffering from AD or VaD has not yet been described. An interesting question is whether any A-profile in serum is comparable to CSF. In addition, it would be relevant to investigate whether significant differences in the A serum profile exist between patients with ©2008 Bentham Science Publishers Ltd.
SELDI-TOF MS Analysis of Amyloid Beta Peptides in Dementia
AD, VaD, and age-matched non-demented control patients and how known variables correlate to A-levels, e.g. age [20], serum creatinine level [21] and presence of the apolipoprotein E (APOE) 4 [22]. A peptides can thus be measured both in CSF and plasma, however the source of the peptides remains unknown. P-glycoprotein (P-gp, ABCB1), a 170 kDa membrane bound efflux pump, is located at the Blood Brain Barrier (BBB) at the apical membrane of endothelial cells and is proposed to act as a flippase and distributes substrates into the extracellular space [23,24]. P-gp is also expressed at the blood-cerebrospinal fluid (BCSF) barrier, that is formed by the choroid plexus [25]. A possible role of P-gp in mechanisms underlying the pathology of Alzheimer’s disease (AD) has been hypothesised. A is an in vitro substrate for P-gp [26] and P-gp deficiency at the BBB increases beta amyloid deposition in an AD mouse model [27]. Vogelgesang et al. [28] showed P-gp expression at the BBB to be inversely correlated to the number of amyloid plaques in the medial temporal lobe in 243 non-demented elderly. The Multi Drug Resistance gene (ABCB1), encoding for P-gp, is highly polymorphic [29]. The three most frequently occurring Single Nucleotide Polymorphisms (SNPs) are C1236T in exon 12, G2677T/A in exon 21 and C3435T in exon 26 [30]. The synonymous SNP C3435T was the first variant to be associated with altered protein expression [29]. As ABCB1 SNPs may be related to P-gp expression and function we hypothesised that ABCB1 genotypes are possibly correlated to serum amyloid levels. This study aims a) to investigate the A-profile in serum of elderly patients with SELDI-TOF MS, b) to evaluate if the serum profile resembles the A CSF profile as investigated with SELDI-TOF MS and c) to investigate if the intensities of the peaks corresponding to A-peptides in serum are correlated to clinical (dementia diagnosis, serum creatinine level), demographic (age, gender) and genetic (APOE 4 allele, ABCB1 SNPs C1236T, G2677T/A, C3435T) variables. METHODS Patients and Diagnosis This prospective study was executed at the geriatric diagnostic day-clinic of the Slotervaart Hospital, a teaching hospital in Amsterdam, the Netherlands. In each patient complete geriatric assessment including Mini Mental State Examination (MMSE) [31], the 7-Minute Neurocognitive Screening Test [32] and laboratory testing, including thyroid function, levels of folic acid, thiamine and vitamin B12, was performed. To assist in dementia diagnosis, patients underwent more extensive neuropsychological assessment and, if necessary, computerised tomography or magnetic resonance imaging was performed. This study included patients suffering from AD or VaD and age-matched non-demented control patients. AD was diagnosed according to the NINCDSADRDA criteria [33] and VaD according to the NINDS/ AIREN criteria [34]. Controls were recruited from the same diagnostic day-clinic. These participants did not show any cognitive impairment. Most of these geriatric patients were
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presented at the diagnostic day-clinic for a somatic screening. The study protocol was approved by the Institutional Review Board of the Slotervaart Hospital, Amsterdam, The Netherlands. Written informed consent was obtained from each participant in this study. Sample Collection From each participant a 2 ml EDTA whole blood sample was obtained by venous puncture and stored at -20 °C until genotype analysis. Serum samples (5 ml) were obtained by venapuncture and directly after clotting centrifuged at 1000 g for 10 min. Supernatants were divided into 0.5 ml aliquots and stored at -20 ºC. After collection of all samples these were stored at -80 ºC. SELDI-TOF MS analysis was performed with serum unexposed to any freeze-thaw cycle. Sample Pre-Treatment and SELDI-TOF MS Analysis SELDI-TOF MS analysis using an antibody-capture assay was performed in duplicate in our serum sample set. 2 l of 6E10 monoclonal antibody (Chemicon International Inc., Temecula, CA, USA) solution in phosphate buffered saline (PBS) (0.25 mg/ml) was added to each spot of a PS20 Protein Chip array (Ciphergen Biosystems Inc., Fremont, CA, USA) used for detection of patient serum samples. Additionally, 2 l of a bovine IgG solution in PBS (0.25 mg/ml) (Ciphergen Biosystems Inc.) was added on spots used for analysis of negative controls. The arrays were incubated for 2 hours at room temperature in a humidity chamber on a platform shaker at 350 r/m. Afterwards 6E10 antibody or IgG were removed from each spot and the arrays were placed in 10 mL polypropylene tubes containing deactivation buffer (0.5M ethanolamine (Sigma, St. Louis, MO, USA) in PBS (Sigma), pH 8.0) to block the residual sites on the array. Tubes were agitated on a shaker for 30 minutes at room temperature. The arrays were then washed twice with wash buffer (0.5% Triton-X-100 (Sigma) in PBS) for 10 minutes and once with PBS for 5 minutes at room temperature. The arrays were transferred to a 96-well format bioprocessor (Ciphergen Biosystems) and 50 l of serum was applied to each well. The bioprocessor was placed on a platform shaker and agitated overnight at 4 ºC. The samples were removed and the arrays were washed in the bioprocessor three times with wash buffer for 15 minutes and three times with PBS for 5 minutes and finally rinsed with 1.0 mM HEPES buffer (Ciphergen Biosystems). As energy absorbing matrix a volume of 0.5 l of a 20% saturated solution of -cyano-4-hydroxycinnamic acid (CHCA) (Ciphergen Biosystems) in 50% v/v acetonitrile and 0.5% v/v TFA was added twice to each spot. Protein profiling of each spot was performed using the PBSIIC Protein Chip Reader (Ciphergen Biosystems). Data were collected between 0 and 15,000 Da. Data collection was optimised, resulting in an average of 100 laser shots per spectrum at laser intensity 150 and detector sensitivity 7. M/z values for detected proteins were calibrated externally with a standard peptide mixture (Ciphergen Biosystems) containing [Arg8] vasopressin (1084.3 Da), somatostatin (1637.9 Da), dynorphine (2147.5 Da), ACTH (2933.5 Da), insuline ß-chain (bovine) (3495.9 Da) and insulin (human recombinant) (5807.7 Da).
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Bioinformatics
Statistics
All acquired SELDI-TOF MS spectra were compiled and analysed as a whole experiment with ProteinChip software (version 3.1, Ciphergen Biosystems). Spectra were baseline subtracted and normalised to the total ion current between 1500 and 15,000. The Biomarker Wizard application (BMW) (Ciphergen Biosystems) was used to autodetect m/z peaks with a signal-to-noise ratio of at least 5. Peak clusters were completed with peaks with a signal-to-noise ratio of at least 2 in a 0.3% cluster mass window. Peak data per spectrum were exported to Excel (Microsoft Inc., Redmond, WA, USA) and mean intensities were calculated from duplicate spectra for each participant.
Univariate linear regression analyses were performed with intensities of selected peaks obtained for each patient by SELDI-TOF MS as dependent variable and age, gender, serum creatinine level, presence or absence of an APOE 4 allele, ABCB1 genotypes C1236T (CC, CT, TT), G2677T/A (GG, GT, TT/TA), C3435T (CC, CT, TT) and diagnostic group (age-matched non-demented controls, AD or VaD) as independent variables. When multiple significant (p
