ORIGINAL CONTRIBUTION. Age but Not Diagnosis Is the Main Predictor of Plasma Amyloid -Protein Levels

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1 ORIGINAL CONTRIUTION Age but Not Diagnosis Is the Main Predictor of Plasma Amyloid -Protein Levels Hiroaki Fukumoto, PhD; Marsha Tennis, RN; Joseph J. Locascio, PhD; radley T. Hyman, MD, PhD; John H. Growdon, MD; Michael C. Irizarry, MD ackground: Plasma amyloid -protein A 42 levels are increased in patients with familial Alzheimer disease (AD) mutations, and high levels reportedly identify individuals at risk to develop AD. Objectives: To determine whether there are characteristic changes in plasma A 4 and A 42 levels in sporadic AD, and to examine the relationship of plasma A measures with clinical, demographic, and genetic variables in a prospectively characterized outpatient clinic population. Patients: A total of 371 outpatients with sporadic AD (n=146), mild cognitive impairment (n=37), or Parkinson disease (n=96) and nondemented control cases (n=92). Methods: We collected plasma samples and determined A 4 and A 42 levels by sandwich enzymelinked immunosorbent assay with the use of the capture antibody NT77 (anti A 11-28) and the detector antibodies horseradish peroxidase linked A27 (anti- A 4) and C5 (anti-a 42). Results: Mean A 4 and A 42 levels increased significantly with age in each diagnostic group. When covaried for age, mean plasma levels of A 4 and A 42 did not differ significantly among the 4 diagnostic groups. Within the mild cognitive impairment and AD groups, A 4 and A 42 levels did not correlate with duration of memory impairment or with cognitive test scores. The A measures were not influenced by family history of AD, apolipoprotein E genotype, or current medication use of cholinesterase inhibitors, vitamin E, statins, nonsteroidal anti-inflammatory drugs, or estrogen. Conclusions: Plasma A measures increase with age, but, in contrast to reports on familial AD, plasma A measures were neither sensitive nor specific for the clinical diagnosis of mild cognitive impairment or sporadic AD. Arch Neurol. 23;6: From the Department of Neurology, Massachusetts General Hospital, oston. AMYLOID -PROTEIN (A )is a major component of amyloid plaques in brain of patients with Alzheimer disease (AD). Amyloid -protein is derived from the -secretase pathway of amyloid precursor protein (APP) processing by the enzymatic activity of the -site APP cleaving enzyme which releases the N-terminus of A from APP and a presenilin-dependent -secretase activity that releases the C-terminus of A from the membrane. 1 The most common forms of A contain 4 (A 4) or 42 (A 42) amino acids. The A 42 is more fibrillogenic and deposits early in amyloid plaques. 2,3 In addition to being deposited in the brain, A can be detected in cerebrospinal fluid (CSF) and plasma, leading to the analysis of A levels in these fluids as biomarkers of the cerebral amyloidosis in AD. Cerebrospinal fluid A 42 level is reduced in AD 4-7 and is inversely proportional to dementia severity in some studies. 8 Plasma A 42 level is increased in patients with familial AD mutations. 9,1 Studies of A 4 and A 42 in plasma of patients with sporadic AD have been equivocal, some suggesting increased A 4 or A 42 levels in AD or preclinical AD, 11,12 but others showing no change. 9,1,13,14 Sensitive measurement of plasma A levels in a large patient group is required to clarify the clinical, demographic, and genetic factors that influence plasma A levels, and as a prerequisite for proposing plasma A as a biomarker for diagnosis, progression, and treatment effects. The principal goal of this study, therefore, was to determine the sensitivity and specificity of plasma A 4 and A 42 levels for the diagnosis of AD. A related goal was to examine the relationship of plasma A measures with disease severity, medication use, apolipoprotein E (APOE) genotype, and other demographic variables in a prospectively characterized outpatient clinic population. (REPRINTED) ARCH NEUROL / VOL 6, JULY Downloaded From: on 7/3/ American Medical Association. All rights reserved.

2 METHODS PATIENTS Plasma samples were collected from patients in the Memory and Movement Disorders Units of Massachusetts General Hospital, oston, with a diagnosis of AD, 15 mild cognitive impairment (MCI), 16 nondemented Parkinson disease (PD), and no dementia. Informed consent was obtained from the patient and caregiver by a staff physician. The study was approved by the Massachusetts General Hospital Institutional Review oard. The following anonymized data were available for each case: (1) subject demographics, including date of birth, age, sex, race, education, family history of AD (defined as first-degree relative with AD), and family history of dementia; (2) clinical characteristics, including diagnosis, onset of disease, disease duration, lessed Dementia Scale Information-Memory-Concentration (DS-IMC) score, 17 the Clinical Dementia Rating Scale score, 18 and Hoehn and Yahr PD severity scale score 19 ; (3) current medication use, including cholinesterase inhibitors, estrogen, carbidopa-levodopa, dopamine agonists, anticholinergics, antiinflammatory medications, hypoglycemic agents, antioxidants, aspirin, and statins; and (4) protocol notes, including last meal, processing details, and protocol violations. LOOD COLLECTION From each patient, 22.5 ml of blood was collected in three 7.5-mL polypropylene sterile plunger tubes (S-Monovette; Sarstedt, Newton, NC), containing potassium EDTA, by a trained phlebotomist. The blood samples were cooled to 4 C for 15 minutes. A serum-plasma separator was added (Sure-Sep II; Organon, West Orange, NJ). In rapid succession, the samples were centrifuged at 33 rpm (138g) for 15 minutes and aliquoted in 96-µL quantities into polypropylene tubes containing 4 µl of a protease inhibitor cocktail (Complete, 1 tablet in 2 ml of phosphatebuffered saline; Roche, Indianapolis, Ind), then frozen on dry ice. The samples were stored at 8 C until ready for use. PLASMA PRETREATMENT To block cross-reaction of unidentified components of human plasma with the enzyme-linked immunosorbent assay (ELISA), plasma was precleared with mouse IgG1 (Sigma-Aldrich Corp, St Louis, Mo) cross-linked to agarose beads (CNr-activated Sepharose 4; Amersham iosciences, Piscataway, NJ). 1 Preclearing was performed by diluting 3 µl of each plasma sample with 525 µl of sample buffer (2mM phosphate, 4mM sodium chloride, 2mM EDTA, 1% blocking agent [lock Ace Liquid; Dainippon Pharmaceutical, Osaka, Japan],.2% bovine serum albumin,.765% 3-{[3-cholamidopropyl]dimethylammonio}-1-propanesulfonate [CHAPS], ph 7.2), and 75 µl of the agarose beads covalently cross-linked to nonspecific mouse IgG1. After incubation for 2 hours at 4 C, the beads were removed by centrifugation. SANDWICH A ELISA For this assay, 1 96-well microtiter plates (Maxisorp lack; Nalge Nunc, Rochester, NY) were coated with the capture antibody 5-µg/mL NT77 (mouse IgA anti A 11-28; Takeda Chemical Industries, Osaka, Japan) and blocked with blocking buffer (25% lock Ace Liquid in phosphate-buffered saline) for 6 hours. Pretreated plasma samples (1 µl, in triplicate) were incubated in NT77-coated wells containing 5 µl of sample buffer overnight at 4 C. The plates were washed 4 times with phosphate-buffered saline, then reacted with horseradish peroxidase conjugated detector antibodies (A27 mouse IgG2 anti-a 4, 1:1; C5 mouse IgG1 anti-a 42, 1:1,.5 µg/ml; Takeda Chemical Industries) in 75 µl of sample buffer for 4 hours at room temperature. After 6 washes with phosphate-buffered saline, horseradish peroxidase enzyme activity was measured with a fluorogenic substrate (Quanta lu; Pierce, Rockford, Ill) on a fluorometer (Wallac Victor Multilabel Counter; Perkin-Elmer, oston, Mass) with a 32-nm excitation filter and 4-nm emission filter. Each plate contained known concentrations of human synthetic A 1-4 and A 1-42 (achem, King of Prussia, Pa) in sample buffer to construct a log-log standard curve. These ELISAs can detect N-terminally truncated -site APP cleaving enzyme cleaved A species (A 11-4/ 42) as well as full-length A (A 1-4/42), but not -secretase cleaved products (p3; A 17-4/42). 2 STATISTICAL ANALYSIS Within groups, A variables were regressed on age, sex, duration of illness, and DS-IMC score. Significant factors (age and sex) were included in an analysis of covariance with A measures as dependent variables comparing diagnostic groups with nondemented controls, as well as other demographic and clinical variables. Most of the analyses in this study were well powered. For continuous factors, with the sample sizes of 92 to 146 in the control, PD, and AD cases, and a 2-tailed test at P=.5, the power was 8% to detect a population correlation of approximately r=.25 to.29. Only for the MCI group with a sample size of 37 was the power weaker, at 7% to detect a correlation of r=.4. Power for between-group comparisons was 8% to detect differences of approximately.4 SD (.5 SD for MCI). There were few or no missing values for medication use and demographic variables, so power was similarly strong for analyses involving them. RESULTS STANDARDIZATION OF ELISA The sensitivity and specificity of the antibodies and the ELISA have been published. 1 In our hands, the ELISA had a sensitivity of 1 pmol/l for A 4 and A 42. The recovery of exogenous A 4 and A 42 added to plasma was greater than 9%, irrespective of the presence or absence of the IgG1 resin, indicating that these ELISAs can detect both free A and A bound to plasma proteins. 1,21 Repeated measures of frozen aliquots of the same sample yielded SDs less than 1%, and correlation of repeated measures of samples showed r DEMOGRAPHICS Plasma samples were collected from 371 outpatients with a diagnosis of sporadic AD (n=146), MCI (n=37), nondemented control cases (n=92), and PD (n=96) (Table). Relative to the control group, the patients with AD were significantly older (P.1), had fewer years of education (P.1), had a greater family history of AD (P=.3), and had a greater APOE 4 allele frequency (.38 vs.11). Relative to the control group, the MCI group had a greater APOE 4 allele frequency (.39 vs.11), and the PD group had a significantly higher proportion of men (P.1). ANALYSIS OF A LEVELS WITH AGE, SEX, DURATION OF ILLNESS, AND DS-IMC y regression analysis, we found that the most robust determinant for A 4 and A 42 levels in each diagnostic (REPRINTED) ARCH NEUROL / VOL 6, JULY Downloaded From: on 7/3/ American Medical Association. All rights reserved.

3 Case Demorgraphics and Plasma Amyloid -Protein Levels Demographics No. of subjects Age, mean ± SD, y 69.4 ± ± ± ± 1. Sex, No. (%) male 37 (4) 66 (45) 18 (49) 66 (69) Education, mean ± SD, y 15.6 ± ± ± ± 3.3 Duration, mean ± SD, y NA 5.3 ± ± ± 5.8 Family history of AD, No./total No. (%) 6/62 (1) 23/91 (25) 6/22 (27) 4/68 (6) DS-IMC score, mean ± SD.9 ± ± ± ± 4.1 APOE ε4 allele frequency Medication use Statin, No. (%) 27 (29) 32 (22) 12 (32) 27 (28) Estrogen, No. (%) of women 13/55 (24) 18/8 (23) 7/19 (37) 12/3 (4) AchE-I, No. (%) () 115 (79) 17 (47) () NSAID, No. (%) 1 (11) 21 (14) 3 (8) 9 (9) Antioxidant, No. (%) 46 (5) 127 (87) 28 (78) 23 (24) Amyloid -protein results, mean ± SD A 4, pmol/l 48.9 ± ± ± ± 17.4 A 42, pmol/l 7. ± ± ± ± 3. A 42/A 12.5 ± ± ± ± 3.5 Abbreviations: AchE-I, acetylcholinesterase inhibitor; AD, Alzheimer disease; APOE, apolipoprotein E gene; DS-IMC: lessed Dementia Scale Information-Memory-Concentration; MCI, mild cognitive impairment; NA, not applicable; NSAID, nonsteroidal anti-inflammatory drug; PD, Parkinson disease. A Men Women Plasma Aβ4, pmol/l Age, y Age, y Figure 1. Amyloid -proteins A 4 (A) and A 42 () levels in relation to age and sex in the nondemented control group. Age had a significant positive relation to A 4 (P.1) and A 42 (P=.5). Sex had a significant relation to A 4 (P=.2) and a marginal relation to A 42 (P=.7), in both cases women having a higher mean than men. Age had similar positive relations to A measures in all diagnostic groups. est-fit lines are indicated for men (solid lines) and women (dashed lines). group was age (Figure 1). Other effects were seen only in single diagnostic groups. Within the AD and MCI groups, there was no association of A measures with duration of illness or severity of dementia, as estimated by the DS- IMC scores (Figure 2). For the control group, the A variables (A 4, A 42, and the ratio of A 42 to total A [A 42/A ]) were regressed on age and sex. Age had a significant positive relation to A 4 (P.1) and A 42 (P=.5). Sex had a significant relation to A 4 (P=.2), with women having a higher mean than men. For the AD group, A variables were regressed on age, sex, duration of illness, and the DS-IMC scores. The only significant effects were that age had a positive relation to A 4 (P=.1) and to A 42 (P=.1). For the MCI group, as for the AD group, A variables were regressed on age, sex, duration of illness, and the DS-IMC. The only significant effect was that age had a positive relation with A 4 (P.1). For the PD group, as for other diagnostic groups, A variables were regressed on age, sex, duration of illness, and the DS-IMC. The only significant effects were that age had a positive relation with A 4 (P.1) and with A 42 (P=.1), and duration of PD had a weak positive relation for A 4 (P.5). Pursuant to these findings, age and sex were included as covariates in the group comparisons that follow. (REPRINTED) ARCH NEUROL / VOL 6, JULY Downloaded From: on 7/3/ American Medical Association. All rights reserved.

4 A AD MCI Plasma Aβ4, pmol/l Duration, y Duration, y C D Plasma Aβ4, pmol/l DS-IMC DS-IMC Figure 2. Amyloid proteins A 4 (A and C) and A 42 ( and D) according to clinical measures of dementia severity. Duration of illness (A and ) and lessed Dementia Scale Information-Memory-Concentration score (DS-IMC, scored from to 37 mistakes) (C and D) had no correlation with A measures in the Alzheimer disease (AD) and mild cognitive impairment (MCI) groups. A variables were regressed on age, sex, duration of illness, and the DS-IMC. est-fit lines are indicated for AD (solid lines) and MCI (dashed lines). ANALYSIS OF A LEVELS ETWEEN GROUPS After covarying for age, there was no significant difference in A measures between diagnostic groups (Table). Analyses of covariance were run with the A variables as dependent variables comparing AD vs control groups crossed with a sex factor and including age as a covariate. The only significant effects involving group comparisons were significant interactions between sex and diagnostic group for A 4 (P=.4) and for A 42 (P=.4). In both cases, the interaction was due to the control group having a higher mean than the AD group among women, with the reverse situation occurring among men. The MCI and control groups as well as the PD and control groups were compared with the same analysis of covariance used to compare the AD and control groups. No significant effects involving diagnostic group were found. SECONDARY ANALYSES To determine whether other genetic or clinical features affect A measures, we evaluated the number of APOE 4 alleles, family history of dementia, family history of AD, and medication use. Number of APOE 4 alleles (, 1, or 2) was crossed with sex and diagnostic group (AD and MCI only; PD and control subjects were not included because there were too few individuals who were homozygotes for APOE 4), and age was covaried. Dependent variables were A 4, A 42, and A 42/A. No significant effects involving APOE 4 were found (Figure 3). Presence or absence of family history of dementia and of family history of AD were crossed with sex and diagnostic group (AD, PD, MCI, and controls), and age was covaried. Dependent variables were A 4, A 42, and (REPRINTED) ARCH NEUROL / VOL 6, JULY Downloaded From: on 7/3/ American Medical Association. All rights reserved.

5 A Plasma Aβ4, pmol/l /3 3/3 3/4 4/4 2/3 3/3 3/4 4/4 2/3 3/3 3/4 4/4 2/3 3/3 3/4 4/4 Diagnosis/APOE Genotype /3 3/3 3/4 4/4 2/3 3/3 3/4 4/4 2/3 3/3 3/4 4/4 2/3 3/3 3/4 4/4 Diagnosis/APOE Genotype Figure 3. Amyloid -proteins A 4 (A) and A 42 () levels (mean±sd) according to apolipoprotein E (APOE) genotype and diagnosis. Number of APOE 4 alleles was crossed with sex and diagnostic group, and age was covaried. No significant effects involving 4 were found. AD indicates Alzheimer disease; MCI, mild cognitive impairment; and PD, Parkinson disease. A FH AD No FH AD 1 12 Plasma Aβ4, pmol/l Diagnosis Diagnosis Figure 4. Amyloid -proteins A 4 (A) and A 42 () levels (mean±sd) according to family history of Alzheimer disease (FH AD). Presence or absence of family history of Alzheimer disease was crossed with sex and diagnostic group, and age was covaried. No effects involving family history of Alzheimer disease were significant except for complex higher-order interactions involving sex and diagnostic group. MCI indicates mild cognitive impairment; PD, Parkinson disease. A 42/A. No effects involving family history were significant except for complex higher-order interactions involving sex and diagnostic group (Figure 4). Whether or not participants were taking various medications was analyzed in relation to A 4, A 42, and A 42/A. In separate analyses, the medications were cholinesterase inhibitors, anti-inflammatory drugs, antioxidants, estrogen, and statins. Only data for women were analyzed in the case of estrogen. Medication use was crossed with diagnostic group and sex, and age was covaried. In each analysis, only diagnostic groups with sufficient numbers of participants taking the medication were included. No effects involving medications were found to be significant (Figure 5). COMMENT Since amyloid plaques are a fundamental feature of AD neuropathology, and A can be detected in CSF and plasma, A measures in biological fluids are compelling candidate biomarkers for AD diagnosis and progression. 22 The combination of low A 42 level and elevated tau protein in CSF has modest sensitivity and specificity for diagnosing AD. 6 Plasma A or A 42 is increased in familial AD with presenilin or APP mutations as well as in Down syndrome with APP triplication, 9,1,23 but, on the basis of our study and others, these plasma measures do not reliably differentiate sporadic AD from control cases. 9,1,13,14 We collected plasma samples from a cohort of 371 patients, and specifically studied patients with MCI and a neurodegenerative control group of nondemented patients with PD, in addition to AD and neurologically normal controls. This large and diverse sample allowed us to examine which genetic, demographic, and clinical factors were significantly associated with the variance in plasma A levels. The results of our study indicate that the primary influence on plasma A 4 and A 42 levels is age rather than diagnosis, with higher A 4 and A 42 levels in older patients regardless of diagnostic category. This effect of age is consistent with the findings of Younkin et al 24 and Mayeux et al. 11 After controlling for age, there was no significant difference in A levels among the diagnoses. Stud- (REPRINTED) ARCH NEUROL / VOL 6, JULY Downloaded From: on 7/3/ American Medical Association. All rights reserved.

6 A Plasma Aβ4, pmol/l With Medication Without Medication Statins Estrogen (Control Women) AchE-I (AD) Medication NSAIDs Antioxidants Statins Estrogen (Control Women) AchE-I (AD) Medication NSAIDs Antioxidants Figure 5. Amyloid -proteins A 4 (A) and A 42 () according to medication use in nondemented control subjects (for statins, estrogen [women only], nonsteroidal anti-inflammatory drugs [NSAIDS], and antioxidants) and patients with Alzheimer disease (AD) (for anticholinesterase inhibitors [AchE-I]). Medication use was crossed with diagnostic group and sex, and age was covaried. No effects involving medications were found to be significant when all diagnostic groups were analyzed, except for an occasional higher-order interaction involving sex and diagnostic group. ies using similar antibodies to our assay (either AN5 or NT77 capture antibodies and A27/C5 detector antibodies) and others (3D6 capture antibody and 21F12 anti- A 42 detector antibody) also found no significant differences between AD and control cases. 9,1,13,14 In contrast, ELISAs using 6E1 capture with R162/R164 or R165 detector antibodies have detected elevated plasma A measures in AD or incipient AD, with a large overlap with non-ad cases. 11,12 Our study did not detect elevated A measures in MCI cases, which could be considered preclinical AD; however, it is important to note that in the study by Mayeux et al, 11 elevated A levels were present before any cognitive impairment in those who subsequently became demented. In secondary analyses, we investigated other factors associated with AD risk and therapy, including education, sex, family history of dementia, family history of AD, APOE genotype, and use of classes of medications. When age was covaried, no significant effects were found for medication use, APOE genotype, or family history of dementia. Within the AD and MCI groups, plasma A level did not correlate with duration or severity of memory impairment. These results indicate that the variance in plasma A levels in late-onset AD is largely related to age, although we cannot rule out other genetic factors besides APOE, PS-1, and APP, since there is evidence that plasma A levels behave like heritable traits (independent of diagnosis or family history of AD). 25,26 Few published studies have correlated plasma A levels with medication use. Our cross-sectional results with statins are consistent with another cross-sectional study finding that plasma levels of A were not associated with statin use, 27 and with a study indicating no association of CSF A 42 levels with statin use 28 ; however, lovastatin reduced serum A levels in a dose-dependent manner during 3 months in a placebo-controlled study of hypercholesterolemic patients, 29 and simvastatin treatment for 26 weeks showed a trend toward reduced CSF A 4 levels. 3 We did not detect significant effects on plasma A by commonly used current medication classes for AD cholinesterase inhibitors and antioxidants (eg, vitamin E) as well as by putative preventive agents against the development of AD estrogen, nonsteroidal anti-inflammatory drugs, and statins. 31 While large class effects on plasma A were not found in this analysis, we cannot rule out individual medication effects. Specific medications within the nonsteroidal anti-inflammatory drug and statin classes may differ in effects on APP processing and AD risk. For instance, in a study of the nonsteroidal anti-inflammatory drugs, ibuprofen, sulindac sulfide, and indomethacin were more effective than naproxen, aspirin, and celecoxib in reducing A 42 production in cell culture. 32 Among the statins, a reduced prevalence of AD was associated with lovastatin and pravastatin but not simvastatin. 33 However, insufficient numbers of our sample population took any single medication to allow subclass analysis of this sort. Some classes of medications may affect AD risk without affecting APP metabolism and A levels, such as cholinesterase inhibitors and antioxidants. esides these clinical, demographic, and genetic factors, the physiologic processes that affect plasma A levels are unknown, in particular where A in plasma is synthesized and metabolized. Studies in APP transgenic mice suggest an equilibrium between A deposited in brain, soluble A in CSF, and A in plasma: in aging Tg2576 APP KN67-1ML mice, age-related A deposition in the brain is associated with a reduction in CSF and plasma A levels. 34 The A injected intraventricularly in rats is cleared into the blood, 35 intravenously administered A can enter mouse brain, 36 and peripheral administration of A antibodies in APP transgenic mice can bind A from the CSFbrain compartment. 37 Studies in humans have failed to demonstrate, however, correlation of CSF A levels and plasma A levels. 38 Alternatively, extracerebral sources such as platelets are a source of A in plasma. 39 The age-related increase of A species in plasma may be a peripheral reflection of increases in A production or reduction in A clearance in the brain leading to increased A deposition and AD with aging; changes in the central or peripheral activity of A synthetic enzymes (eg, -secretase or -secretase) or A catabolic enzymes (eg, insulin-degrading enzyme or neprilysin) with aging remain to be clarified. This study demonstrates that age is the principal correlate of plasma A levels, rather than diagnosis, medication use, or APOE genotype. Therefore, plasma A is not a reliably sensitive or specific biomarker of AD or MCI di- (REPRINTED) ARCH NEUROL / VOL 6, JULY Downloaded From: on 7/3/ American Medical Association. All rights reserved.

7 agnosis in cross-sectional study. Longitudinal analysis of plasma in the course of a double-blind placebo-controlled study of specific drugs could detect more sensitive effects of medications on plasma A measures in individual patients. Clinical follow-up of individuals in our study is also under way to determine whether these baseline levels of A 4, A 42, or A 42/A predict future cognitive decline, as suggested by the results of Mayeux et al 11 and the studies of familial AD with PS-1 and APP mutations Serial measurement of population-based samples could also determine whether the pattern of change in A levels in plasma is predictive of conversion to AD or progression of established AD. 4 Accepted for publication November 14, 22. Author contributions: Study concept and design (Drs Fukumoto, Hyman, Growdon, and Irizarry and Ms Tennis); acquisition of data (Drs Fukumoto, Hyman, and Irizarry and Ms Tennis); analysis and interpretation of data (Drs Fukumoto, Locascio, Hyman, Growdon, and Irizarry); drafting of the manuscript (Drs Fukumoto, Locascio, Hyman, and Irizarry); critical revision of the manuscript for important intellectual content (Drs Fukumoto, Locascio, Hyman, Growdon, and Irizarry and Ms Tennis); statistical expertise (Drs Locascio, Hyman, and Irizarry); obtained funding (Drs Hyman, Growdon, and Irizarry); administrative, technical, and material support (Drs Fukumoto, Growdon, and Irizarry and Ms Tennis); study supervision (Drs Fukumoto, Hyman, Growdon, and Irizarry and Ms Tennis). This study was supported by grants AG793 and AG5134 from the National Institutes of Health, ethesda, Md, and the Lawrence J. and Anne Cable Rubenstein Foundation. Dr Fukumoto s salary is supported by Takeda Chemical Industries, Osaka, Japan. We thank Marisa Dreisbach, Kerri Anne Giglio, onnie Cheung, Sarah McKenzie Hallen, Lue Davis, and Ellen Valentine for phlebotomy collection, sample processing, and administrative support. Corresponding author and reprints: Michael C. Irizarry, MD, Alzheimer Disease Research Unit, Massachusetts General Hospital East, , 11416th St, Charlestown, MA 2129 ( mirizarry@partners.org). REFERENCES 1. Walter J, Kaether C, Steiner H, Haass C. The cell biology of Alzheimer s disease: uncovering the secrets of secretases. Curr Opin Neurobiol. 21;11: Gravina SA, Ho L, Eckman C, et al. Amyloid beta protein (A ) in Alzheimer s disease brain. J iol Chem. 1995;27: Iwatsubo T, Mann DM, Odaka A, Suzuki N, Ihara Y. Amyloid beta protein (A ) deposition. Ann Neurol. 1995;37: Tamaoka A, Sawamura N, Fukushima T, et al. Amyloid beta protein 42(43) in cerebrospinal fluid of patients with Alzheimer s disease. 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