ORIGINAL CONTRIBUTION

ORIGINAL CONTRIBUTION Plasma Levels of -Amyloid (1-4), -Amyloid (1-42), and Total -Amyloid Remain Unaffected in Adult Patients With Hypercholesterolemia After Treatment With Statins Kina Höglund, MSc; Olov Wiklund, MD, PhD; Hugo Vanderstichele, PhD; Oliver Eikenberg, PhD; Eugeen Vanmechelen, PhD; Kaj Blennow, MD, PhD Background: Epidemiological studies suggest that statins reduce the risk of developing Alzheimer disease. Cell and animal experiments have revealed a connection between cholesterol metabolism and the processing of amyloid precursor protein. To our knowledge, the mechanism for statins in risk reduction of Alzheimer disease is unknown. Objective: To test the effect of statin treatment on -amyloid (A ) metabolism in humans. Design: A prospective, randomized, dose-finding 36- week treatment trial with statins. Plasma samples were taken at baseline (week ) and at weeks 6, 12, and 36. Setting: Outpatient clinical study at a university hospital. Patients: Thirty-nine patients who met the criteria for hypercholesterolemia. Interventions: Patients were randomized to oral treatment with either simvastatin or atorvastatin calcium according to the following regimen: simvastatin, 4 mg/d, or atorvastatin, 2 mg/d, for 6 weeks; followed by simvastatin, 8 mg/d, or atorvastatin, 4 mg/d, for 6 weeks; and finally, simvastatin, 8 mg/d, or atorvastatin, 8 mg/d, for 24 weeks. Main Outcome Measures: Plasma levels of A (1-4) and A (1-42) were measured using 2 enzyme-linked immunosorbent assays, and total A was quantified by Western blotting. Results: Treatment with both statins reduced total plasma cholesterol levels by 56% (P=.). The plasma levels of A (1-4), A (1-42), and total A were stable in individual patients during the treatment period. No significant change in the level of A (1-4), A (1-42), or total A was found. Conclusion: This study questions the effect of statins on the processing of amyloid precursor protein in humans. Arch Neurol. 24;61:333-337 From the Institute of Clinical Neuroscience (Ms Höglund and Dr Blennow) and the Wallenberg Laboratory (Dr Wiklund), The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden; INNOGENETICS NV, Zwijnaarde, Belgium (Drs Vanderstichele and Vanmechelen); and ABETA GmbH, Heidelberg, Germany (Dr Eikenberg). PREVIOUS STUDIES SUPPORT THE theory that the formation and aggregation of -amyloid (A ), the major constituent of senile plaques in brain and blood vessels, is critical for the initiation of Alzheimer disease (AD). The A peptide is produced in 2 major forms, A 4 and A 42, during the processing of the integral plasma membrane protein, amyloid precursor protein (APP). Secreted soluble A is a product of normal cell metabolism, 1 and is found in various body fluids, including cerebrospinal fluid (CSF) and plasma. 2,3 Several studies have suggested an association between AD, A, and cholesterol metabolism. Clinical studies have revealed that patients with AD have elevated levels of cholesterol in the plasma 4 and that the plasma level of apolipoprotein E, 5 the main lipid transporter protein in the brain, 6 is related to AD. Longitudinal studies 7,8 have suggested that an elevated midlife level of cholesterol is a risk factor for AD, and recent retrospective clinical studies 9-11 have indicated that patients treated with statins have a reduced prevalence of AD. Statins reduce de novo cholesterol synthesis by inhibiting the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase, which leads to a decrease in low-density lipoprotein cholesterol level in human plasma. In vitro studies 12,13 have shown that cholesterol modulates the processing of APP. The hypothesis that lowering cholesterol levels favors the nonamyloidogenic pathway has further been supported by animal studies. 12,14 However, there are also 2 prospective studies 15,16 that do not support the connection between statin treatment and reduced prevalence of AD. 333

Levels of Total Cholesterol, A (1-4), and A (1-42) at Baseline and After 6, 12, and 36 Weeks of Treatment With Simvastatin or Atorvastatin Calcium* Treatment Total Cholesterol, mg/dl It is believed that cholesterol in the brain mainly results from de novo synthesis of cholesterol in the cells. 17 The efflux of cholesterol is in the form of 24Shydroxycholesterol. 18 Two recent studies 18,19 have demonstrated that patients with early-onset AD and vascular dementia have elevated concentrations of 24Shydroxycholesterol in their plasma, indicating an overproduction of cholesterol in the brain. Support for this hypothesis has been given in 2 studies 2,21 showing that cholesterol metabolism in the human brain was affected by treatment with statins and effects were seen after 6 weeks of treatment. To test the effect of statins on A metabolism in humans, we examined the plasma levels of A (1-4), A (1-42), and total A in longitudinal samples from patients with hypercholesterolemia who were treated with statins. We observed that pharmacologically attainable concentrations of atorvastatin calcium or simvastatin did not affect the plasma levels of A (1-4),A (1-42),or total A. METHODS A (1-4), pg/ml A (1-42), pg/ml Atorvastatin (n = 2) Baseline 263 (244-35) 141 (117-163) 23 (21-31) 6 wk, 2 mg/d 17 (155-182) 138 (124-169) 25 (21-28) 12 wk, 4 mg/d 151 (143-17) 147 (124-178) 25 (22-28) 36 wk, 8 mg/d 143 (135-155) 143 (121-175) 25 (23-28) Simvastatin (n = 19) Baseline 325 (244-35) 148 (137-161) 35 (21-46) 6 wk, 4 mg/d 21 (155-182) 153 (144-184) 34 (23-48) 12 wk, 8 mg/d 186 (174-25) 163 (146-179) 36 (27-48) 36 wk, 8 mg/d 186 (135-25) 157 (135-178) 3 (24-44) Abbreviation: A, -amyloid. SI conversion factor: To convert total cholesterol to millimoles per liter, multiply by.259. *Data are given as median (interquartile range). The interquartile range is from the 25th percentile to the 75th percentile. This study is an extension of a substudy of a larger multicenter investigation. 22 In the present study, 39 subjects (27 men and 12 women) were included, and all had completed the entire treatment; 19 patients were treated with simvastatin, and 2 with atorvastatin. The mean age of the 39 subjects was 55 years (range, 28-67 years). Eight patients were excluded from the multicenter investigation because no samples were available. Inclusion criteria were the same as for the main study, 23 published separately. To qualify, all patients were required to have a low-density lipoprotein cholesterol level of 162.2 mg/dl or more ( 4.19 mmol/l) and a triglycerides level of 351.5 mg/dl or less ( 3.97 mmol/l). At baseline, the mean (SD) plasma level of total cholesterol was 31.5 (54.2) mg/dl (7.8 [1.4] mmol/l). The medical history of all patients was studied, and all patients underwent a thorough clinical examination to exclude cognitive impairment. Following the start of a 4-week run-in period, the patients were randomized to 1 of 2 treatments: simvastatin, 4 mg/d, or atorvastatin, 2 mg/d, for 6 weeks; followed by simvastatin, 8 mg/d, or atorvastatin, 4 mg/d, for 6 weeks; and finally, simvastatin, 8 mg/d, or atorvastatin, 8 mg/d, for 24 weeks. Other ongoing lipid-lowering treatment was discontinued at least 8 weeks before randomization. The EDTA-plasma samples were collected at baseline (week ) and at the end of each treatment period (weeks 6, 12, and 36), and then frozen. All samples analyzed by an enzyme-linked immunosorbent assay (ELISA) were analyzed on the same day. The study was approved by the local ethics committees, and patients were included after giving informed consent. The study was performed according to the Declaration of Helsinki. BIOCHEMICAL ANALYSIS The plasma A (1-42) concentration was determined using the highsensitivity format of one assay (INNOTEST -AMYLOID (1-42) ; INNOGENETICS NV) with a detection limit of 7.8 pg/ml. The assay and its characteristics have previously been described in detail. 24 The method is based on the antibody 21F12, specific for the carboxyl terminus of A 42 ; and the biotinylated detection antibody 3D6, specific for the amino terminus, including amino acid D1 of A (1-42). The concentration of A 4 plasma levels was determined by a research prototype version of another assay (INNOTEST -AMYLOID (1-4) ; INNOGENETICS NV) with a detection limit of 7.8 pg/ml. The method is based on the antibody R293, specific to the carboxyl terminus of A (1-4) ; and the biotinylated detection antibody 3D6. The total levels of A were measured using an advanced version of the sensitive Western blot assay according to an internally developed protocol (ABETA GmbH). The method is based on the monoclonal antibody W-2 (ABETA GmbH), which recognizes the 4-1 residue of A. Quantification was performed using computer software (Quantity One; BioRad Laboratories, Hercules, Calif). The synthetic peptide from the assay used (INNOTEST -AMYLOID (1-42) ) was used as a standard. A control sample was loaded onto all gels, and was quantified and used subsequently as an internal quality control. Wiklund et al 22 provide further information on the characterization of lipids and lipoproteins. STATISTICAL ANALYSIS All statistical procedures were performed using a commercially available software program (SPSS for Windows; SPSS Inc, Chicago, Ill). The Pearson product moment correlation coefficient was used for correlations. Comparison between groups was performed using the nonparametric Kruskal-Wallis test. We used the Wilcoxon matched-pair signed rank test to compare differences at baseline and after 6, 12, or 36 weeks of treatment. P.5 was considered significant. RESULTS LIPIDS Treatment with either atorvastatin or simvastatin for 36 weeks produced, as expected, significant effects on total cholesterol levels (even after just 6 weeks of treatment). For the combined groups, total cholesterol levels were reduced by 56% (P=.) after 36 weeks of treatment. In the group treated with atorvastatin, the reduction was 53% and in the simvastatin group, 57% (P=. for both). Changes in total cholesterol levels are given in the Table. The major adverse effect was gastrointestinal symptoms; however, no patient in either treatment group experienced myopathy. 334

A (1-4) AND A (1-42) Neither simvastatin nor atorvastatin administration significantly affected the plasma levels of A (1-4) (P=.99) or A (1-42) (P=.1). At baseline, the mean (SD) plasma level of A (1-4) for the combined groups (N=39) was 148 (44) pg/ml; after 36 weeks of treatment, it was 152±36 pg/ml. Mean (SD) levels of A (1-42) for the combined groups (N=39) were as follows: 33 (23) pg/ml at baseline and 32 (22) pg/ml after 36 weeks of treatment. Values of A (1-4) and A (1-42) in plasma at baseline and after 6, 12, and 36 weeks of treatment are given in the Table. Figure 1 depicts the levels of A (1-4) and A (1-42) for the combined groups at baseline and after 6, 12, and 36 weeks of treatment. There were broad variations between individuals, but the levels were stable between samples taken at baseline and after 6, 12, or 36 weeks of treatment in individual subjects. Individual plasma levels of cholesterol and A (1-42) before and during treatment are illustrated in Figure 2. The levels of A (1-42) and A (1-4) corresponded, ie, patients with higher levels of A (1-42) also had higher levels of A (1-4), as recognized by the correlation coefficients (r week =.42 [P.1], r week 6 =.4 [P.1], r week 12 =.45 [P.4], and r week 36 =.35 [P.1]). TOTAL A Because of the limited sample volume (due to an earlier analysis), selected samples only (n=14) were analyzed by Western blotting. Neither simvastatin nor atorvastatin treatment significantly changed the levels of total A (P=.59). For the combined treatment groups (n=14), the mean±sd baseline level of A was 228 (15) pg/ml; after 36 weeks of treatment, it was 219 (172) pg/ml. Broad variations between individuals were seen, but the levels were relatively stable between samples taken at baseline and after 6, 12, or 36 weeks of treatment in individual subjects. COMMENT Aβ Level, pg/ml 25 2 15 1 5 6 12 36 Aβ(1-42) Aβ (1-4) Figure 1. Histogram illustrating the levels of 2 -amyloids (A s), A (1-4) and A (1-42), in plasma, measured by an enzyme-linked immunosorbent assay, at baseline and after 6, 12, and 36 weeks of treatment with atorvastatin calcium or simvastatin. Data are given as mean (SD) levels for the combined groups (N=39). At several time points after treatment with 2 different statins, simvastatin or atorvastatin, no significant change in the levels of either A (1-4) or A (1-42) was observed compared with baseline levels. The levels of A (1-4) and A (1-42) measured at baseline and after 6, 12, or 36 weeks of treatment were remarkably stable within each patient, and there was no significant difference between the 2 treatment groups. There was no control group in this study. However, the CSF levels of A 42 have been shown to be stable in untreated patients with AD. 25 Although ELISA is sensitive, one potential drawback when measuring A is that the measurement can be affected if other proteins bind to A 26-28 and mask the epitope of antibodies used in the assay. Therefore, selected plasma samples were also quantified using an advanced sensitive Western blot; where possible, protein complexes were separated by subjecting the samples to sodium dodecyl sulfate and heat treatment. The results corresponded well with the ELISA measurements, and no significant change in plasma A level was seen during treatment with statins. Thus, it is unlikely that the lack of effect on plasma A (1-4) and A (1-42) measured by the present ELISA (INNOGENETICS NV) is due to methodological shortcomings. To our knowledge, this is the first longitudinal treatment study of patients with hypercholesterolemia in which the plasma levels of A have been studied. In a case-control study, 29 the CSF level of A in patients with hypercholesterolemia, treated with statins or untreated, was compared with that of healthy controls. In agreement with our findings, no effect on A metabolism was seen. It was demonstrated earlier 3 that levels of A (1-42) in the CSF and plasma were unchanged in patients with AD treated with simvastatin for 12 weeks. However, the levels of sapp ( -cleaved soluble APP) and sapp ( -cleaved soluble APP) were significantly reduced, which could indicate that altered APP processing is not reflected in plasma or CSF A levels. In a placebo-controlled double-blind study, 31 patients with AD were treated with simvastatin for 26 weeks. In the total AD group, simvastatin did not significantly alter CSF levels of A (1-4) or A (1-42), in agreement with our results. However, a post hoc analysis revealed that the levels of A (1-4) were slightly ( 5.7%±6.5%), but significantly (n=8; P.5), reduced in patients with mild AD. In the present study, the plasma level of A was measured. Buxbaum and colleagues 32 recently published a study on serum levels of A in patients with hypercholesterolemia who were treated for 3 months with controlreleased lovastatin. They observed a concentrationdependent decrease in A using a different ELISA, based on different antibodies: 4G8, specific to amino acids 17 to 24 of the A peptide; and 6E1, specific to amino acids5to1ofthea peptide. The serum level of A has 335

A B 387 16 348 14 Total Cholesterol Level, mg/dl 39 271 232 193 155 116 77 Aβ (1-42) Level, pg/ml 12 1 8 6 4 39 2 6 12 36 6 12 36 C D 541 16 464 14 Total Cholesterol Level, mg/dl 387 39 232 155 Aβ (1-42) Level, pg/ml 12 1 8 6 4 77 2 6 12 36 6 12 36 Figure 2. The reduction of total cholesterol levels in plasma is shown after treatment with atorvastatin calcium (n=2) (A) or simvastatin (n=19) (C). The reduction can already be seen after 6 weeks of treatment. To convert total cholesterol to millimoles per liter, multiply by.259. The stability of -amyloid (1-42) (A (1-42) ) levels in the plasma is also shown, measured by an enzyme-linked immunosorbent assay, after treatment with atorvastatin (n=2) (B) or simvastatin (n=19) (D). been considered to be under the detection limit using our assay (INNOTEST -AMYLOID (1-42) ). 24 In agreement with the results in the present study, there are 2 prospective studies 15,16 that do not support the connection between statin treatment and reduced prevalence of AD. In these 2 studies, no effect on developing dementia was seen after statin treatment. However, in both of these studies, patients with cognitive impairment or dementia were excluded, by excluding either patients with a Mini-Mental State Examination score below 24 15 or patients with dementia. 16 The dosages of statins given in the clinical studies mentioned were between 2 and 8 mg/d. However, in animal studies 12 and cell culture experiments, 12,13,33 the dosage of statins exceeds these dosages many times over. The differences in dosage between animal/cell culture studies and human studies may explain the lack of effect of pharmacological dosages of statins on A in the present study. Although cell culture and animal studies suggest that statins reduce A production, the present and other clinical studies show no effect or only a minor effect on plasma and CSF levels of A after statin treatment. Because statins also have other nonlipid effects, such as antiinflammatory or neuroprotective effects, 34 the potential protective effect of statins on AD seen in epidemiological studies may not be through the inhibition of cholesterol synthesis. The origin of A in plasma is still unclear. No correlation between CSF and plasma levels of A has been seen, 23,35 nor is there a correlation between CSF, A 42, and blood-brain barrier deficits, which suggests that plasma A 42 is not derived from the brain but is secreted 336

peripherally. Despite a marked reduction in cholesterol levels, we did not observe any change in plasma levels of A. It is possible that statins may affect A metabolism in the brain, but not peripherally. However, several studies 28-3 have found no change in CSF levels or only minor effects in mild AD cases after statin treatment. In conclusion, further studies are needed to examine the mechanism by which statins may reduce the risk of developing AD. Accepted for publication October 28, 23. Author contributions: Study concept and design (Ms Höglund and Drs Wiklund and Blennow); acquisition of data (Ms Höglund and Drs Wiklund and Eikenberg); analysis and interpretation of data (Ms Höglund and Drs Vanderstichele, Eikenberg, Vanmechelen, and Blennow); drafting of the manuscript (Ms Höglund and Drs Vanderstichele and Blennow); critical revision of the manuscript for important intellectual content (Ms Höglund and Drs Wiklund, Eikenberg, Vanmechelen, and Blennow); statistical expertise (Dr Blennow); obtained funding (Ms Höglund and Drs Wiklund, Vanmechelen, and Blennow); administrative, technical, and material support (Drs Vanderstichele, Eikenberg, Vanmechelen, and Blennow); study supervision (Drs Wiklund and Blennow). This study was supported by projects 1156, 1213, and 142 of the Swedish Medical Research Council, Stockholm, Sweden; Alzheimerfonden, Lund, Sweden; and Stiftelsen för Gamla Tjänarinnor, Stockholm. We thank Pankaj Mehta, MD, PhD, for the polyclonal antibodies against Aβ 4 ; Tobias Hartmann, PhD, for introducing us to the blotting method for Aβ, and for sharing his scientific knowledge; and Inge Tomic, Mareike Grees, and Monique Beumer for their skillful technical assistance. Corresponding author: Kina Höglund, MSc, Experimental Neuroscience Section, Institute of Clinical Neuroscience, Neurolab, SU/Mölndal, S-4318 Mölndal, Sweden (e-mail: kina.hoglund@neuro.gu.se). REFERENCES 1. Seubert P, Vigo-Pelfrey C, Esch F, et al. Isolation and quantification of soluble Alzheimer s -peptide from biological fluids. Nature. 1992;359:325-327. 2. Galasko D, Chang L, Motter R, et al. High cerebrospinal fluid tau and low amyloid 42 levels in the clinical diagnosis of Alzheimer disease and relation to apolipoprotein E genotype. Arch Neurol. 1998;55:937-945. 3. Mayeux R, Tang MX, Jacobs DM, et al. Plasma amyloid -peptide 1-42 and incipient Alzheimer s disease. Ann Neurol. 1999;46:412-416. 4. 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