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Amyloid β protein 1–40 and 1–42 levels in matched cerebrospinal fluid and plasma from patients with Alzheimer disease
Neuroscience Letters 304 (2001) 102±106
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Amyloid b protein 1±40 and 1±42 levels in matched cerebrospinal ¯uid and plasma from patients with Alzheimer disease P.D. Mehta a,*, T. Pirttila b, B.A. Patrick a, M. Barshatzky a, S.P. Mehta a a
Department of Immunology, Institute for Basic Research in Developmental Disabilities, Forest Hill Road, Staten Island, New York 10314-6399, USA b Department of Neurology, Kuopio University Hospital, Kuopio, Finland Received 11 January 2001; received in revised form 20 March 2001; accepted 20 March 2001
Abstract We quantitated amyloid b proteins 1±40 (Ab40) and 1±42 (Ab42), and a1- antichymotrypsin (ACT) in matched cerebrospinal ¯uid (CSF) and plasma of 50 patients with probable Alzheimer disease, and analyzed the relationships with age, sex, Mini-Mental State Examination (MMSE), and apolipoprotein E phenotype. There was no relation between CSF Ab40 and Ab42 levels with those of plasma. CSF and plasma Ab40 and Ab42 levels showed no association with age, sex, and MMSE score. There was a signi®cant correlation between CSF ACT and plasma ACT levels. The data suggest that plasma ACT crosses the blood±brain barrier. However, a lack of correlation between CSF Ab40 and Ab42 levels with those of plasma suggests that Ab in CSF and plasma originates from different sources. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer disease; Amyloid b protein; a1-antichymotrypsin; Apolipoprotein E; Enzyme linked immunosorbent assay
The major component of neuritic plaques is the amyloid b protein (Ab), a small 39±42 amino acid residue protein derived through proteolytic processing of a larger membrane-bound glycoprotein, the amyloid b-precursor protein (AbPP) [16]. Secreted, soluble Ab is a product of normal cell metabolism and found in various body ¯uids, including plasma and cerebrospinal ¯uid (CSF) [15]. Ab ending at residue 42 (Ab42) is mainly found in senile plaques; whereas Ab ending at residue 40 (Ab40) is prominent in vascular amyloid deposits [6]. Investigators showed that deposition of Ab42 in brain might be the initial step in amyloid formation [20]. a1-antichymotrypsin (ACT), a serpin serine proteinase inhibitor, is a component of senile plaques, and is characteristic of acute phase in¯ammation [1]. Although the physiological role of ACT is not well established, investigators have reported increased concentrations of ACT in sera from patients with AD [9]. Apolipoprotein E (Apo E) is a 34-kd polymorphic protein that is involved in the transportation and redistribution of lipids among various tissues. The possession of two variants
* Corresponding author. Tel.: 11-718-494-5159; fax: 11-718982-6345. E-mail address: [email protected] (P.D. Mehta).
of Apo E 14 allele is a signi®cant risk factor for the development of sporadic and familial late-onset AD [14]. Investigators quantitated Ab40 and Ab42 in CSF or plasma in sporadic AD patients and reported that the levels were signi®cantly higher in CSF than plasma [4,12,17,18]. However, none examined the levels in matched CSF and plasma. Thus it is not known whether higher Ab levels found in CSF re¯ect intrathecal synthesis or damage in the blood±brain barrier. The aim of this study is to quantitate Ab40 and Ab42 levels in matched CSF and plasma from 50 AD patients and correlate the levels with age, sex, MiniMental State Examination (MMSE) score, and Apo E phenotype. In addition to Ab, we also quantitated ACT levels in the same samples, since our previous studies suggested that CSF ACT is mainly derived from blood [13]. The study included 50 patients with sporadic AD from the Department of Neurology, Kuopio University Medical Center, Kuopio, Finland. The diagnosis of probable AD was made according to the NINCDS-ADRDA criteria [11]. AD patients ranged in age from 57 to 83 years (mean 73 years). There were 16 men (11 with Apo E 14 allele and ®ve without) and 34 women (23 with Apo E 14 allele and 11 without). The MMSE scores ranged from 10 to 29 (median 21). Blood was collected in tubes containing K3-EDTA (B-D Vacutainer Systems, Franklin Lakes, NJ).
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 01) 0 17 54- 2
P.D. Mehta et al. / Neuroscience Letters 304 (2001) 102±106
103
Table 1 CSF and Plasma Ab40, Ab42 and ACT levels a Specimen (n)
Ab40 Median (Range)
Ab42, pg/ml Median (Range)
ACT, mg/ml Median (Range)
CSF (50) With Apo E 14 (34) Without Apo E 14 (16) Plasma (50) With Apo E 14 (34) Without Apo E 14 (16)
9.7 (1.6±17.1), ng/ml 9.0 (1.6±15.8) 10.2 (4.5±17.1) 143 (45±288), pg/ml 136 (45±288) 143 (68±201)
623 (287±1949) 495 (287±1347)* 862 (418±1949) 8.6 (5.0±23) 9.3 (5.0±23) 6.9 (5.0±16)
2.5 (1.0±5.3) 2.5 (1.2±5.3) 2.5 (1.0±4.0) 312 (203±752) 409 (209±541) 323 (203±752)
a
*P , 0:0002, analysis of variance with Bonferroni correction.
Plasma were separated by centrifuging blood at 3000 rpm at 48C for 20 min, aliquoted and frozen at 2808C. CSF were also aliquoted, and stored frozen at 2808C. All CSF were tested for cell counts, glucose and total proteins. Those contaminated with blood were excluded. Apo E phenotypes of AD plasma were determined using isoelectric focusing in agarose gel containing 3M urea followed by immunoblotting with speci®c goat antihuman Apo E antiserum [7]. Ab32±40 and Ab33±42 peptides synthesized commercially (Ana Spec, San Jose, CA) were conjugated to keyhole limpet hemocyanin in PBS with 0.5% glutaraldehyde and immunized in rabbits. The speci®city of rabbit antisera R209 produced against Ab40, and R226 raised against Ab42 was examined in a sandwich enzyme linked immu-
nosorbent assay (ELISA). There was a strong response of R209 with 1 ng/ml of Ab40 but no detectable response with 10 ng/ml of Ab42. Similarly R226 was found speci®c to Ab42 but showed no reactivity to Ab40. Western blot also showed that R209 was speci®c to Ab40, and R226 was speci®c to Ab42 (data not shown). Ab levels were measured using monoclonal antibody 6E10 (speci®c to an epitope present on 1±16 amino acid residues of Ab) and R209 and R226 in a double antibody sandwich ELISA [12]. The detection limit was 10 pg/ml for Ab40 and 5 pg/ml for Ab42. The percent coef®cients of variation ranged from 8 to 14% (intra-assay) and 10 to 18% (inter-assay). ACT levels were measured in a double antibody sandwich ELISA [13]. The detection limit of ACT was 12.5 ng/ml.
Fig. 1. Box plots of Ab 40 and Ab42 levels in CSF and plasma of AD patients with and without Apo E 14 allele.
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P.D. Mehta et al. / Neuroscience Letters 304 (2001) 102±106
Fig. 2. Relationship of Ab40 and Ab42 levels in CSF (A) with Apo E 14 allele and (B) without Apo E 14 allele and plasma (C) with Apo E 14 allele and (D) without Apo E 14 allele.
The relationship between variables was determined by using Pearson product-moment correlation with Bonferroni correction. The level of P , 0:01 was considered signi®cant. Relationship between CSF and plasma Ab40, Ab42 and ACT levels. There was no relation between CSF Ab40 and plasma Ab40 levels (r 20:13, P , 0:47), and CSF Ab42 and plasma Ab42 levels (r 0:01, P , 0:93). However, there was a signi®cant relation between CSF ACT and plasma ACT levels (r 0:53, P , 0:001). Relationship of CSF and plasma Ab40, Ab42 and ACT levels with Apo E 14 allele. CSF Ab40 levels were similar in AD patients with and without Apo E 14 allele. However, CSF Ab42 levels were signi®cantly lower in AD patients with Apo E 14 allele than those without (P , 0:0002) (Table 1) (Fig. 1). Plasma Ab40 and Ab42 levels were similar in AD patients with and without Apo E 14 allele. There was a signi®cant association between CSF Ab40 and Ab42 levels in AD with Apo E 14 allele (r 0:56, P , 0:0005), but not in those without (r 0:42, P , 0:1) (Fig. 2). There was a signi®cant association between plasma Ab40 and Ab42 levels in AD with Apo E 14 allele (r 0:69, P , 0:0001) and those without (r 0:67, P , 0:005) (Fig. 2). Levels of CSF and plasma ACT were similar in AD patients with and without Apo E 14 allele. There was a signi®cant association between CSF ACT and
plasma ACT levels in AD with Apo E 14 allele (r 0:5, P , 0:003) and those without (r 0:69, P , 0:003) (data not shown). Relationship with age, sex and MMSE score. CSF and plasma Ab40 and Ab42 levels showed no association with age, sex, and MMSE score. However, there was a relation between CSF Ab40 and CSF ACT levels but with borderline signi®cance (r 0:29, P , 0:039). There was a significant association between age and levels of plasma ACT (r 0:37, P , 0:009) and CSF ACT (r 0:37, P , 0:008). However, there was no relation between sex and MMSE score with CSF or plasma ACT levels. Ab levels in CSF re¯ect the net result of the increased production and diminished clearance of Ab in AD brain. Studies of an animal model of AD suggested that Ab in CSF is produced by the neuronal source of APP rather than local production or Ab uptake from the blood [2]. It is possible that some CSF Ab originates from blood, since it has been reported that Ab injected intravenously enters the brain through the blood±brain barrier [8]. However, the lack of correlation between matched CSF Ab40 and Ab42 levels with those of plasma suggests that a large portion of Ab in CSF may have originated in the brain rather than plasma. Investigators have reported that plasma Ab42 levels were elevated in patients with the early-onset familial AD-linked
P.D. Mehta et al. / Neuroscience Letters 304 (2001) 102±106
APP, PS1, and PS2 mutations [15]. The data support the idea that this effect is related to the pathogenesis of AD. However, in most sporadic AD cases, plasma Ab42 levels were similar to those of elderly non-demented controls. Thus, in sporadic AD, cerebral deposition of Ab42 is caused by other factors, such as synthesis or secretion of Ab42, or alterations of Ab-binding proteins [15]. The origin of Ab in plasma is unknown. Previous studies have shown that Ab injected into ventricular CSF in rats cleared rapidly into blood by subependymal capillaries and the choroid plexus [5]. Histological studies have shown that Ab accumulated in periarterial interstitial ¯uid drainage pathways of the brain [19]. The results suggested that some Ab in brain parenchyma can be eliminated through the perivascular spaces and may in¯uence Ab levels in blood. However, studies have showed that anti-Ab immunoreactivity in blood was associated with platelets. The data suggested that platelets are one of the major sources of Ab in human plasma [3]. ACT forms a non-covalent complex with Ab, thus inducing multiple effects that favor the propagation of in¯ammation and synthesis of Ab42 ®brils. However, the strong correlation between CSF ACT and plasma ACT suggests that plasma ACT crosses the blood±brain barrier and that CSF ACT originates mainly from blood. The ®ndings are consistent with our previous studies [13]. Although in our study most AD patients had mild dementia, levels of Ab40 and Ab42 in plasma and CSF did not correlate with MMSE score. In contrast, other investigators have shown a weak correlation between these levels and dementia severity [4]. The reason for the discrepancy between the studies is not clear. Further longitudinal studies using a large number of samples will be necessary to determine conclusively whether there is a relationship between plasma Ab levels and progression of AD. In summary, the lack of correlation between Ab40 and Ab42 levels in CSF and plasma in AD suggests that the higher levels found in CSF are not re¯ected in plasma and that the source of the synthesis of Ab in these two compartments is different. Our results suggest that plasma Ab levels do not re¯ect the pathological changes seen in AD brain. The question remains why do about 10 to 20% of sporadic AD patients show increased Ab42 concentrations in plasma [12,15,17]? In addition, investigators have also reported higher plasma Ab42 levels in the ®rst-degree relatives of patients with AD, and several cognitively normal individuals in whom AD later developed [10]. The signi®cance of the relationship between Ab levels in plasma and higher concentrations of Ab in CSF and brain is unknown. The work in part was supported by a grant from P®zer, Inc. and in part by the funds provided by NY State through its Of®ce of Mental Retardation and Developmental Disabilities. We thank Dr Eugene A. Sersen for statistical analyses.
105
[1] Abraham, C.R., Selkoe, D.J. and Potter, H., Immunochemical identi®cation of serine protease inhibitor alpha-1-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease, Cell, 52 (1988) 487±501. [2] Calhoun, M., Burgermeister, P., Phinney, A., Stadler, M., Tolnay, M., Wiederhold, K.H., Abramowski, D., StruchlerPierrat, C., Sommer, B., Staufenbiel, M. and Jucker, M., Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid, PNAS, 96 (1999) 14088±14093. [3] Chen, M., Inestrosa, N.C.K., Ross, G.S. and Fernandez, H.L., Platelets are the primary source of amyloid b-peptide in human blood, Biochem. Biophys, Res. Comm., 213 (1995) 96±103. [4] Galasko, D., Chang, L., Motter, R., Clark, C.M., Kaye, J., Knopman, D., Thomas, R., Khodolenko, D., Schenk, D., Lieberburg, I., Miller, B., Green, R., Basherad, R., Kertilles, L., Boss, M.A. and Seubert, P., High cerebrospinal ¯uid tau and low amyloid beta 42 levels in the clinical diagnosis of Alzheimer disease and relation to apolipoprotein E genotype, Arch. Neurol., 55 (1998) 937±945. [5] Ghersi-Egea, J.F., Gorevic, P.D., Ghiso-Frangione, B., Pattlak, C.S. and Fenstermacher, J.D., Fate of cerebrospinal ¯uid-borne amyloid beta-peptide: rapid clearance into blood and appreciable accumulation by cerebral arteries, J. Neurochem., 67 (1996) 880±883. [6] Gravina, S.A., Libin, H., Eckman, C.B., Long, K.E., Otvos Jr, L., Younkin, L.H., Suzuki, N. and Youkin, S.G., Amyloid b protein (Ab) in Alzheimer's disease brain: biochemical and immunocytochemical analysis with antibodies speci®c for forms of Ab40 and Ab42(43), J. Biol. Chem., 270 (1995) 7013±7016. [7] Kataoka, S., Paidi, M. and Howard, B.V., Simpli®ed isoelectric focusing/immunoblotting determination of apolipoprotein E phenotype, Clin. Chem., 40 (1994) 11±13. [8] Maness, L.M., Banks, W.A., Podlinsky, M.B., Selkoe, D.J. and Kastin, A.J., Passage of human amyloid beta-protein 1±40 across the murine blood±brain barrier, Life Sci., 55 (1994) 1643±1650. [9] Matsubara, E., Hirai, S., Amari, M., Shoji, M., Yamaguchi, H., Okamoto, K., Ishiguro, K., Harigaya, Y. and Wakabayashi, K., a1-Antichymotrypsin as a possible biochemical marker for Alzheimer type dementia, Ann. Neurol., 28 (1990) 561±567. [10] Mayeux, R., Tang, M.X., Jacobs, S.M., Manly, J., Bell, K., Merchant, C., Small, S.A., Stern, Y., Wisniewski, H.M. and Mehta, P.D., Plasma amyloid beta-peptide 1±42 and incipient Alzheimer's disease, Ann. Neurol., 46 (1999) 412±416. [11] McKhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D. and Stadlan, E.M., Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease, Neurology, 34 (1984) 939±944. [12] Mehta, P.D., Pirttila, T., Mehta, S.P., Sersen, E.A., Aisen, P. and Wisniewski, H.M., Plasma and cerebrospinal ¯uid levels of amyloid b proteins 1±40 and 1±42 in Alzheimer disease, Arch. Neurol., 57 (2000) 100±105. [13] Pirttila, T., Mehta, P.D., Frey, H. and Wisniewski, H.M., a1Antichymotrypsin and IL-1b are not increased in CSF or serum in Alzheimer's disease, Neurobiol. of Aging., 15 (1994) 313±317. [14] Saunders, A.M., Strittmatter, W.J., Schmechel, D., St. George-Hyslop, P.H., Pericak-Vance, M.A., Joo, S.H., Rosi, B.L., Gusella, J.F., Crapper-MacLachlan, D.R., Alberts, M.J., Hulette, C., Crain, B., Goldgaber, D. and Roses, A.D., Asso-
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ciation of apolipoprotein E allele 14 with late-onset familial and sporadic Alzheimer's disease, Neurology, 43 (1993) 1467±1472. [15] Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T.D., Hardy, J., Hutton, M., Kukull, W., Larson, E., Levy-Lahad, E., Vittanen, M., Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco, W., Lannfelt, L., Selkoe, D. and Younkin, S., Secreted amyloid b-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease, Nature Med., 2 (1996) 864±870. [16] Selkoe, D., Normal and abnormal biology of the b-amyloid, Ann. Rev. Neurosci., 17 (1994) 489±517. [17] Tamaoka, A., Fukushima, T., Sawamura, N., Ishikawa, K.,
Oguni, E., Komatsuzaki, Y. and Shoji, S., Amyloid b protein in plasma from patients with sporadic Alzheimer's disease, J. Neurol. Sci., 151 (1996) 65±68. [18] Tamaoka, A., Sawamura, N., Fukushima, T., Shoji, S., Matsubara, E., Shoji, M., Hirai, S., Furiya, Y., Endoh, R. and Mori, H., Amyloid b protein 42(43) in cerebrospinal ¯uid of patients with Alzheimer's disease, J. Neurol. Sci., 148 (1997) 41±45. [19] Weller, R.O., Massey, A., Newman, T.A., Hutchings, M., Kuo, Y.M. and Roher, A.E., Cerebral amyloid angiopathy: amyloid beta accumulates in putative interstitial ¯uid drainage pathways in Alzheimer's disease, Am. J. Pathol., 153 (1998) 725±733. [20] Younkin, S.G., Evidence that Ab42 is the real culprit in Alzheimer's disease, Ann. Neurol., 37 (1995) 287±288.
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Amyloid b protein 1±40 and 1±42 levels in matched cerebrospinal ¯uid and plasma from patients with Alzheimer disease P.D. Mehta a,*, T. Pirttila b, B.A. Patrick a, M. Barshatzky a, S.P. Mehta a a
Department of Immunology, Institute for Basic Research in Developmental Disabilities, Forest Hill Road, Staten Island, New York 10314-6399, USA b Department of Neurology, Kuopio University Hospital, Kuopio, Finland Received 11 January 2001; received in revised form 20 March 2001; accepted 20 March 2001
Abstract We quantitated amyloid b proteins 1±40 (Ab40) and 1±42 (Ab42), and a1- antichymotrypsin (ACT) in matched cerebrospinal ¯uid (CSF) and plasma of 50 patients with probable Alzheimer disease, and analyzed the relationships with age, sex, Mini-Mental State Examination (MMSE), and apolipoprotein E phenotype. There was no relation between CSF Ab40 and Ab42 levels with those of plasma. CSF and plasma Ab40 and Ab42 levels showed no association with age, sex, and MMSE score. There was a signi®cant correlation between CSF ACT and plasma ACT levels. The data suggest that plasma ACT crosses the blood±brain barrier. However, a lack of correlation between CSF Ab40 and Ab42 levels with those of plasma suggests that Ab in CSF and plasma originates from different sources. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer disease; Amyloid b protein; a1-antichymotrypsin; Apolipoprotein E; Enzyme linked immunosorbent assay
The major component of neuritic plaques is the amyloid b protein (Ab), a small 39±42 amino acid residue protein derived through proteolytic processing of a larger membrane-bound glycoprotein, the amyloid b-precursor protein (AbPP) [16]. Secreted, soluble Ab is a product of normal cell metabolism and found in various body ¯uids, including plasma and cerebrospinal ¯uid (CSF) [15]. Ab ending at residue 42 (Ab42) is mainly found in senile plaques; whereas Ab ending at residue 40 (Ab40) is prominent in vascular amyloid deposits [6]. Investigators showed that deposition of Ab42 in brain might be the initial step in amyloid formation [20]. a1-antichymotrypsin (ACT), a serpin serine proteinase inhibitor, is a component of senile plaques, and is characteristic of acute phase in¯ammation [1]. Although the physiological role of ACT is not well established, investigators have reported increased concentrations of ACT in sera from patients with AD [9]. Apolipoprotein E (Apo E) is a 34-kd polymorphic protein that is involved in the transportation and redistribution of lipids among various tissues. The possession of two variants
* Corresponding author. Tel.: 11-718-494-5159; fax: 11-718982-6345. E-mail address: [email protected] (P.D. Mehta).
of Apo E 14 allele is a signi®cant risk factor for the development of sporadic and familial late-onset AD [14]. Investigators quantitated Ab40 and Ab42 in CSF or plasma in sporadic AD patients and reported that the levels were signi®cantly higher in CSF than plasma [4,12,17,18]. However, none examined the levels in matched CSF and plasma. Thus it is not known whether higher Ab levels found in CSF re¯ect intrathecal synthesis or damage in the blood±brain barrier. The aim of this study is to quantitate Ab40 and Ab42 levels in matched CSF and plasma from 50 AD patients and correlate the levels with age, sex, MiniMental State Examination (MMSE) score, and Apo E phenotype. In addition to Ab, we also quantitated ACT levels in the same samples, since our previous studies suggested that CSF ACT is mainly derived from blood [13]. The study included 50 patients with sporadic AD from the Department of Neurology, Kuopio University Medical Center, Kuopio, Finland. The diagnosis of probable AD was made according to the NINCDS-ADRDA criteria [11]. AD patients ranged in age from 57 to 83 years (mean 73 years). There were 16 men (11 with Apo E 14 allele and ®ve without) and 34 women (23 with Apo E 14 allele and 11 without). The MMSE scores ranged from 10 to 29 (median 21). Blood was collected in tubes containing K3-EDTA (B-D Vacutainer Systems, Franklin Lakes, NJ).
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 01) 0 17 54- 2
P.D. Mehta et al. / Neuroscience Letters 304 (2001) 102±106
103
Table 1 CSF and Plasma Ab40, Ab42 and ACT levels a Specimen (n)
Ab40 Median (Range)
Ab42, pg/ml Median (Range)
ACT, mg/ml Median (Range)
CSF (50) With Apo E 14 (34) Without Apo E 14 (16) Plasma (50) With Apo E 14 (34) Without Apo E 14 (16)
9.7 (1.6±17.1), ng/ml 9.0 (1.6±15.8) 10.2 (4.5±17.1) 143 (45±288), pg/ml 136 (45±288) 143 (68±201)
623 (287±1949) 495 (287±1347)* 862 (418±1949) 8.6 (5.0±23) 9.3 (5.0±23) 6.9 (5.0±16)
2.5 (1.0±5.3) 2.5 (1.2±5.3) 2.5 (1.0±4.0) 312 (203±752) 409 (209±541) 323 (203±752)
a
*P , 0:0002, analysis of variance with Bonferroni correction.
Plasma were separated by centrifuging blood at 3000 rpm at 48C for 20 min, aliquoted and frozen at 2808C. CSF were also aliquoted, and stored frozen at 2808C. All CSF were tested for cell counts, glucose and total proteins. Those contaminated with blood were excluded. Apo E phenotypes of AD plasma were determined using isoelectric focusing in agarose gel containing 3M urea followed by immunoblotting with speci®c goat antihuman Apo E antiserum [7]. Ab32±40 and Ab33±42 peptides synthesized commercially (Ana Spec, San Jose, CA) were conjugated to keyhole limpet hemocyanin in PBS with 0.5% glutaraldehyde and immunized in rabbits. The speci®city of rabbit antisera R209 produced against Ab40, and R226 raised against Ab42 was examined in a sandwich enzyme linked immu-
nosorbent assay (ELISA). There was a strong response of R209 with 1 ng/ml of Ab40 but no detectable response with 10 ng/ml of Ab42. Similarly R226 was found speci®c to Ab42 but showed no reactivity to Ab40. Western blot also showed that R209 was speci®c to Ab40, and R226 was speci®c to Ab42 (data not shown). Ab levels were measured using monoclonal antibody 6E10 (speci®c to an epitope present on 1±16 amino acid residues of Ab) and R209 and R226 in a double antibody sandwich ELISA [12]. The detection limit was 10 pg/ml for Ab40 and 5 pg/ml for Ab42. The percent coef®cients of variation ranged from 8 to 14% (intra-assay) and 10 to 18% (inter-assay). ACT levels were measured in a double antibody sandwich ELISA [13]. The detection limit of ACT was 12.5 ng/ml.
Fig. 1. Box plots of Ab 40 and Ab42 levels in CSF and plasma of AD patients with and without Apo E 14 allele.
104
P.D. Mehta et al. / Neuroscience Letters 304 (2001) 102±106
Fig. 2. Relationship of Ab40 and Ab42 levels in CSF (A) with Apo E 14 allele and (B) without Apo E 14 allele and plasma (C) with Apo E 14 allele and (D) without Apo E 14 allele.
The relationship between variables was determined by using Pearson product-moment correlation with Bonferroni correction. The level of P , 0:01 was considered signi®cant. Relationship between CSF and plasma Ab40, Ab42 and ACT levels. There was no relation between CSF Ab40 and plasma Ab40 levels (r 20:13, P , 0:47), and CSF Ab42 and plasma Ab42 levels (r 0:01, P , 0:93). However, there was a signi®cant relation between CSF ACT and plasma ACT levels (r 0:53, P , 0:001). Relationship of CSF and plasma Ab40, Ab42 and ACT levels with Apo E 14 allele. CSF Ab40 levels were similar in AD patients with and without Apo E 14 allele. However, CSF Ab42 levels were signi®cantly lower in AD patients with Apo E 14 allele than those without (P , 0:0002) (Table 1) (Fig. 1). Plasma Ab40 and Ab42 levels were similar in AD patients with and without Apo E 14 allele. There was a signi®cant association between CSF Ab40 and Ab42 levels in AD with Apo E 14 allele (r 0:56, P , 0:0005), but not in those without (r 0:42, P , 0:1) (Fig. 2). There was a signi®cant association between plasma Ab40 and Ab42 levels in AD with Apo E 14 allele (r 0:69, P , 0:0001) and those without (r 0:67, P , 0:005) (Fig. 2). Levels of CSF and plasma ACT were similar in AD patients with and without Apo E 14 allele. There was a signi®cant association between CSF ACT and
plasma ACT levels in AD with Apo E 14 allele (r 0:5, P , 0:003) and those without (r 0:69, P , 0:003) (data not shown). Relationship with age, sex and MMSE score. CSF and plasma Ab40 and Ab42 levels showed no association with age, sex, and MMSE score. However, there was a relation between CSF Ab40 and CSF ACT levels but with borderline signi®cance (r 0:29, P , 0:039). There was a significant association between age and levels of plasma ACT (r 0:37, P , 0:009) and CSF ACT (r 0:37, P , 0:008). However, there was no relation between sex and MMSE score with CSF or plasma ACT levels. Ab levels in CSF re¯ect the net result of the increased production and diminished clearance of Ab in AD brain. Studies of an animal model of AD suggested that Ab in CSF is produced by the neuronal source of APP rather than local production or Ab uptake from the blood [2]. It is possible that some CSF Ab originates from blood, since it has been reported that Ab injected intravenously enters the brain through the blood±brain barrier [8]. However, the lack of correlation between matched CSF Ab40 and Ab42 levels with those of plasma suggests that a large portion of Ab in CSF may have originated in the brain rather than plasma. Investigators have reported that plasma Ab42 levels were elevated in patients with the early-onset familial AD-linked
P.D. Mehta et al. / Neuroscience Letters 304 (2001) 102±106
APP, PS1, and PS2 mutations [15]. The data support the idea that this effect is related to the pathogenesis of AD. However, in most sporadic AD cases, plasma Ab42 levels were similar to those of elderly non-demented controls. Thus, in sporadic AD, cerebral deposition of Ab42 is caused by other factors, such as synthesis or secretion of Ab42, or alterations of Ab-binding proteins [15]. The origin of Ab in plasma is unknown. Previous studies have shown that Ab injected into ventricular CSF in rats cleared rapidly into blood by subependymal capillaries and the choroid plexus [5]. Histological studies have shown that Ab accumulated in periarterial interstitial ¯uid drainage pathways of the brain [19]. The results suggested that some Ab in brain parenchyma can be eliminated through the perivascular spaces and may in¯uence Ab levels in blood. However, studies have showed that anti-Ab immunoreactivity in blood was associated with platelets. The data suggested that platelets are one of the major sources of Ab in human plasma [3]. ACT forms a non-covalent complex with Ab, thus inducing multiple effects that favor the propagation of in¯ammation and synthesis of Ab42 ®brils. However, the strong correlation between CSF ACT and plasma ACT suggests that plasma ACT crosses the blood±brain barrier and that CSF ACT originates mainly from blood. The ®ndings are consistent with our previous studies [13]. Although in our study most AD patients had mild dementia, levels of Ab40 and Ab42 in plasma and CSF did not correlate with MMSE score. In contrast, other investigators have shown a weak correlation between these levels and dementia severity [4]. The reason for the discrepancy between the studies is not clear. Further longitudinal studies using a large number of samples will be necessary to determine conclusively whether there is a relationship between plasma Ab levels and progression of AD. In summary, the lack of correlation between Ab40 and Ab42 levels in CSF and plasma in AD suggests that the higher levels found in CSF are not re¯ected in plasma and that the source of the synthesis of Ab in these two compartments is different. Our results suggest that plasma Ab levels do not re¯ect the pathological changes seen in AD brain. The question remains why do about 10 to 20% of sporadic AD patients show increased Ab42 concentrations in plasma [12,15,17]? In addition, investigators have also reported higher plasma Ab42 levels in the ®rst-degree relatives of patients with AD, and several cognitively normal individuals in whom AD later developed [10]. The signi®cance of the relationship between Ab levels in plasma and higher concentrations of Ab in CSF and brain is unknown. The work in part was supported by a grant from P®zer, Inc. and in part by the funds provided by NY State through its Of®ce of Mental Retardation and Developmental Disabilities. We thank Dr Eugene A. Sersen for statistical analyses.
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