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Altered levels of acetylcholinesterase in Alzheimer plasma.

García-Ayllón MS, Riba-Llena I, Serra-Basante C, Alom J, Boopathy R, Sáez-Valero J - PLoS ONE (2010)

Bottom Line: Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem.The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE.We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, San Juan de Alicante, Spain.

ABSTRACT

Background: Many studies have been conducted in an extensive effort to identify alterations in blood cholinesterase levels as a consequence of disease, including the analysis of acetylcholinesterase (AChE) in plasma. Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem.

Principal findings: Here we have estimated the levels of AChE activity in human plasma by first immunoprecipitating BuChE and measuring AChE activity in the immunodepleted plasma. Human plasma AChE activity levels were approximately 20 nmol/min/mL, about 160 times lower than BuChE. The majority of AChE species are the light G(1)+G(2) forms and not G(4) tetramers. The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE. We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis. Finally, a selective increase of AChE activity was detected in plasma from Alzheimer's disease (AD) patients compared to age and gender-matched controls. This increase correlates with an increase in the G(1)+G(2) forms, the subset of AChE species which are increased in Alzheimer's brain. Western blot analysis demonstrated that a 78 kDa immunoreactive AChE protein band was also increased in Alzheimer's plasma, attributed in part to AChE-T subunits common in brain and CSF.

Conclusion: Plasma AChE might have potential as an indicator of disease progress and prognosis in AD and warrants further investigation.

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Related in: MedlinePlus

AChE molecular form and lectin-binding profile in human plasma, liver, RBCs, CSF and brain (frontal cortex).(A) Representative profiles of AChE molecular forms (G4 = tetramers; G1+G2 = monomers and dimers). (B) Comparison of Con A and LCA binding of AChE. Plasma, CSF and total extracts from liver, RBCs and brain (n = 6 for each group) were incubated with immobilized lectins, AChE activity was assayed in the supernatants and the percentage of (%) AChE activity unbound to lectins was calculated. For total CSF and brain extracts, both rich in tetramers, the % AChE unbound to Con A (%Unb Con ACSF = 3±1; %Unb Con ABrain = 4±1) and to LCA (%Unb LCACSF = 4±1; %Unb LCABrain = 17±2) were determined. Additionally, individual G4 and G1+G2 fractions, separated by sucrose gradient centrifugation, from CSF and brain extracts (n = 5), were also pooled, dialyzed against Tris-saline-Triton X-100 buffer, and concentrated by ultrafiltration. AChE peaks were then assayed by incubation with immobilized lectins, and the percentages of unbound enzymatic activity were calculated. For CSF tetramers, the %Unb Con A was 0.9±0.5; and the %Unb LCA was 0.3±0.1. For brain tetramers, the %Unb Con A was 1.3±0.2; and the %Unb LCA 2.4±0.2. Please note differences in lectin binding for total AChE from brain or CSF, or its enriched G4 fractions, when compared to the respective G1+G2 peaks (see Figure), revealing distinct glycosylation patterns for different AChE molecules. Values are means± SEM *p<0.05, significantly different from plasma samples, as assessed by one-way analysis of variance with Bonferroni posttest.
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pone-0008701-g002: AChE molecular form and lectin-binding profile in human plasma, liver, RBCs, CSF and brain (frontal cortex).(A) Representative profiles of AChE molecular forms (G4 = tetramers; G1+G2 = monomers and dimers). (B) Comparison of Con A and LCA binding of AChE. Plasma, CSF and total extracts from liver, RBCs and brain (n = 6 for each group) were incubated with immobilized lectins, AChE activity was assayed in the supernatants and the percentage of (%) AChE activity unbound to lectins was calculated. For total CSF and brain extracts, both rich in tetramers, the % AChE unbound to Con A (%Unb Con ACSF = 3±1; %Unb Con ABrain = 4±1) and to LCA (%Unb LCACSF = 4±1; %Unb LCABrain = 17±2) were determined. Additionally, individual G4 and G1+G2 fractions, separated by sucrose gradient centrifugation, from CSF and brain extracts (n = 5), were also pooled, dialyzed against Tris-saline-Triton X-100 buffer, and concentrated by ultrafiltration. AChE peaks were then assayed by incubation with immobilized lectins, and the percentages of unbound enzymatic activity were calculated. For CSF tetramers, the %Unb Con A was 0.9±0.5; and the %Unb LCA was 0.3±0.1. For brain tetramers, the %Unb Con A was 1.3±0.2; and the %Unb LCA 2.4±0.2. Please note differences in lectin binding for total AChE from brain or CSF, or its enriched G4 fractions, when compared to the respective G1+G2 peaks (see Figure), revealing distinct glycosylation patterns for different AChE molecules. Values are means± SEM *p<0.05, significantly different from plasma samples, as assessed by one-way analysis of variance with Bonferroni posttest.

Mentions: The cellular origin of circulating AChE remains controversial. We performed a comparative analysis of AChE from different human tissues and fluids obtained from non-diseased subjects. The different AChE molecular species are cell type-dependent, with differences in developmental and adult tissues and in different species [7]. As the molecular pattern of AChE in human tissues is unclear, we have analyzed the different molecular forms of AChE in liver, RBCs, CSF and brain extracts and compared these to the pattern obtained from plasma which is rich in light species and contains only trace amounts of tetramers (Fig 2A). Sedimentation analysis of AChE from liver (total AChE activity levels, 2.2±0.3 mU/mg) and RBC extracts (491±40 mU/mg) showed similar profiles to that of plasma, whereas CSF (16±2 mU/mL) and frontal cortical extracts (10±2 mU/mg) displayed abundant amounts of G4 and small amounts of the G1+G2 species.


Altered levels of acetylcholinesterase in Alzheimer plasma.

García-Ayllón MS, Riba-Llena I, Serra-Basante C, Alom J, Boopathy R, Sáez-Valero J - PLoS ONE (2010)

AChE molecular form and lectin-binding profile in human plasma, liver, RBCs, CSF and brain (frontal cortex).(A) Representative profiles of AChE molecular forms (G4 = tetramers; G1+G2 = monomers and dimers). (B) Comparison of Con A and LCA binding of AChE. Plasma, CSF and total extracts from liver, RBCs and brain (n = 6 for each group) were incubated with immobilized lectins, AChE activity was assayed in the supernatants and the percentage of (%) AChE activity unbound to lectins was calculated. For total CSF and brain extracts, both rich in tetramers, the % AChE unbound to Con A (%Unb Con ACSF = 3±1; %Unb Con ABrain = 4±1) and to LCA (%Unb LCACSF = 4±1; %Unb LCABrain = 17±2) were determined. Additionally, individual G4 and G1+G2 fractions, separated by sucrose gradient centrifugation, from CSF and brain extracts (n = 5), were also pooled, dialyzed against Tris-saline-Triton X-100 buffer, and concentrated by ultrafiltration. AChE peaks were then assayed by incubation with immobilized lectins, and the percentages of unbound enzymatic activity were calculated. For CSF tetramers, the %Unb Con A was 0.9±0.5; and the %Unb LCA was 0.3±0.1. For brain tetramers, the %Unb Con A was 1.3±0.2; and the %Unb LCA 2.4±0.2. Please note differences in lectin binding for total AChE from brain or CSF, or its enriched G4 fractions, when compared to the respective G1+G2 peaks (see Figure), revealing distinct glycosylation patterns for different AChE molecules. Values are means± SEM *p<0.05, significantly different from plasma samples, as assessed by one-way analysis of variance with Bonferroni posttest.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806824&req=5

pone-0008701-g002: AChE molecular form and lectin-binding profile in human plasma, liver, RBCs, CSF and brain (frontal cortex).(A) Representative profiles of AChE molecular forms (G4 = tetramers; G1+G2 = monomers and dimers). (B) Comparison of Con A and LCA binding of AChE. Plasma, CSF and total extracts from liver, RBCs and brain (n = 6 for each group) were incubated with immobilized lectins, AChE activity was assayed in the supernatants and the percentage of (%) AChE activity unbound to lectins was calculated. For total CSF and brain extracts, both rich in tetramers, the % AChE unbound to Con A (%Unb Con ACSF = 3±1; %Unb Con ABrain = 4±1) and to LCA (%Unb LCACSF = 4±1; %Unb LCABrain = 17±2) were determined. Additionally, individual G4 and G1+G2 fractions, separated by sucrose gradient centrifugation, from CSF and brain extracts (n = 5), were also pooled, dialyzed against Tris-saline-Triton X-100 buffer, and concentrated by ultrafiltration. AChE peaks were then assayed by incubation with immobilized lectins, and the percentages of unbound enzymatic activity were calculated. For CSF tetramers, the %Unb Con A was 0.9±0.5; and the %Unb LCA was 0.3±0.1. For brain tetramers, the %Unb Con A was 1.3±0.2; and the %Unb LCA 2.4±0.2. Please note differences in lectin binding for total AChE from brain or CSF, or its enriched G4 fractions, when compared to the respective G1+G2 peaks (see Figure), revealing distinct glycosylation patterns for different AChE molecules. Values are means± SEM *p<0.05, significantly different from plasma samples, as assessed by one-way analysis of variance with Bonferroni posttest.
Mentions: The cellular origin of circulating AChE remains controversial. We performed a comparative analysis of AChE from different human tissues and fluids obtained from non-diseased subjects. The different AChE molecular species are cell type-dependent, with differences in developmental and adult tissues and in different species [7]. As the molecular pattern of AChE in human tissues is unclear, we have analyzed the different molecular forms of AChE in liver, RBCs, CSF and brain extracts and compared these to the pattern obtained from plasma which is rich in light species and contains only trace amounts of tetramers (Fig 2A). Sedimentation analysis of AChE from liver (total AChE activity levels, 2.2±0.3 mU/mg) and RBC extracts (491±40 mU/mg) showed similar profiles to that of plasma, whereas CSF (16±2 mU/mL) and frontal cortical extracts (10±2 mU/mg) displayed abundant amounts of G4 and small amounts of the G1+G2 species.

Bottom Line: Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem.The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE.We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, San Juan de Alicante, Spain.

ABSTRACT

Background: Many studies have been conducted in an extensive effort to identify alterations in blood cholinesterase levels as a consequence of disease, including the analysis of acetylcholinesterase (AChE) in plasma. Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem.

Principal findings: Here we have estimated the levels of AChE activity in human plasma by first immunoprecipitating BuChE and measuring AChE activity in the immunodepleted plasma. Human plasma AChE activity levels were approximately 20 nmol/min/mL, about 160 times lower than BuChE. The majority of AChE species are the light G(1)+G(2) forms and not G(4) tetramers. The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE. We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis. Finally, a selective increase of AChE activity was detected in plasma from Alzheimer's disease (AD) patients compared to age and gender-matched controls. This increase correlates with an increase in the G(1)+G(2) forms, the subset of AChE species which are increased in Alzheimer's brain. Western blot analysis demonstrated that a 78 kDa immunoreactive AChE protein band was also increased in Alzheimer's plasma, attributed in part to AChE-T subunits common in brain and CSF.

Conclusion: Plasma AChE might have potential as an indicator of disease progress and prognosis in AD and warrants further investigation.

Show MeSH
Related in: MedlinePlus