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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

Plasma AChE levels in healthy controls (wild-type) after BuChE immunodepletion and in BuChE-silent individuals.(A) Control plasma was immunoprecipitated with anti-BuChE antibody and cholinesterase activity levels determined beforeb and aftera immunoprecipitation (n = 6; 46±4 yrs). AChE activity level in plasma from BuChE-silent individuals is also shown (n = 3; 30±5 yrs). The anti-BuChE antibody does not immunoprecipitate AChE in BuChE-silent plasma (not shown). Values are means ± SEM. (B) Immunoprecipitation of control plasma with antibody, followed by immunoblotting with the anti-AChE antibody, N-19. The presence (+) or absence (−) of the anti-BuChE antibody linked to the resin is indicated in the top margin. Prior to electrophoretic analysis, proteins abundant in plasma were depleted by immunoaffinity-based protein subtraction chromatography with IgY microbeads (Seppro™). The anti-BuChE antibody does not immunoprecipitate AChE. Extracts incubated with protein A-Sepharose, in the absence of the antibody, were analyzed in parallel as negative controls. (C) Representative profiles of AChE and (D) BuChE molecular forms (G4 = tetramers; G1+G2 = monomers and dimers) in control plasma samples before (•) and after (○) BuChE-immunoprecipitation, and in BuChE-silent plasma (▴). (E) Representative immunoblot of individual AChE G4 and G1+G2 peak-fractions separated by sucrose gradient centrifugation from control plasma and detected with the N-19 antibody (a similar volume for both the G4 and G1+G2 peaks was loaded in each lane). (F) Comparison of the AChE-banding pattern detected with the N-19 antibody, for fractions bound and unbound to the anti-AChE antibody HR2 and to the Fas2-Sepharose affinity matrix.
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pone-0008701-g001: Plasma AChE levels in healthy controls (wild-type) after BuChE immunodepletion and in BuChE-silent individuals.(A) Control plasma was immunoprecipitated with anti-BuChE antibody and cholinesterase activity levels determined beforeb and aftera immunoprecipitation (n = 6; 46±4 yrs). AChE activity level in plasma from BuChE-silent individuals is also shown (n = 3; 30±5 yrs). The anti-BuChE antibody does not immunoprecipitate AChE in BuChE-silent plasma (not shown). Values are means ± SEM. (B) Immunoprecipitation of control plasma with antibody, followed by immunoblotting with the anti-AChE antibody, N-19. The presence (+) or absence (−) of the anti-BuChE antibody linked to the resin is indicated in the top margin. Prior to electrophoretic analysis, proteins abundant in plasma were depleted by immunoaffinity-based protein subtraction chromatography with IgY microbeads (Seppro™). The anti-BuChE antibody does not immunoprecipitate AChE. Extracts incubated with protein A-Sepharose, in the absence of the antibody, were analyzed in parallel as negative controls. (C) Representative profiles of AChE and (D) BuChE molecular forms (G4 = tetramers; G1+G2 = monomers and dimers) in control plasma samples before (•) and after (○) BuChE-immunoprecipitation, and in BuChE-silent plasma (▴). (E) Representative immunoblot of individual AChE G4 and G1+G2 peak-fractions separated by sucrose gradient centrifugation from control plasma and detected with the N-19 antibody (a similar volume for both the G4 and G1+G2 peaks was loaded in each lane). (F) Comparison of the AChE-banding pattern detected with the N-19 antibody, for fractions bound and unbound to the anti-AChE antibody HR2 and to the Fas2-Sepharose affinity matrix.

Mentions: As the elevated levels of BuChE in human plasma were expected to interfere in the determination of AChE, two cycles of BuChE immunoprecipitation were first performed in plasma aliquots from healthy individuals (46±4 years). Such immunoprecipitation reduced the levels of BuChE ∼190 times, from 3242±286 to 17±3 mU/mL (Fig 1A). We have utilized a polyclonal antibody raised against highly purified human plasma BuChE that has previously been demonstrated to be effective in immunoprecipitating human BuChE [38]. Western blot analysis with the anti-AChE antibody N-19 confirmed the specificity of the immunoprecipitation, with no AChE immunoreactivity detected in BuChE immunoprecipitates (Fig 1B). The remaining AChE activity in the BuChE depleted supernatant was 20±1 mU/mL, much lower than the levels measured before BuChE depletion (Fig 1A).


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)

Plasma AChE levels in healthy controls (wild-type) after BuChE immunodepletion and in BuChE-silent individuals.(A) Control plasma was immunoprecipitated with anti-BuChE antibody and cholinesterase activity levels determined beforeb and aftera immunoprecipitation (n = 6; 46±4 yrs). AChE activity level in plasma from BuChE-silent individuals is also shown (n = 3; 30±5 yrs). The anti-BuChE antibody does not immunoprecipitate AChE in BuChE-silent plasma (not shown). Values are means ± SEM. (B) Immunoprecipitation of control plasma with antibody, followed by immunoblotting with the anti-AChE antibody, N-19. The presence (+) or absence (−) of the anti-BuChE antibody linked to the resin is indicated in the top margin. Prior to electrophoretic analysis, proteins abundant in plasma were depleted by immunoaffinity-based protein subtraction chromatography with IgY microbeads (Seppro™). The anti-BuChE antibody does not immunoprecipitate AChE. Extracts incubated with protein A-Sepharose, in the absence of the antibody, were analyzed in parallel as negative controls. (C) Representative profiles of AChE and (D) BuChE molecular forms (G4 = tetramers; G1+G2 = monomers and dimers) in control plasma samples before (•) and after (○) BuChE-immunoprecipitation, and in BuChE-silent plasma (▴). (E) Representative immunoblot of individual AChE G4 and G1+G2 peak-fractions separated by sucrose gradient centrifugation from control plasma and detected with the N-19 antibody (a similar volume for both the G4 and G1+G2 peaks was loaded in each lane). (F) Comparison of the AChE-banding pattern detected with the N-19 antibody, for fractions bound and unbound to the anti-AChE antibody HR2 and to the Fas2-Sepharose affinity matrix.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806824&req=5

pone-0008701-g001: Plasma AChE levels in healthy controls (wild-type) after BuChE immunodepletion and in BuChE-silent individuals.(A) Control plasma was immunoprecipitated with anti-BuChE antibody and cholinesterase activity levels determined beforeb and aftera immunoprecipitation (n = 6; 46±4 yrs). AChE activity level in plasma from BuChE-silent individuals is also shown (n = 3; 30±5 yrs). The anti-BuChE antibody does not immunoprecipitate AChE in BuChE-silent plasma (not shown). Values are means ± SEM. (B) Immunoprecipitation of control plasma with antibody, followed by immunoblotting with the anti-AChE antibody, N-19. The presence (+) or absence (−) of the anti-BuChE antibody linked to the resin is indicated in the top margin. Prior to electrophoretic analysis, proteins abundant in plasma were depleted by immunoaffinity-based protein subtraction chromatography with IgY microbeads (Seppro™). The anti-BuChE antibody does not immunoprecipitate AChE. Extracts incubated with protein A-Sepharose, in the absence of the antibody, were analyzed in parallel as negative controls. (C) Representative profiles of AChE and (D) BuChE molecular forms (G4 = tetramers; G1+G2 = monomers and dimers) in control plasma samples before (•) and after (○) BuChE-immunoprecipitation, and in BuChE-silent plasma (▴). (E) Representative immunoblot of individual AChE G4 and G1+G2 peak-fractions separated by sucrose gradient centrifugation from control plasma and detected with the N-19 antibody (a similar volume for both the G4 and G1+G2 peaks was loaded in each lane). (F) Comparison of the AChE-banding pattern detected with the N-19 antibody, for fractions bound and unbound to the anti-AChE antibody HR2 and to the Fas2-Sepharose affinity matrix.
Mentions: As the elevated levels of BuChE in human plasma were expected to interfere in the determination of AChE, two cycles of BuChE immunoprecipitation were first performed in plasma aliquots from healthy individuals (46±4 years). Such immunoprecipitation reduced the levels of BuChE ∼190 times, from 3242±286 to 17±3 mU/mL (Fig 1A). We have utilized a polyclonal antibody raised against highly purified human plasma BuChE that has previously been demonstrated to be effective in immunoprecipitating human BuChE [38]. Western blot analysis with the anti-AChE antibody N-19 confirmed the specificity of the immunoprecipitation, with no AChE immunoreactivity detected in BuChE immunoprecipitates (Fig 1B). The remaining AChE activity in the BuChE depleted supernatant was 20±1 mU/mL, much lower than the levels measured before BuChE depletion (Fig 1A).

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