Particulate Acetylcholinesterase in Amniotic Fluid and its Implications for Neural Tube Defect Screening

Webb, Brian A.; Richardson, Sara; Garry, Ray; Atkins, Jessica

doi:10.1177/000456328101800506

Cited by 5 publications

(2 citation statements)

References 14 publications

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“…2 The concentration of methanol in the reaction mixture (0·1 %) had no effect on enzyme activity, increase in background absorption, or pH of the assay system. As shown in Fig.…”

Section: Resultsmentioning

confidence: 96%

A Potential Systematic Error in Using Lysivane as Inhibitor in the Measurement of Amniotic Fluid Acetylcholinesterase by the Ellman Method

Webb¹,

Richardson²,

Garry

et al. 1983

Ann Clin Biochem

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examined in an attempt to improve the diagnostic accuracy of the method. Factors which bring about changes in background absorption are shown to include a pH-related effect caused by the addition of lysivane solution.DETERMINATION OF ACETYLCHOLINESTERASE Acetylcholinesterase was measured by the method of Ellman! in the presence of Lysivane (1·42 x lO-OM) at 412 mu, Determinations were made on amniotic fluid (50 !Jol) at 30°C and pH 8·0 in a total volume of 3·0 ml.Lysivane (10-(2-diethylaminopropyl) phenothiazine hydrochloride) was obtained from Rhone Poulenc Sante, Paris, France, under the trade name Parsidol.It was obtained as a fine white powder of 99·9 % purity. The material and other reagents were those previously described.P SUMMARY The measurement of low levels of cholinesterase or acetylcholinesterase by the Ellman method requires correction for a non-enzymatic increase in absorption at 412 mu that is due both to non-enzymatic hydrolysis of the acetylthiocholine substrate and to modification of the colour reagent. The rate ofincrease in absorption is dependent on temperature and pH. Addition of an acidic solution of lysivane to the assay solution for selective measurement of amniotic fluid acetylcholinesterase gives rise to a shift in pH; the use of methanol is suggested as an easier method of dissolving the inhibitor and does not affect the pH of the assay, obviating any need to redetermine the background absorption. There is, however, no improvement in ability of the method to predict pregnancies associated with neural-tube defects.A sensitive quantitative technique for measuring low levels of cholinesterase and acetylcholinesterase using the substrate analogue acetylthiocholine has been described by Ellman.' Enzymatically generated thiocholine reacts with 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) to liberate the coloured anion 5-thio-2-nitrobenzoic acid which can be determined Material and methods spectrophotometrically at 412 rnu, Accurate determination of enzyme activity relies on the application of a correction for significant increase in absorption which has been attributed to non-enzymatic hydrolysis of the substrate.' Measurement of acetylcholinesterase in the presence of serum cholinesterase depends on the use of the selective acetylcholinesterase inhibitor BW 284 C5l dibromide or the pseudocholinesterase inhibitor lysivane.The Ellman method is used by several laboratories for quantitative determination of cholinesterase and acetylcholinesterase in amniotic fluid in connection with screening procedures for prenatal detection of neural-tube defects. Recent published work has demonstrated a significant false-positive rate when the quantitative method is used for cholinesterase! DETERMINATION OF NON-ENZYMATIC DEGRADATION OF acetylcholinesterase measurement in amniotic fluid, SUBSTRATE AND COLOUR REAGENT and this has led to an unfavourable comparison being Non-enzymatic hydrolysis of acetylthiocholine was drawn between this method and the electrophoretic measured at 412 mf! at a concentration of ...

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“…2 The concentration of methanol in the reaction mixture (0·1 %) had no effect on enzyme activity, increase in background absorption, or pH of the assay system. As shown in Fig.…”

Section: Resultsmentioning

confidence: 96%

A Potential Systematic Error in Using Lysivane as Inhibitor in the Measurement of Amniotic Fluid Acetylcholinesterase by the Ellman Method

Webb¹,

Richardson²,

Garry

et al. 1983

Ann Clin Biochem

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“…Red cell lysates and even a few normal amniotic fluid samples contain a particulate fraction of AChE. As shown by Webb et al (1981) this particle-associated fraction of AChE may be responsible for the discrepancy between results obtained by a quantitative spectrophotometric method and a qualitative electrophoretic technique (PAG-gel). This particulate or membrane-bound AChE is found at the application end (origin) of the electrophoretic pattern due to its size related exclusion from the gel pores ( Figure 3a).…”

Section: Discussionmentioning

confidence: 98%

Identification of acetylcholinesterase and cholinesterase in amniotic fluid by immune absorption technique

1982

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A method for immunological detection of acetylcholinesterase (AChE) and cholinesterase (ChE) in amniotic fluid is described. By addition of a small amount of antihuman-erythrocyte membrane antibody or anti-pseudocholinesterase antibody to the sample before electrophoresis the two esterase bands on polyacrylamide gel (PAG) can be absorbed away. Similar staining results can also be obtained by specific inhibition of the two esterases with either BW 284C51 (AChE inhibitor) or Lysivane (ChE inhibitor). In cases with a faint AChE band and in cases with blood contamination the immune absorption technique makes interpretation easier. Nearly identical staining results have been obtained by the immune absorption technique and the inhibition technique in the following samples with an AChE band: 34 samples from pregnancies with severe fetal malformation or intrauterine death (2 cases), 4 fetal serum samples, 4 samples of cerebrospinal fluid, 4 samples of fetal erythrolysate and 4 samples of adult erythrolysate. It can be concluded that an antibody prepared against erythrocyte AChE cross-reacts with AChE in cerebrospinal fluid, and that this antibody can be used for demonstration of AChE in amniotic fluid.

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Cholinesterase evaluation in the prenatal diagnosis of open neural tube defects

Troccoli

Biagioni

Stella

et al. 1985

La Ricerca Clin. Lab.

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Particulate Acetylcholinesterase in Amniotic Fluid and its Implications for Neural Tube Defect Screening

Cited by 5 publications

References 14 publications

A Potential Systematic Error in Using Lysivane as Inhibitor in the Measurement of Amniotic Fluid Acetylcholinesterase by the Ellman Method

A Potential Systematic Error in Using Lysivane as Inhibitor in the Measurement of Amniotic Fluid Acetylcholinesterase by the Ellman Method

Identification of acetylcholinesterase and cholinesterase in amniotic fluid by immune absorption technique

Cholinesterase evaluation in the prenatal diagnosis of open neural tube defects

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