Normal range of human red blood cell acetylcholinesterase activity.

The normal range of human erythrocyte acetylcholinesterase (RBC-AChE) activity is important when monitoring exposure to pesticides and chemical warfare agents. A modification of Michel's method measured RBC-AChE activities from 991 individuals (818 males and 173 females) presumably unexposed to nerve agents. Median age was 42 (range, 18-76) years. RBC-AChE (mean +/- SD) was 0.74 +/- 0.06 delta pH units/hour. Multivariate linear regression showed an association with age (slope +0.0008 delta pH units/hour for each year; P < 0.001) unlikely to be clinically significant. The findings represent the largest study of human RBC-AChE to date providing measures of central tendency and variation.

Advantages of the WRAIR whole blood cholinesterase assay: comparative analysis to the micro-Ellman, Test-mate ChE, and Michel (DeltapH) assays.

Red blood cell AChE (RBC-AChE) and plasma BChE can be used as sensitive biomarkers to detect exposure to OP nerve agents, pesticides, and cholinergic drugs. In a comparative study, RBC-AChE and serum BChE activities in whole blood was obtained from forty seven healthy male and female human volunteers, and then exposed separately ex vivo to three OP nerve agents (soman (GD), sarin (GB) and VX) to generate a wide range of inhibition of AChE and BChE activity (up to 90% of control). These samples were measured using four different ChE assays: (i) colorimetric microEllman (using DTNB at 412 nm), (ii) Test-mate ChE field kit (also based on the Ellman assay), (iii) Michel (delta pH), and (iv) the Walter Reed Army Institute of Research Whole Blood (WRAIR WB) cholinesterase assay. The WRAIR assay is a modified Ellman method using DTP at 324 nm (which minimizes hemoglobin interference and improves sensitivity), and determines AChE and BChE in a small whole blood sample simultaneously. Scatter plots of RBC-AChE activities were determined using the WRAIR ChE assay versus the micro-Ellman, Test-mate and Michel after exposure to varying concentrations of soman, sarin and VX. Regression analyses yielded mostly linear relationships with high correlations (r2 = 0.83-0.93) for RBC-AChE values in the WRAIR assay compared to the alternate methods. For the plasma BChE measurements, individual human values were significantly more variable (as expected), resulting in lower correlations using WRAIR ChE versus the alternate assays (r2 values 0.5 - 0.6). To circumvent the limitations of simple correlation analysis, Bland and Altman analysis for comparing two independent measurement techniques was performed. For example, a Bland and Altman plot of the ratio of the WRAIR whole blood AChE and Michel AChE (plotted on the y-axis) vs. the average of the two methods (x-axis) shows that the majority of the individual AChE values are within +/- 1.96 S.D. of the mean difference, indicating that the two methods may be used interchangeably with a high degree of confidence. The WRAIR ChE assay can be thus be used as a reliable inter-conversion assay when comparing results from laboratory-based (Michel) and field-based (Test-mate ChE kit), which use different methodology and report in different units of AChE activity.

Intraindividual stability of human erythrocyte cholinesterase activity.

BACKGROUND: Erythrocyte cholinesterase (RBC-ChE) activities serve as useful and sensitive biomarkers to monitor exposure to cholinesterase-inhibiting substances, such as chemical warfare nerve agents and pesticides. Although the interindividual variation of RBC-ChE is well characterized, the magnitude of intraindividual variation for RBC-ChE remains controversial. An accurate measure of intraindividual variation is critical for establishing the appropriate frequency of RBC-ChE testing. METHODS: We retrospectively tracked the intraindividual variation of RBC-ChE activities among 46 male nerve agent workers from a single US Army depot that participated in a medical surveillance program requiring periodic RBC-ChE monitoring. All RBC-ChE analysis was performed by the same medical laboratory technician by the delta pH method. RESULTS: A mean of 38 and a median of 37 RBC-ChE measurements were available for each worker. The mean duration of employment for these workers was 20 years (median, 21 years). The mean CV for RBC-ChE in this set of 46 workers was 3.9%. Linear regression analysis of the data for each worker resulted in a mean slope of 0.0010 delta pH units/h per year. CONCLUSIONS: RBC-ChE activities increased in each person by a mean of 0.01 delta pH units/h every 10 years, which is a negligible rate. These findings highlight the stability of RBC-ChE activities over time in a given individual and may have important policy implications regarding the appropriate frequency of RBC-ChE testing.

Sulfur mustard-induced arachidonic acid release is mediated by phospholipase D in human keratinocytes.

Sulfur mustard (2,2(')-dichloroethyl sulfide) is a chemical warfare agent that causes incapacitating skin blisters in humans 12-24h post-exposure following a variable asymptomatic phase. Recent reports demonstrate that inflammation plays a vital role in sulfur mustard toxicity. One of the key biochemical pathways involved in inflammation is the arachidonic acid cascade. In this report, we demonstrate that arachidonic acid is released in response to sulfur mustard and investigate the mechanisms of arachidonic acid release. Exposure to sulfur mustard caused a 5- to 8-fold increase in arachidonic acid release from human keratinocytes that had been radiolabeled with arachidonic acid. Maximal arachidonic acid release occurred between 12 and 24h. Several enzymatic pathways can lead to arachidonic acid release. Treatment with 2.0% (v/v) ethanol, an inhibitor of phospholipase D, decreased sulfur mustard-induced arachidonic acid release 40+/-7%. Additionally, 100 microM (+/-)-propranolol, an inhibitor of phosphatidic acid phosphohydrolase, blocked sulfur mustard-induced arachidonic acid release by 62+/-3%. These findings suggest that arachidonic acid release is mediated by phospholipase D and phosphatidic acid phosphohydrolase in human keratinocytes following sulfur mustard exposure. Due to the 12-24h delay in arachidonic acid release following sulfur mustard exposure, delayed therapeutic intervention may be possible. Indeed, we found that the addition of 100 microM (+/-)-propranolol up to 18 h after sulfur mustard exposure was still able to block arachidonic acid release by 30+/-3%.