Acetylcholinesterase activity measurements

In cases where the mode of action is the strong or irreversible inhibition of an enzyme system, the assay may measure the extent of inhibition of this enzyme. This may be accomplished by first measuring the activity of the inhibited enzyme and then making comparison with the uninhibited enzyme. This practice is followed when studying acetylcholinesterase inhibition by organophosphates (OP). Acetylcholinesterase activity is measured in a sample of tissue of brain from an animal that has been exposed to an OP. Activity is measured in the same way in tissue samples from untreated controls of the same species, sex, age, etc. Comparison is then made between the two activity measurements, and the percentage inhibition is estimated. 

Because cholinesterase inhibition is a very sensitive biomarker for other chemicals, it is not always conclusive evidence of disulfoton exposure. However, depression of cholinesterase activity can alert a physician to the possibility of more serious neurological effects. Erythrocyte acetylcholinesterase activity more accurately reflects the degree of synaptic cholinesterase inhibition in nervous tissue, while serum cholinesterase activity may be associated with other sites (Goldfrank et al. 1990). In addition, a recent study showed that after rats received oral doses of disulfoton for 14 days, acetylcholinesterase levels in circulating lymphocytes correlated better with brain acetylcholinesterase activity than did erythrocyte cell cholinesterase activities during exposure (Fitzgerald and Costa 1993). However, recovery of the activity in lymphocytes was faster than the recovery of activity in the brain, which correlated better with the activity in erythrocytes. Animal studies have also demonstrated that brain acetylcholinesterase depression is a sensitive indicator of neurological effects (Carpy et al. 1975 Costa et al. 1984 Schwab and Murphy 1981 Schwab et al. 1981, 1983) however, the measurement of brain acetylcholinesterase in humans is too invasive to be practical. 

More recently, B. Goldstein and McDonagh demonstrated that the native protein fluorescence (280-nm excitation, 330-nm emission) of red-cell membranes exposed in vitro to ozone at 1 ppm was a somewhat more sensitive indicator of ozone effect than other characteristics measured in the same em, including oxidation of cell-membrane sulfhydiyl groups, loss of acetylcholinesterase activity, and formation of lipid peroxide breakdown products. 

FIGURE 4.2 Mortality of fathead minnows in relation to acetylcholinesterase activity.3 a As measured in the brain tissue following (A) a 14-day exposure to a ternary mixture of azinphos-methyl, diazinon, and chlorpyrifos in a concentration-response study and (B) a 7-day exposure to a ternary mixture of azinphos-methyl, diazinon, and chlorpyrifos applied as equipotent (toxic equivalent [TE]) mixtures. Note Dashed lines correspond to 50% reduction in AChE activity the dashed-dotted line corresponds to 50% mortality. Source Redrawn from Sibley et al. (2000). 

Table 1. The effect of 1 mM NaF + 20 pM A1C13 on the acetylcholinesterase activity (AChE) in freshly prepared intact RBC and in hemolysate of patients with AD (mean age 72.5 5.1 years), age-matched healthy controls (AM-HS) (72.1 1.6 years), and the group of young healthy subjects (YS) (35.9 8.5 years). Whole venous blood samples were drawn from each subject after overnight fasting., always at 07 30 AM. Red blood cells (RBC) were isolated from the blood of patients with AD, AM-HS, and YS by centrifugation [68], RBC AChE activity was evaluated in intact freshly prepared RBC or hemolyzate following the spectrophotometric method [45] with modifications. Buffer was Tris-HCl, pH 7.5 in the solution of 154 mmol L 1 NaCl, acetylthiocholine iodide was a substrate. Measurement of enzymatic activity was performed in fluorimeter polystyrene cuvettes for 3 min (UV/VIS spectrophotometer Shimadzu, Japan). The effects of 1 mmol L-1 NaF in the presence of 20 pmol L 1 A1C13 were measured. Data are expressed in percentage of the AChE activity in the absence of aluminum and fluoride ions. No differences between the AChE activity were found between the investigated groups...

Hlley and Burgen (58) measured also the choline acetylase and acetylcholinesterase activities of selected areas of the brain. In an attempt to determine whether the concentration of muscarinic receptors in a location was related to either of these enzyme activities. The amount of Che labeled prppylbenzllylchollne mustard bound was not well correlated with either the choline acetylase or the acetylcholinesterase activity there was a modest correlation between binding of the model agonist by synapcosomes and membranes prepared from nine areas of brain and the choline acetylase activities of the same areas, but It was far from precise. 

For detection of intoxication with OPr, many different analytical methods have been reported. One alternative is to measure the cholinesterase activity, because an acute intoxication is characterized by a 20% decrease in acetylcholinesterase activity. Another approach includes the determination of the unmetabolized OPi in blood or other tissues of... 

Mohammad FK, Faris GA, al-Kassim NA. A modified electrometric method for measurement of erythrocyte acetylcholinesterase activity in sheep. Vet Hum Toxicol l991 39-.337-9. 

Poisoned patients may require monitoring of vital signs, measurement of ventilatory adequacy such as blood gases and pulse oximetry, leukocyte count with differential to assess development of pneumonia, and chest radiographs to assess the degree of pulmonary edema or development of hydrocarbon pneumonitis. Workers involved in the formulation and application of pesticides should be monitored by periodic measurement of cholinesterase activity in their bloodstream. Untreated, anticholinesterase-depressed acetylcholinesterase activity returns to normal values in approximately 120 days. 

Rinne and colleagues in Finland found decreased acetylcholinesterase activity in the hippocampus in patients with mild cognitive impairment and early Alzheimer s disease. Yet, they conduded that the value of in vivo acetylcholinesterase measurements in detecting the early Alzheimer process is limited. It is not known whether destruction of the cells that make ACh is a cause or a consequence of Alzheimer s disease. 

The activities of two enzymes have been used as biomarkers of effects for OPs, namely acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase, sometimes known as pseudocholinesterase (EC 3.1.1.8). The structure and function of these enzymes has been reviewed. " In humans the former is present in red blood cells and the latter in plasma, but such distribution is not true of all species. In dogs, both enzymes are present in plasma with a ratio of butyrylcholinesterase to acetylcholinesterase of 7 1, while in the rat, plasma cholinesterase activity comprises more acetylcholinesterase with a butyrylcholinesterase to acetylcholinesterase activity of 1 3 in males and 2 1 in females in neither blood compartment are the functions of the enzymes fully understood.Because of the possibility of confusion, the terms plasma cholinesterase and erythrocyte cholinesterase as synonyms for butyrylcholinesterase and acetylcholinesterase are to be deprecated, especially when used of enzymes in animals where serious confusion may result. It is often necessary to look in detail at animal studies to see what activity has been measured in each matrix. In particular, it is necessary to look at the substrate(s) used in the assay together with any inhibitors used. Methods for measuring acetylcholinesterase have been reviewed and acetylcholinesterase and butyrylcholinesterase activities can be measured separately. In almost all cases it is the enzyme activity, rather than protein concentration, that is measured and many of the procedures used are variants of the Ellman method. Correct storage of blood samples is important as reactivation of inhibited enzymes ex vivo can occur. 

Individuals with hereditary low plasma cholinesterase levels (Kalow 1956 Lehman and Ryan 1956) and those with paroxysmal nocturnal hemoglobinuria, which is related to abnormally low levels of erythrocyte acetylcholinesterase (Auditore and Hartmann 1959), would have increased susceptibility to the effects of anticholinesterase agents such as methyl parathion. Repeated measurements of plasma cholinesterase activity (in the absence of organophosphate exposure) can be used to identify individuals with genetically determined low plasma cholinesterase. 

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