Cholinesterases from plasma

In vitro studies have shown that tetrachlorvinphos is not mutagenic in bacteria and is a weak inducer of chromosomal aberrations in human lymphocytes. Studies with plasma and erythrocyte cholinesterases from different species have revealed that the... 

Benzoylcholine has been shown to be hydrolyzed by the plasma cholinesterases from many species, but not to be hydrolyzed by acetylcholinesterase. It is important to check whether the plasma of a given species does hydrolyze benzoylcholine before assuming that the plasma of that species has negligible enzymic activity in general. Acetyl-/3-methylcholine is, in contrast, readily hydrolyzed by aeetylcholinesterase but very slowly by the plasma enzyme (M12). These two substrates may therefore be used in complementary ways to characterize a given enzyme. 

Augustinsson (A25) studied the cholinesterase activities of the plasmas of many vertebrate species. Subsequent workers have confirmed that the highest activity of the enzyme is found in equine and human plasma or serum. These sources have been used consequently by most workers as starting materials for the purification of cholinesterase. Outdated plasma from national blood banks is valuable for large-scale preparations of the human enzyme. 

Fig. 1. Elution pattern of human plasma cholinesterase from DEAE-cellulose column solid line, absorbance at 280 nm broken line, enzyme activity. Partially purified enzyme preparation was placed on a DEAE-cellulose column (1.2 x 10 cm) and equilibrated with 0.02 mol/liter acetate buffer, pH 6.0. The column was washed with the same buffer containing 0.06 mmol/liter sodium chloride (up to 400 ml of effluent). Then (at single arrow) the enzyme was eluted with a linear gradient of sodium chloride and choline chloride ranging in concentration from 0.06 mmol/liter NaCl to 0.03 mmol/liter NaCl + 0.03 mol/liter choline chloride. At the double arrow, the buffer was changed to 0.02 mol/liter acetate buffer, pH 6.0, containing 0.2 mol/liter NaCl flow rate 25 ml per hour. (After Yoshida, Y2.)...

It has already been mentioned that there are some doubts A26) about the existence of an anionic site in human or horse cholinesterase. Comparative kinetic studies using a series of pyridylcarbinol acetates as substrates have shown that acetylcholinesterase from T. marmorata electric organ and the plasma cholinesterases from horse and man have similar esteratic sites. It was also shown that the electric eel organ enzyme has an anionic site, whereas the second site of butyrylcholine... 

Fig. 6. Autoradiogram of diisopropyl fluorophosphate-labeled tryptic peptides of normal and variant plasma cholinesterases U, enzyme from plasma of genotype EYEY A, enzyme from plasma of genotype EfEf UA, enzyme from plasma of genotype EYEY AS, enzyme from plasma of genotype EfEl and Cs, enzyme from Q positive plasma with usual kinetic properties. Electrophoresis was carried out in silica gel sheets with pyridine-acetate buffer (10% acetic acid adjusted to pH 3.5 with p)nidine) at 40 V/cm for 3 hours. (After Muensch et al., M21.)...

Recent investigations of the reactivation of OP-inhibited CaE have suggested that it may be possible to increase its potential as an OP scavenger by exploiting its turnover of OP compounds. Maxwell et al. observed that OP-inhibited CaE does not undergo the aging process that prevents oxime reactivation of OP-inhibited cholinesterases, while Jokanovic et al. found that OP-inhibited CaE from plasma... 

Neurological effects related to cholinesterase depression occurred in seven children acutely exposed to methyl parathion by inhalation as well as orally and dermally (Dean et al. 1984). The children were admitted to a local hospital with signs and symptoms of lethargy, increased salivation, increased respiratory secretions, and miosis. Two of the children were in respiratory arrest. Two children died within several days of each other. All of the children had depressed plasma and erythrocyte cholinesterase levels (Table 3-2). These effects are similar to those occurring in methyl parathion intoxication by other routes (see Sections 3.2.2.4 and 3.2.3.4). Three adults exposed in the same incident had normal plasma (apart from one female) and red blood cell cholinesterase, and urinary levels of 4-nitrophenol (0.46-12.7 ppm) as high as some of the ill children. 

Neurologic signs did not occur over a 30-day period in male prisoner volunteers in California who ingested daily doses of methyl parathion ranging from 1.0 to 19 mg. There were no uniform changes in plasma or erythrocyte cholinesterase levels at any of these doses (Rider et al. 1969). By increasing concentrations of methyl parathion administered to the same experimental population and using the same protocol, a dose that inhibited cholinesterase values was established. These additional studies were published nearly 20 years ago in abstract form only therefore, they are not discussed in this section. 

There is a second type of cholinesterase called butyrylcholinesterase, pseudocholinesterase, or cholinesterase. This enzyme is present in some nonneural cells in the central and peripheral nervous systems as well as in plasma and serum, the liver, and other organs. Its physiologic function is not known, but is hypothesized to be the hydrolysis of esters ingested from plants (Lefkowitz et al. 1996). Plasma cholinesterases are also inhibited by organophosphate compounds through irreversible binding this binding can act as a detoxification mechanism as it affords some protection to acetylcholinesterase in the nervous system (Parkinson 1996 Taylor 1996). 

Released ACh is broken down by membrane-bound acetylcholinesterase, often called the true or specific cholinesterase to distinguish it from butyrylcholinesterase, a pseudo-or non-specific plasma cholinesterase. It is an extremely efficient enzyme with one molecule capable of dealing with something like 10000 molecules of ACh each second, which means a short life and rapid turnover (100 ps) for each molecule of ACh. It seems that about 50% of the choline freed by the hydrolysis of ACh is taken back into the nerve. There is a wide range of anticholinesterases which can be used to prolong and potentiate the action of ACh. Some of these, such as physostigmine, which can cross the blood-brain barrier to produce central effects and neostigmine, which does not readily... 

The initial enthusiasm for tacrine and velnacrine, which are the anticholinesterases most studied clinically, has been tempered by the fact that not all patients respond. Most show the peripheral parasympathomimetic effects of cholinesterase inhibition, e.g. dyspepsia and diarrhoea, as well as nausea and vomiting, and about half of the patients develop hepatotoxicity with elevated levels of plasma alanine transaminase. While some peripheral effects can be attenuated with antimuscarinics that do not enter the brain, these add further side-effects and the drop-out rate from such trials is high (<75%) in most long-term studies. Donepezil appears to show less hepatotoxicity but its long-term value remains to be determined. 

However, there are some data on interactions of phosphate esters with other compounds. Cocaine undergoes metabolism by three major routes one of these routes involves hydrolysis by liver and plasma cholinesterases to form ecgonine methyl ester. It has been suggested that cocaine users with serious complications tend to have lower plasma cholinesterase levels. Thus, it is possible that individuals with decreased plasma cholinesterase levels (such as resulting from organophosphate ester exposure) may be highly sensitive to cocaine (Cregler and Mark 1986 Hoffman et al. 1992). However, there are no experimental data to support this hypothesis. 

Based on the data from controlled human studies, the NOEL for plasma cholinesterase inhibition for a single dose of chlorpyrifos is between 0.1 and 0.5 mg/kg bw/day, and the more conservative 0.1 mg/kg bw/day (100 pg/kg bw/day) is used in this assessment as the acute NOEL for chlorpyrifos. The repeated dose NOEL in humans is 0.03 mg/kg bw/day (30 pg/kg bw/day), based on plasma cholinesterase activity, and this is the basis for the establishment of the reference dose of 0.003 mg/kg bw/day (3 pg/kg bw/day) used by the EPA in assessing dietary risk to chlorpyrifos. For the work described here, both NOELs are used as bases for assessing risks to persons who have the potential for non-dietary exposure to chlorpyrifos. For exposures that are infrequent or of short duration, the 100 pg/kg bw/day NOEL is assumed to be the more appropriate value, and the lower 30 pg/kg bw/day will be used in those situations in which exposure may be considered to be more frequent. ... 

The results from using the Student s f-test for a distributional analysis are presented in Table 4. These results indicate the probability of a given worker in the listed scenario exceeding the NOEL of the toxicity endpoint. The probability of exceeding the LOEL and of thus experiencing a depression of plasma cholinesterase activity is not given (except for chronic exposure scenarios in the "100 ug/kg bw/day" column). Hence, even these probabilities may be considered to be conservative and not fully representative of the probability of a worker actually experiencing a toxic effect. 

Blood specimens of approximately 5 mL were collected on two separate days during the week preceding the study. Additional blood specimens of approximately 5 mL each were collected approximately 24 and 48 hr after the start of the study. These blood specimens were drawn and assayed for plasma cholinesterase activity by personnel from the Michigan Division Medical Department of The Dow Chemical Company. 

Pharmacologically, carbofuran inhibits cholinesterase, resulting in stimulation of the central, parasympathetic, and somatic motor systems. Sensitive biochemical tests have been developed to measure cholinesterase inhibition in avian and mammalian brain and plasma samples and are useful in the forensic assessment of carbamate exposure in human and wildlife pesticide incidents (Bal-lantyne and Marrs Hunt and Hooper 1993). Acute toxic clinical effects resulting from carbofuran exposure in animals and humans appear to be completely reversible and have been successfully treated with atropine sulfate. However, treatment should occur as soon as possible after exposure because acute carbofuran toxicosis can be fatal younger age groups of various species are more susceptible than adults (Finlayson et al. 1979). Carbofuran labels indicate that application is forbidden to streams, lakes, or ponds. In addition, manufacturers have stated that carbofuran is poisonous if swallowed, inhaled, or absorbed through the skin. Users are cautioned not to breathe carbofuran dust, fumes, or spray mist and treated areas should be avoided for at least 2 days (Anonymous 1971). Three points are emphasized at this juncture. First, some carbofuran degradation... 

A comprehensive approach to a states response to a chemical terrorism includes having a plan not only for the crisis and consequence management phases of the incident, but also for all elements required for complete resolution of the event. This may include the necessity to definitively establish whether chemical agents were used, to provide supporting evidence to confirm other analyses, or to provide the forensic proof required to support a criminal prosecution. The collection and analysis of biomedical samples - blood, urine or other tissue from affected humans or animals - is one of the means for providing such information. Although current capabilities such as urinary thiodyglycol excretion or plasma cholinesterase activity can be performed, there is scope for far more sensitive and specific assessments that overcome the limitations of these approaches. 

The maximum level of HMMA in the urine occurred 72 hours after exposure, which coincides with the time period for maximum urine catecholamine levels. There was a direct relationship between blood cholinesterase inhibition and catecholamine (adrenaline and noradrenaline) levels in the urine and blood (Brzezinski and Ludwicki 1973). Maximum inhibition of cholinesterase activity and maximum plasma catecholamine occurred during the first I-2 hours after exposure. However, catecholamine levels returned to normal more rapidly than cholinesterase activity. It was proposed that high levels of acetylcholine, which are normally associated with cholinesterase activity inhibition, caused a release of catecholamines from the stores in the adrenals. 

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