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The effect of exposure to pesticides among children in a Nicaraguan community in the path of rain water runoff from a large crop-dusting airport was evaluated by measuring plasma cholinesterase. Mean cholinesterase activity in 17 children in the path of runoff was 2.4 international units/ml blood/min, lower than the 2.9 IU/ml/min measured in a group of 43 children from an unexposed community (difference=0.49 IU/ml/min; 95% C.I. 0.24, 0.76). Six (35%) of the 17 exposed children had abnormally low cholinesterase levels. A possible explanation for this physiological effect of exposure to pesticides is the dermal absorption which may have occurred among children playing barefoot in puddles grossly contaminated by runoff from the airport. Drinking water from a well in the exposed community demonstrated low level residues of cholinesterase-inhibiting pesticides, although contamination with toxaphene (not a cholinesterase inhibitor) exceeded by over 8-fold the United States Environmental Protection Agency maximum permissible concentration in drinking water. The difficulty in measuring health effects resulting from environmental pesticide contamination, and in controlling exposure resulting from the rapidly increasing use of pesticides, is a growing problem for developing countries like Nicaragua.

Cholinesterase activity was determined in extracts of muscle tissue from 28 species of fish. Flatfish and sea bass muscle were found to be particularly high in activity. Fish muscle cholinesterase from 11 high-activity species was separated from a small amount (1–10%) of acetylcholinesterase and further characterized kinetically. The K M s ranged from 0.077 to 0.93 mM using acetylthiocholine substrate, and from 0.036 to 0.323 mM using butyrylthiocholine substrate. The acetylthiocholine/butyrylthiocholine V max ratio ranged from 0.5 to 3.3. These cholinesterases were extremely sensitive to organophosphate, hypochlorite-activated organothiophosphate and carbamate pesticides.

In studies in developing countries, closed systems for mechanically mixing and loading hazardous pesticides have been shown to reduce exposure to workers. To evaluate the efficacy of closed systems in preventing worker exposure in the developing world, a cross sectional study was conducted at rural crop dusting airports in the cotton growing region of Nicaragua. Worker exposure was evaluated by measuring the activity of erythrocyte cholinesterase in the field with a new design battery operated colorimeter. The 10 mixer loaders at four airstrips with closed systems were compared with the 16 mixer loaders at four airstrips where pesticides were hand poured. Paradoxically, cholinesterase activity was 1.1 IU/ml blood (95% Cl 0.49-1.8) lower (inhibited) among workers in airstrips with closed systems than among workers hand pouring insecticides, after adjusting for weight of organophosphates sprayed in the past 14 days, and for prior training in safe use of pesticides. Mixer loaders with prior training had cholinesterase activity 0.83 IU (95% Cl 0.30-1.4) higher than untrained workers, and the weight of organophosphates sprayed was also a statistically significant predictor in the model. Unfortunately, management viewed the closed systems primarily as a production tool, rather than as a way to protect workers. Airstrips with closed systems were able to apply an average of 3250 lb organophosphates per worker in the 14 days before the survey compared with 849 lb per worker in airstrips without closed systems. Only three of 10 mixer-loaders at airstrips with closed systems had received formal training in safer use of pesticides. Because of shortage of personnel and transport, it was difficult for the responsible government agencies to train workers adequately and to enforce pesticide health and safety standards at multiple dispersed worksites.

A semiquantitative tintometric field kit has been used in the developing world for almost 30 years to measure whole blood cholinesterase levels in persons exposed to organophosphate pesticides. The validity of this screening kit was evaluated among 79 workers heavily exposed to organophosphates by comparison with a reference assay for erythrocyte cholinesterase. Overall correlation between the two methods was good. However, either sensitivity or specificity of the tintometric kit was less than 75% for each of the three tintometric categories commonly used to define the limit of normal. Because baseline erythrocyte cholinesterase levels were not available for this population, the true sensitivity and specificity of the tintometric assay may be even lower.

A cross-sectional medical survey was conducted among 63 Nicaraguan aviation mechanics exposed to organophosphate and other toxic pesticides. Thirty-one (49 percent) reported having been acutely poisoned on the job. Also, seven of 14 novice mechanics, with less than one year on the job, reported that they had been poisoned. Thirty-eight (61 percent) had cholinesterase levels below the lower limit of normal, including three workers with levels less than 20 percent of the lower limit of normal. Risk factors for low cholinesterase included recent hire and recent poisoning. Workers did not use protective equipment, nor were there facilities for bathing on site. As a result of this survey, the government has prohibited the mixing and loading of pesticides at this airport and requires the washing of planes prior to maintenance work; coveralls and thin, pesticide impermeable gloves are to be issued to mechanics handling pesticide-contaminated parts. Closed system mixing and loading systems have been installed at satellite airstrips.

We describe here a field method we developed for colorimetry of erythrocytic acetylcholinesterase (EC 3.1.1.7) in capillary blood samples. Three stable, premixed assay reagents and de-ionized water (but no centrifuge or balance) are required. This method, adapted for a microplate format, is essentially that of Ellman et al. (Biochem Pharmacol 1961;7:88-95) as modified by George and Abernethy (Clin Chem 1983;29:365-8). Assays were quantified and corrected for hematocrit by using a battery-powered colorimeter with a silicon carbide (blue) light-emitting-diode source. Advantages over existing field methods include better portability, ruggedness, greater precision, and lower cost per sample.

Incubation of trypsin with m-guanidinobenzenesulfonic acid methyl ester (mGBSOM) under mild conditions resulted in its quantitative and specific conversion to N-3-methylhistidinyl-57-trypsin (methyltrypsin). The interactions of alpha-2-plasmin inhibitor (alpha 2PI) and alpha-1-proteinase inhibitor (alpha 1PI) with the active-site modified enzymes methyltrypsin and dehydroalanyl-195-trypsin (anhydrotrypsin) were studied by thionine difference spectroscopy. For methyltrypsin the KA with alpha 1PI and alpha 2PI was 2.7 X 10(5) M-1 and 1.3 X 10(5) M-1, respectively, and with anhydrotrypsin, 7.0 X 10(3) M-1 and 3.2 X 10(5) M-1, respectively.

We describe microplate methods for measurement of human urinary albumin (HUA) by competitive enzyme-linked immunosorbant assay (ELISA) and creatinine with a modified commercial enzymatic kit. Incorporation of substrate mixing into the competitive ELISA changes the dynamic absorbance-concentration response, greatly simplifying calculations and improving sensitivity and accuracy. Measurement of creatinine in urine and plasma samples with a commercially available enzymatic kit modified for analysis by use of an inexpensive microplate reader produced values comparable in precision and accuracy to those obtained by an automated kinetic Jaffé method.

A method for the determination of the molar concentration of active tissue plasminogen activator (TPA) and urokinase (UK) has been developed. The method employs the principle of back-titration of a calibrated trypsin standard with a calibrated standard solution of a chloromethyl ketone inhibitor reactive with both trypsin and activator. Both trypsin and chloromethyl ketone standards are calibrated using a guanidinobenzoyl ester active-site titrant. Less than 20 ng of activator can be accurately determined by this method. The method is used to assay commercial samples of TPA and UK, to calculate the specific activity of such samples, and to determine kinetic constants of plasma activator inhibitor.

We have optimized Ellman-type plasma and erythrocyte cholinesterase assays for the field determination of pesticide exposure. The assays employ stable, premixed, inexpensive colorimetric reagents and a simplified kinetic measurement procedure using a novel battery-operable light-emitting-diode-source colorimeter (LEDSC). These methods were compared to a field tintometric method for cholinesterase currently in widespread use, using samples from organophosphate-intoxicated hospitalized patients. In comparison to the colorimetric method, the tintometric method was less precise and consistently underestimated the extent of pesticide exposure. Methods for the effective field determination of pesticide exposure using cholinesterase assays are critically evaluated with regard to their practical, biochemical, and medical relevance.

The reaction between plasmin and alpha 2PI (alpha-2-plasmin inhibitor), which constitutes a major regulatory step in fibrinolysis, involves both interactions between the kringle structures of plasmin and a complementary site on the inhibitor, and also between the inhibitor reactive center and the enzyme active site. An attempt was made to distinguish calorimetrically the two functional domains of alpha 2PI. Only one transition was seen, both in the DSC and UV experiments, contrary to the expected two. This transition was unaffected by K4 of plasminogen but was abolished in the presence of anhydrotrypsin. This suggests the major domain structure in alpha 2PI is associated with the reactive center.