Comparative study of acetylcholinesterase and butyrylcholinesterase in brain and serum of several freshwater fish: specific activities and in vitro inhibition by DDVP, an organophosphorus pesticide

A comparative study of specific activities and in vitro inhibition of brain and serum acetylcholinesterase (AChE; EC 3.1.1.7) and serum butyrylcholinesterase (BChE; EC 3.1.1.8) by DDVP, an organophosphorus pesticide, was conducted in 11 freshwater teleost species belonging to four families (Cyprinidae; common carp Cyprinus carpio, bream Abramis brama, blue bream A. ballerus, white bream Blicca bjoerkna, roach Rutilus rutilus, bleak Alburnus alburnus, ide Leuciscus idus; Percidae: perch Perca fluviatilis, pikeperch Stizostedion lucioperca; Esocidae: pike Esox lucius and Coregonidae: whitefish Coregonus albula). Specific AChE and BChE activities in brain and serum of fish were determined. Brain AChE activity varied among fish species approximately 10-fold, ranging from 192.6 to 1353.2 micromol g(-1) h(-1), respectively in perch and whitefish. All cyprinids had higher brain AChE activity than those of other fish families. Serum AChE activity was 100-fold lower than in brain. Serum BChE activity was found only in cyprinids with the exception of the common carp. It varied from 163.8 to 970.3 micromol g(-1) h(-1), respectively in roach and bleak. The bimolecular enzyme inhibition rate constants (kIIs) and pI50) values for DDVP were calculated. Sensitivity of fish AChEs both in brain and serum is similar to those of typical AChEs in mammals. The range of kIIs was 3.4-51.7 x 10(3) mol(-1) 1 min(-1) (pI50s were 5.3-6.5), respectively in white bream and ide. In contrast, fish serum BChE was more sensitive to inhibition than typical BChE and AChE in mammals. Values of kII for BChE were 1.0-2.5 x 10(7) mol(-1) 1 min(-1) (pI50 was 8.8-9.2), respectively in ide and bleak.     

Age-dependent changes in plasma and brain cholinesterase activities of house wrens and European starlings

We determined age-dependent changes in plasma and brain cholinesterase (ChE) activity for two species of passerines: house wren (Troglodytes aedon) and European starling (Sturnus vulgaris, starling). In plasma from nestlings of both species, total ChE activity increased with age, acetycholinesterase (AChE, EC 3.1.1.7) activity declined rapidly immediately after hatching, and butyrylcholinesterase (BChE, EC 3.1.1.8) activity increased steadily. For both species, total ChE and BChE activities and the BChE:AChE ratio in plasma were significantly greater in adults than nestlings suggesting trends observed in nestlings continue post fledging. In older nestlings and adults, AChE activity in plasma was significantly greater and BChE:AChE ratio less in house wrens than starlings. For house wrens as compared with starlings, ChE activity in brain increased at a significantly greater rate with age in nestlings and was significantly greater in adults. However, ChE activity in brain was similar at fledging for both species suggesting that the increase in ChE in brain is more directly related to ontogeny than chronologic age in nestlings of passerines. For both species, ChE activity increased significantly with brain weight of nestlings but not adults. House wrens hold similar patterns of age-dependent change in ChE activity in common with starlings but also exhibit differences in AChE activity in plasma that should be considered as a factor potentially affecting their relative toxicologic response to ChE inhibitors.     

Plasma B-esterase activities in European raptors

B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and Strigidae families were characterized by a BChE contribution that dominated the total ChE activity, while in the species of the Falconidae family, AChE activity dominated. With the exception of the barn owl, CbE activity (eserine-insensitive alpha-naphthyl acetate esterase [alpha-NAE] activity) in all species was almost absent or very low. The values obtained in this study for ChE, AChE, and BChE activities and the AChE:BChE ratios for buzzard, kestrel, barn owl, and tawny owl provide a good estimate of the normal values in free-living individuals of these European species. They can be used as a baseline to evaluate the effect of anticholinesterase insecticides in the field.     

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in Human Plasma and Cerebrospinal Fluid

The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120-500-fold) between total BChE activity in the CSF and the BChE-rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF--both lumbar and ventricular--was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.     

Unusual physiology of scale-less carp,Gymnocypris przewalskii,in Lake Qinghai: a high altitude alkaline saline lake

The scale-less carp (Gymnocypris przewalskii) inhabits Lake Qinghai located on the Qinghai-Tibet plateau (elevation, 3200 m) in western China. The lake waters are alkaline (pH 9.4, titratable alkalinity=30 mmol l(-1)), Mg(2+)-rich (18.7 mmol l(-1)), Ca(2+)-poor (0.30 mmol l(-1)) and saline (9 per thousand ). These fish make annual spawning migrations into freshwater rivers. We investigated the physiology of nitrogen excretion and ionoregulation of fish from the lake and river. Fish from both waters were ammonotelic, although ammonia-N excretion rates were lower in lake fish (175 vs. 344 micromol kg(-1) h(-1), P<0.05) resulting in unusually high levels of ammonia in blood plasma (2.23 vs. 0.32 mmol l(-1)), bile, liver, muscle and brain. Exposure to 0.4 mmol l(-1) total ammonia in lake water ([NH(3)]=0.16 mmol l(-1)) killed fish within 8 h. River fish survived exposure to 1.0 mmol l(-1) total ammonia in river water at pH 8.0 ([NH(3)]=0.023 mmol l(-1)) for 24 h suggesting high ammonia tolerance in lake fish. High glutamate dehydrogenase and glutamine synthetase activities in tissues probably allow the fish to alleviate ammonia toxicity by amino acid accumulation. Neither lake nor river fish relied on urea excretion to remove excess N. Urea-N excretion rates were below 20 micromol kg(-1) h(-1) for both groups, and levels of urea in plasma and tissues were moderate. When exposed to elevated ammonia, urea-N excretion increased slightly (approximately 50 micromol kg(-1) h(-1)) and liver and muscle urea levels increased in the river fish. Plasma ion levels were within the range typical of cyprinids, but river fish had significantly higher plasma [Na(+)] and [Cl(-)] and lower [K(+)] than fish from the lake. During 48-h lake-to-river water transfer, plasma Na(+) and Cl(-) levels rose significantly. Significantly higher Na(+)/K(+)-ATPase activity in the gills of river fish may be related to the higher plasma ion levels. Plasma [Mg(2+)] and [Ca(2+)] were tightly regulated despite the great differences in the lake and river water levels.     

Cerebrospinal fluid gradients of acetylcholinesterase and butyrylcholinesterase activity in healthy aging

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in 13 sequential 2 ml aliquots of cerebrospinal fluid (CSF) obtained by lumbar puncture from 7 young and 7 elderly healthy normal subjects. The slopes of the rostrocaudal gradients of AChE and BChE were calculated and compared to those of total protein concentration and the major dopaminergic metabolite homovanillic acid (HVA), for which a pronounced rostrocaudal gradient (with highest concentrations of HVA in more rostral CSF) is consistent with HVA originating primarily from the brain. AChE activity was higher in more caudal fractions of young, but not elderly subjects and there was a significant difference between the mean AChE gradient slopes in the young and old groups. These results suggest that the spinal cord makes an important contribution to AChE activity in lumbar CSF. Furthermore, the absence of a negative AChE gradient in elderly subjects may be the result of a greater rate of entry of cerebral AChE into CSF, possibly as a consequence of an increased ventricular surface area and shorter diffusion distances in atrophic elderly brains. In contrast to AChE, BChE activity and total protein concentrations were higher in more caudal CSF fractions of not only young but also old subjects. In addition, there was a significant correlation between the gradient slopes of BChE activity and total protein concentrations, suggesting that the majority of BChE activity in lumbar CSF derives from the same source as the majority of total protein, namely plasma. The diffuse (i.e. brain and spinal cord) origin of AChE in lumbar CSF would explain the relatively modest changes in lumbar CSF AChE activity in diseases involving certain central cholinergic systems, most notably Alzheimer's disease.     

Organophosphorus Compound O,O-Dimethyl-O-(2,2-dichlorovynyl)-Phosphate as Selective Inhibitor for Separate Determination of Acetylcholinesterase and Butyrylcholinesterase Activities in the Roach Rutilus rutilus

A comparative study is carried out on dependence of degree of activity inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) of a freshwater bony fish, the roach Rutilus rutilus L., on concentration of organophosphates: O,O-dimethyl-O-(2,2-dichlorovynyl)phosphate (DDVP) and tetraisopropylamidopyrophosphate (iso-OMPA). It has been shown that both in roach and in horse the both inhibitors are selective for BChE in comparison with AChE. Their selectivity degree was 2000-fold and 80-fold, respectively. The ranges of effective DDVP concentrations are overlapped for horse AChE and BChE, while they do not for the roach enzymes. A similar regularity is revealed at action of iso-OMPA. It is established that DDVP has a higher inhibitory potency and selectivity in relation to roach BChE, than iso-OMPA. It is suggested to use DDVP as a new selective inhibitor for separate evaluation of AChE and BChE activities in fish tissues.     

Inherited and acquired interactions between ACHE and PON1 polymorphisms modulate plasma acetylcholinesterase and paraoxonase activities

The 5.5 Mb chromosome 7q21-22 ACHE/PON1 locus harbours the ACHE gene encoding the acetylcholine hydrolyzing, organophosphate (OP)-inhibitable acetylcholinesterase protein and the paraoxonase gene PON1, yielding the OP-hydrolyzing PON1 enzyme which also displays arylesterase activity. In search of inherited and acquired ACHE-PON1 interactions we genotyped seven polymorphic sites and determined the hydrolytic activities of the corresponding plasma enzymes and of the AChE-homologous butyrylcholinesetrase (BChE) in 157 healthy Israelis. AChE, arylesterase, BChE and paraoxonase activities in plasma displayed 5.4-, 6.5-, 7.2- and 15.5-fold variability, respectively, with genotype-specific differences between carriers of distinct compound polymorphisms. AChE, BChE and arylesterase but not paraoxonase activity increased with age, depending on leucine at PON1 position 55. In contrast, carriers of PON1 M55 displayed decreased arylesterase activity independent of the - 108 promoter polymorphism. Predicted structural consequences of the PON1 L55M substitution demonstrated spatial shifts in adjacent residues. Molecular modelling showed substrate interactions with the enzyme variants, explaining the changes in substrate specificity induced by the Q192R substitution. Intriguingly, PON1, but not BChE or arylesterase, activities displayed inverse association with AChE activity. Our findings demonstrate that polymorphism(s) in the adjacent PON1 and ACHE genes affect each other's expression, predicting for carriers of biochemically debilitating ACHE/PON1 polymorphisms adverse genome-environment interactions.     

Nippostrongylus brasiliensis: Infection decreases plasma butyrylcholinesterase activity in rats

Plasma butyrylcholinesterase (BChE) hydrolyzes ester-containing compounds such as succinylcholine, as well as acting as a scavenger against neurotoxic organophosphates (OPs). We previously found that Nippostrongylus brasiliensis infection makes rats more susceptible to OP toxicity by decreasing serum paraoxonase-1 (PON1) activity. In the present study, we examined the effects of N.brasiliensis infection on acetylcholinesterase (AChE) activity in plasma, red blood cells (RBCs), brain and diaphragm, as well as serum PON1 activity, in rats at day 7 after infection. N.brasiliensis infection significantly decreased plasma BChE and PON1 activities without significantly altering AChE activity in RBCs, brain and diaphragm. These results provide further insight into the unusual deleterious effects of intestinal nematode infections on body homeostasis.     

Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants

Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.     

Quantitative Development and Molecular Forms of Acetyl-and Butyrylcholinesterase During Morphogenesis and Synaptogenesis of Chick Brain and Retina

The embryonic development of total specific activities as well as of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and of butyrylcholinesterase (BChE, EC 3.1.1.8) have been studied in the chick brain. A comparison of the development in different brain parts shows that cholinesterases first develop in diencephalon, then in tectum and telencephalon; cholinesterase development in retina is delayed by about 2-3 days; and the development in rhombencephalon [not studied until embryonic day 6 (E6)] and cerebellum is last. Both enzymes show complex and independent developmental patterns. During the early period (E3-E7) first BChE expresses high specific activities that decline rapidly, but in contrast AChE increases more or less constantly with a short temporal delay. Thereafter the developmental courses approach a late phase (E14-E20), during which AChE reaches very high specific activities and BChE follows at much lower but about parallel levels. By extraction of tissues from brain and retina in high salt plus 1% Triton X-100, we find that both cholinesterases are present in two major molecular forms, AChE sedimenting at 5.9S and 11.6S (corresponding to G2 and G4 globular forms) and BChE at 2.9S and 10.3S (G1 and G4, globular). During development there is a continuous increase of G4 over G2 AChE, the G4 form reaching 80% in brain but only 30% in retina. The proportion of G1 BChE in brain remains almost constant at 55%, but in retina there is a drastic shift from 65% G1 before E5 to 70% G4 form at E7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phenytoin on acetylcholinesterase activity and cell protein in cultured chick embryonic skeletal muscle

Cultured pectoral muscle from 11-day-old chick embryos was treated for 48 h with phenytoin (diphenylhydantoin, DPH) in concentrations ranging from 15 to 270 microgram/ml on days 7-9 in vitro. Acetylcholinesterase (AChE, EC 3.1.1.7), creatine phosphokinase (CPK, EC 2.7.3.2), and lactic dehydrogenase (LDH, EC 1.1.1.27) activities, [3H]leucine incorporation into protein, and total protein of the cultures decreased in a dose-related manner with DPH concentrations of 30 microgram/ml and greater. Total AChE activity and AChE activity released into the medium were specifically decreased with 15 microgram DPH per millilitre. In cultures treated chronically with 15 microgram DPH per millilitre on days 5-13 in vitro, total AChE activity and AChE activity released into the medium were 66.0 +/- 13.2 and 64.7 +/- 11.8% of untreated controls, respectively, but cellular AChE activity, cell protein, and [3H]leucine incorporation into protein were unaffected. The results indicate that DPH specifically decreases the total net synthesis of AChE activity by a direct action on cultured chick embryo muscle.     

Changes in acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease resemble embryonic development--a study of molecular forms.

The pattern of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) separated by density gradient centrifugation was investigated in the brain and cerebrospinal fluid in Alzheimer's disease (AD), in human embryonic brain and in rat brain after experimental cholinergic deafferentation of the cerebral cortex. While a selective loss of the AChE G4 form was a rather constant finding in AD, a small but significant increase of G1 for both AChE and BChE was found in the most severely affected cases. Both in normal human brain and in AD a significant relationship could be established between the AChE G4/G1 ratio in different brain regions and the activity of choline acetyltransferase (ChAT). A similar decrease of the AChE G4 form as observed in AD can be induced in rat by experimental cholinergic deafferentation of the cerebral cortex. The increase in G1 of both AChE and BChE in different brain regions in AD is quantitatively related to the local density of neuritic plaques which are histochemically reactive for both enzymes. In human embryonic brain, a high abundance of G1 and a low G4/G1 ratio for both AChE and BChE was found resembling the pattern observed in AD. Furthermore, both in embryonic brain and in AD AChE shows no substrate inhibition which is a constant feature of the enzyme in the adult human brain. It is, therefore, concluded that the degeneration of the cholinergic cortical afferentation in AD as reflected by a decrease of AChE G4 is accompanied by the process of a neuritic sprouting response involved in plaque formation which is probably associated with the expression of a developmental form of the enzyme.     

Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse

We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.     

Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity

Central cholinergic systems are involved in a plethora of brain functions and are severely and selectively damaged in neurodegenerative diseases such as Alzheimer's disease and dementia with Lewy bodies. Cholinergic dysfunction is treated with inhibitors of acetylcholinesterase (AChE) while the role of butyrylcholinesterase (BChE) for brain cholinergic function is unclear. We have used in vivo microdialysis to investigate the regulation of hippocampal acetylcholine (ACh) levels in mice that are devoid of AChE (AChE-/- mice). Extracellular ACh levels in the hippocampus were 60-fold elevated in AChE-/- mice compared with wild-type (AChE+/+) animals. In AChE-/- mice, calcium-free conditions reduced hippocampal ACh levels by 50%, and infusion of tetrodotoxin by more than 90%, indicating continuous ACh release. Infusion of a selective AChE inhibitor (BW284c51) caused a dose-dependent, up to 16-fold increase of extracellular ACh levels in AChE+/+ mice but did not change ACh levels in AChE-/- mice. In contrast, infusion of a selective inhibitor of BChE (bambuterol) caused up to fivefold elevation of ACh levels in AChE-/- mice, but was without effect in AChE+/+ animals. These results were corroborated with two other specific inhibitors of AChE and BChE, tolserine and bis-norcymserine, respectively. We conclude that lack of AChE causes dramatically increased levels of extracellular ACh in the brain. Importantly, in the absence of AChE, the levels of extracellular ACh in the brain are controlled by the activity of BChE. These results point to a potential usefulness of BChE inhibitors in the treatment of central cholinergic dysfunction in which brain AChE activity is typically reduced.     

Neospora caninum: Activity of cholinesterases during the acute and chronic phases of an experimental infection in gerbils

Neosporosis is an infectious disease primarily of dogs and cattle which has been found in many countries around the world. Neospora caninum causes an important immune response (cellular and humoral) in animals that it infects. Since the participation of the cholinergic system in the immune response is well documented, the aim of this study was to evaluate the relationship between N. caninum infection and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) during the acute and chronic phase of infection. For that, tachyzoites of N. caninum (Nc-1 strain) were inoculated intraperitoneally in gerbils (Meriones unguiculatus), which were separated in two experiments, I and II, differing in infective doses of tachyzoites, aiming to reach an acute phase as well as chronic phase, respectively. Samples were collected on day 7 post infection (PI) for Experiment I and on days 15 and 30 PI for Experiment II. AChE activity was evaluated on whole blood and brain, while BChE was evaluated in plasma. On day 7 a reduction of AChE in total blood and brain was observed, along with reduction of BChE in plasma of infected animals when compared with non-infected. In Experiment II, AChE activity increased in total blood on day 30 PI; however, maintaining, during the same period, the AChE activity has a reduced in brain tissue. BChE activity was significantly increased on day 30 PI. Based on the results obtained, it was possible to observe a response of the cholinergic system, providing different grades of AChE and BChE activities, in response to the acute and chronic infection of gerbils experimentally infected with N. caninum. These results will serve as initial points to further studies of our research group about the relationship between the infection/disease and the cholinergic system.     

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in the Aged Human Central Nervous System

The distribution of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) molecular forms and their solubility characteristics were examined, using density gradient centrifugation, in various regions of the postmortem human CNS. Total AChE activity varied extensively (50-fold) among the regions investigated, being highest in the telencephalic subcortical structures (caudate nucleus and nucleus of Meynert); intermediate in the substantia nigra, cerebellum, and spinal cord; and least in the fornix and cortical regions (hippocampus and temporal and parietal cortex). Total BChE activity was, in contrast, much more evenly distributed, with only a threefold variation between the regions studied. Although the patterns of molecular forms of each enzyme were broadly similar among the different areas, regional variations in the distribution and abundance of the various forms of AChE were much greater than those of BChE. Thus, although the tetrameric G4 form of AChE constituted the majority of the total AChE activity in all regions examined, the ratio of the G4 form to the monomeric G1 form, the latter of which constituted the majority of the remaining activity, varied markedly, ranging from 21 in the caudate nucleus to 1.7 in the temporal cortex. In addition to the G4 and G1 forms of AChE, the dimeric G2 form was observed in the nucleus of Meynert and a fast-sedimenting (16S) species was found in samples of both the parietal cortex and spinal cord. In contrast, the G4 and G1 forms of BChE were the only molecular species observed in the different areas and the G4:G1 ratio varied from 3.3 in the substantia nigra to 0.9 in the temporal cortex. Regarding the solubility characteristics of the individual AChE and BChE molecular forms, the majority of the G4 form of AChE was extractable only in the presence of detergent, indicating a predominantly membrane-bound localization of this species. The smaller AChE forms (G1 and G2) and both the G1 and G4 forms of BChE were all relatively evenly distributed between soluble and membrane-bound species. These findings are discussed in relation to neurochemical and neuroanatomical, particularly cholinergic, features of the regions examined.     

Comparison of methods used for the determination of cholinesterase activity in whole blood

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20muM ethopropazine and 500nM (-) huperzine A can be used for measuring AChE and BChE activities across species.     

Interactions of butane, but-2-ene or xylene-like linked bispyridinium para-aldoximes with native and tabun-inhibited human cholinesterases

Kinetic parameters were evaluated for inhibition of native and reactivation of tabun-inhibited human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) and human plasma butyrylcholinesterase (BChE, EC 3.1.1.8) by three bispyridinium para-aldoximes with butane (K074), but-2-ene (K075) or xylene-like linker (K114). Tested aldoximes reversibly inhibited both cholinesterases with the preference for binding to the native AChE. Both cholinesterases showed the highest affinity for K114 (K(i) was 0.01mM for AChE and 0.06mM for BChE). The reactivation of tabun-inhibited AChE was efficient by K074 and K075. Their overall reactivation rate constants were around 2000min(-1)M(-1), which is seven times higher than for the classical bispyridinium para-aldoxime TMB-4. The reactivation of tabun-inhibited AChE assisted by K114 was slow and reached 90% after 20h. Since the aldoxime binding affinity of tabun-inhibited AChE was similar for all tested aldoximes (and corresponded to their K(i)), the rate of the nucleophilic displacement of the phosphoryl-moiety from the active site serine was the limiting factor for AChE reactivation. On the other hand, none of the aldoximes displayed a significant reactivation of tabun-inhibited BChE. Even after 20h, the reactivation maximum was 60% for 1mM K074 and K075, and only 20% for 1mM K114. However, lower BChE affinities for K074 and K075 compared to AChE suggest that the fast tabun-inhibited AChE reactivation by these compounds would not be obstructed by their interactions with BChE in vivo.     

Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase

As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 microg doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48h following the final and highest (400 microg) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30-40% after 100 microg to >50% at 400 microg, and peaking 1.5h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5h after the final 400 microg dose (5.47+/-2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected). Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C(18) reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning.