Comparison of different diluents and chromophores for spectrophotometric determination of livestock blood cholinesterase activity.
Effects of seven different blood diluents (distilled water, Triton X-100, saponin, isotonic saline solution, pH 7.5 and 8 phosphate buffers and bovine serum albumin) and two chromophores: 5, 5'-dithiobis 2-nitrobenzoic acid (DTNB) and 2,2'-dithiodipyridine (2- PDS) on blood cholinesterase determination in four domestic species (cow, sheep, goat and horse) are described and compared. Haemolytic diluents (distilled water, Triton X-100 and saponin) gave the best precision results when fresh blood was assayed. However, Triton X-100 induced lower ChE activity values in horses, and saponin yielded very high backgrounds in all species tested; so distilled water was recommended as diluent for fresh blood cholinesterase determination. In frozen samples all diluents (except Triton X-100) gave homogeneous final ChE results and showed good between-run precision. Use of 2- PDS as chromophore allowed to do kinetic measurements with approximately 1/3 less haemoglobin interference than when DTNB was employed. This fact allows the use of more concentrated whole blood samples, improving measurements accuracy and decreasing the possible reactivation of inhibited ChE. On the basis of these results, distilled water as diluent and 2- PDS as chromophore are recommended for ChE determination in whole blood.
Cerón
J J
JJ
Department of Animal Pathology, University of Murcia. 30100 Murcia, Spain.
Tecles
F
F
Espín
J C
JC
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives; D000818:Animals; D000829:Animals, Domestic / Q000097:blood*; D002021:Buffers; D002417:Cattle; D002802:Cholinesterases / Q000097:blood*; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D006041:Goats; D006736:Horses; D017830:Octoxynol; D012503:Saponins; D012756:Sheep; D013054:Spectrophotometry, Atomic; D013439:Sulfhydryl Reagents; D014867:Water
D002021:Buffers; D004220:Disulfides; D012503:Saponins; D013439:Sulfhydryl Reagents; D014867:Water; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D017830:Octoxynol; D004228:Dithionitrobenzoic Acid; D002802:Cholinesterases