This study investigates the chemical synthesis and physiological activities of leukotoxin (9: 10-epoxy-12-octadecenoic acid), a toxic substance known to occur naturally in polymorphonuclear leukocytes. The leukotoxin used in this study was synthesized from linoleic acid with peracetic acid and was separated from the side product of this synthesis (an isomer of leukotoxin, 12: 13-epoxy-9-octadecenoic acid) by thin layer chromatography and high performance liquid chromatography. The chemical structure of the leukotoxin was confirmed by gas chromatography-mass spectrometry. In order to examine the physiological effects of leukotoxin, this fatty acid was injected into guinea pigs. The leukotoxin initially increased blood pressure and eventually caused cardiac arrest. The results of this preliminary study indicate that leukotoxin synthesized via the above method is physiologically active and can be used for experiments investigating toxic and/or physiological effects of leukotoxin.
A new method for measuring the activity of serum pyruvate kinase (PK: EC2. 7. 1. 40) was developed by using a thermostable glucokinase (Glck) from a thermophilic bacterium, Bacillus stearothermophilus. The ATP formed by the reaction of PK is finally converted to NADPH via glucose-6-phosphate by the action of Glck and glucose-6-phosphate dehydrogenase. The change in absorbance at 340nm was found to be linear up to about 5000U/I of PK. The within-run and day-to-day coefficients of variation were1.13% at 85.4U/l and 2.08% at 45.7U/l, respectively. The influence of various coexistents and anticoagulants, such as bilirubin, ascorbate, glucose, EDTA, sodium fluoride, etc., on the assay was negligible. The reagent was stable in solution for about one month at 10°. A method for simultaneously measuring the activities of PK and creatine kinase (CK) in a single specimen was also developed. This was based on the fact that the assay conditions for both enzymes were similar. This method was found to have a high degree of precision and a good correlation with respective PK and CK assay methods. This simultaneous measurement may be useful for the accurate differential diagnosis of myocardial infarction.
A simple and sensitive method for the determination of estrogen 3-sulfates by highperformance liquid chromatography with electrochemical detection is described. The sulfates were separated on a reversed-phase column and subsequently hydrolyzed with an immobilized sulfatase reactor in a continuous flow mode. Estrogens liberated were then detected with a coulometric detector. A linear response was observed for four sulfates, estrone sulfate, 16 a -hydroxyestrone 3-sulfate, estradiol 3-sulfate and estriol 3-sulfate in the range 25-1000 ng as an injected amount. The detection limits (S/N=10) were 20 ng for estriol 3-sulfate and 16 a -hydroxyestrone 3-sulfate, and 50 ng for estrone sulfate and estradiol 3-sulfate, respectively.