A new inhibitive heavy metals determination method using trypsin has been developed. The enzyme was assayed using the casein-Coomassie-dye-binding method. In the absence of inhibitors, casein was hydrolysed to completion and the Coomassie-dye was unable to stain the protein and the solution became brown. In the presence of metals, the hydrolysis of casein was inhibited and the solution remained blue. The bioassay was able to detect zinc and mercury with IC50 (concentration causing 50% inhibition) values of 5.78 and 16.38 mg l(-1) respectively. The limits of detection (LOD), for zinc and mercury were 0.06 mg l(-1) (0.05-0.07, 95% confidence interval) and 1.06 mg l(-1) (1.017-1.102, 95% confidence interval), respectively. The limits of quantitation (LOQ) for zinc and mercury were 0.61 mg l(-1) (0.51-0.74 at a 95% confidence interval) and 1.35 mg l(-1) (1.29-1.40 at a 95% confidence interval), respectively. The IC50 value for zinc was much higher than the IC50 values for papain and Rainbow trout, but was within the range of Daphnia magna and Microtox. The IC50 value for zinc was only lower than those for immobilized urease. Other toxic heavy metals, such as lead, silver arsenic, copper and cadmium, did not inhibit the enzyme at 20 mg l(-1). Using this assay we managed to detect elevated zinc concentrations in several environmental samples. Pesticides, such as carbaryl, flucythrinate, metolachlor glyphosate, diuron, diazinon, endosulfan sulphate, atrazine, coumaphos, imidacloprid, dicamba and paraquat, showed no effect on the activity of trypsin relative to control (One-way ANOVA, F(12,26)= 0.3527, p> 0.05). Of the 17 xenobiotics tested, only (sodium dodecyl sulphate) SDS gave positive interference with 150% activity higher than that of the control at 0.25% (v/v).
Serum samples were obtained from 43 children 14 years old or younger in Malaysia and Guatemala. The levels of the serum glycoprotein alpha 2-macroglobulin (alpha 2-M) were assayed by two methods: the trypsin-binding assay of Ganrot (Clin. Chim. Acta 14:493, 1960) and a radial immunodiffusion assay against alpha 2-M antiserum. The two methods gave the same results. When serum alpha 2-M levels were plotted against serum vitamin A concentrations, they were significantly correlated (r = 0.505, P less than 0.001); children with serum vitamin A levels greater than 40 micrograms/100 ml had alpha 2-M levels of 3.71 +/- 0.79 mg/ml (mean +/- SD, n = 13), while those with level less than 40 micrograms/100 ml had alpha 2-M levels of 2.78 +/- 0.51 mg/ml (n = 30); the difference was significant (P less than 0.001). Normal, apparently healthy children had alpha 2-M levels of 3.90 +/- 0.39 mg/ml. Most of the children sampled suffered from a variety of infections; of these, measles appeared to counteract the effect of vitamin A deficiency by elevating alpha 2-M levels. Vitamin A-deficient children with measles had alpha 2-M levels not significantly lower than those of normal children. The difference between deficient and normal values of alpha 2-M was still significant (P less than 0.05) when expressed per milligram of serum protein, showing that the effect was not caused by lowered serum protein concentrations associated with protein-calorie malnutrition, from which most of the deficiency children suffered.
Alpha-1 antitrypsin (AAT) augmentation therapy involves infusion of plasma-purified AAT to AAT deficient individuals. Whether treatment affects microRNA expression has not been investigated. This study's objectives were to evaluate the effect of AAT augmentation therapy on altered miRNA expression in monocytes and investigate the mechanism. Monocytes were isolated from non-AAT deficient (MM) and AAT deficient (ZZ) individuals, and ZZs receiving AAT. mRNA (qRT-PCR, microarray), miRNA (miRNA profiling, qRT-PCR), and protein (western blotting) analyses were performed. Twenty one miRNAs were differentially expressed 3-fold between ZZs and MMs. miRNA validation studies demonstrated that in ZZ monocytes receiving AAT levels of miR-199a-5p, miR-598 and miR-320a, which are predicted to be regulated by NFκB, were restored to levels similar to MMs. Validated targets co-regulated by these miRNAs were reciprocally increased in ZZs receiving AAT in vivo and in vitro. Expression of these miRNAs could be increased in ZZ monocytes treated ex vivo with an NFκB agonist and decreased by NFκB inhibition. p50 and p65 mRNA and protein were significantly lower in ZZs receiving AAT than untreated ZZs. AAT augmentation therapy inhibits NFκB and decreases miR-199a-5p, miR-598 and miR-320a in ZZ monocytes. These NFκB-inhibitory properties may contribute to the anti-inflammatory effects of AAT augmentation therapy.