Alternative method for quantification of alfa-amylase activity / Alternativa para quantificação de alfa-amilase

A modification of the sensitive agar diffusion method was developed for macro-scale determination of alfa-amylase. The proposed modifications lower costs with the utilisation of starch as substrate and agar as supporting medium. Thus, a standard curve was built using alfa-amylase solution from Aspergillus oryzae, with concentrations ranging from 2.4 to 7,500 U.mL-1. Clear radial diffusion zones were measured after 4 hours of incubation at 20 °C. A linear relationship between the logarithm of enzyme activities and the area of clear zones was obtained. The method was validated by testing α-amylase from barley at the concentrations of 2.4; 60; 300 and 1,500 U.mL-1. The proposed method turned out to be simpler, faster, less expensive and able to determine on a macro-scale α-amylase over a wide range (2.4 to 7,500 U.mL-1) in scientific investigation as well as in teaching laboratory activities.

Modificações foram propostas ao método sensível de difusão em ágar para a macrodeterminação de alfa-amilase. As modificações propostas diminuem os custos, com a utilização de amido como substrato e ágar como meio solidificante. Assim, foi construída uma curva padrão utilizando uma solução de alfa-amilase de Aspergillus oryzae com concentrações variando de 2,4 a 7.500 U.mL-1. Em seguida, as zonas claras de difusão radial foram mensuradas depois de 4 horas de incubação a 20 °C. Foi obtida uma relação linear entre o logaritmo da atividade enzimática e os diâmetros das zonas claras. O método foi validado utilizando-se soluções de alfa-amilase de cevada nas concentrações de 2,4; 60; 300 e 1.500 U.mL-1. O método tornou-se mais simples, rápido, com baixo custo e passível de ser utilizado para macrodeterminação de alfa-amilase em ampla faixa (2,4 a 7.500 U.mL-1) na investigação científica e para fins didáticos em aulas práticas.

A re-evaluation of plasma sialic acid determination using the periodate resorcinol method versus the enzymatic method.

The concentration of plasma sialic acid was estimated using the modified chemical method and the more sensitive enzymatic method in 20 subjects with impaired glucose tolerance and 20 control subjects. The mean sialic acid concentration values of the control subjects and subjects with impaired glucose tolerance using the enzymatic method were 1.747 +/- 0.047 and 2.583 +/- 0.070 mmole/l and 1.753 +/- 0.067 and 2.591 +/- 1.02 mmole/l for the chemical method. The intra-assay coefficient of variation for the control subjects and for the subjects with impaired glucose tolerance were 1.963% and 1.583%, respectively, for the enzymatic assay and 2.728% and 2.431%, respectively, for the chemical assay. The inter-assay coefficient of variation for the control subjects and for the subjects with impaired glucose tolerance were 2.686% and 2.723% for the enzymatic assay, and 3.819% and 3.95% for the chemical assay. Since the values do not differ significantly, the chemical assay is a cost effective method that can be used in large epidemiological studies.