| Research Abstract |
Increasing the sensitivity for chemiluminescence, and a chemiluminescence-FIA of serum 3-hydroxybutyrate (3-HBA) and 1,5-anhydroglucitol (1,5-AG) using a bioreactor were tried. The detection sensitivity for H_2O_2 was increased up to about 0.1 mu mol/mu 1 of sample by the introduction of a non-pulse flow pumps, an improvement of a flow cell, and intercepting the light which goes into the detecktor along the tubes from the outside For the determination of serum 3-HBA,the coimmobilized 3-hydroxybutyrate dehydrogenase (3-HBADH)/NADH oxidase (NOX) column with a weight ratio 5 : 1 was the most efficient for determining serum 3-HBA.The column hydrolyzed all the 1.5 mM 3-HBA,and showed linearity of the data up to 1.5 mM 3-HBA.Forever, the chemiluminescence intensity obtained by the column was about twice as much as those by the sequential and the mixed-bed 3-HBADH/NOX columns, because 3-HBADH and NOX in the coimmobilized 3-HBADH/NOX column were in such close proximity that both progresses proceeded quickly.
Pyranose oxidase (PROD) oxidized not only glucose but also 1,5-AG to produce H_2O_2. The oxidation rate for 1,5-AG was far slower than that for glucose, but because the column contained a large quantity of PROD,the oxidation rate for 1,5-AG increased to 70% that for glucose. When the coimmobilized glucokinase (GK)/pyruvate kinase (PK) column was used for the removal of serum glucose, GK phosphorylated not only glucose but also 1,5-AG.But, the phosphorylation rate for 1,5-AG was far lower compared with that for glucose. The most efficient column for 1,5-AG was the coimmobilized GK/PK column with a weight ratio GK 1000U : PK 0.28ml. The column removed 99.99% glucose in a sample of 1 mu 1 injected. The results from serum 1,5-AG correlated satisfactorily with those by other method.
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