Optimization studies of components in enzymatic cholesterol reagents containing cholesterol oxidase from Nocardia erythropolis, Streptomyces sp, or Pseudomonas fluorescens.
Although enzymatic methods for serum cholesterol determination are widely used in clinical laboratories, little is known about the optimization of each component in enzymatic reagents. We investigated the optimal components in the reagents containing cholesterol oxidase isolated from Nocardia erythropolis, Streptomyces sp, or Pseudomonas fluorescens. The optimal components in the reagents are: cholesterol oxidase 250 (Nocardia erythropolis), 250 (Streptomyces sp), or 300 (Pseudomonas fluorescens) U/L, cholesterol esterase 200 U/L, peroxidase 10,000 U/L, sodium cholate 3 mmol/L, 4-aminoantipyrine 0.5 mmol/L, phenol 20 mmol/L, Triton X-100 2 mL/L, and phosphate buffer, pH 7.0. Lower reaction sensitivity and lower cholesterol linearity, < 18.1 mmol/L (700 mg/dL), could be obtained by using lower components than those suggested above. Pseudomonas fluorescens were an improper source for cholesterol oxidase; either Nocardia erythropolis or Streptomyces was suitable cholesterol oxidase. We prefer using Streptomyces sp cholesterol oxidase because of its economical cost and longest reagent stability. Sodium cholate must be included in the enzymatic reagent to prevent turbidity. However, sodium cholate of > 5 mmol/ L will suppress the reaction resulting in low cholesterol linearity.
Lolekha
P H
PH
Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
Teerajetkul
Y
Y
D000675:Ampyrone / Q000737:chemistry; D000818:Animals; D002021:Buffers; D002624:Chemistry, Clinical / Q000379:methods; D002784:Cholesterol / Q000032:analysis*; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000378:metabolism; D019826:Cholic Acid; D002793:Cholic Acids / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry; D009615:Nocardia / Q000201:enzymology*; D017830:Octoxynol / Q000737:chemistry; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism; D019800:Phenol; D010636:Phenols / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D002787:Sterol Esterase / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*
D002021:Buffers; D002793:Cholic Acids; D007202:Indicators and Reagents; D010636:Phenols; D010710:Phosphates; D000675:Ampyrone; D019800:Phenol; D017830:Octoxynol; D002784:Cholesterol; D002789:Cholesterol Oxidase; D009195:Peroxidase; D002787:Sterol Esterase; D019826:Cholic Acid