An automatic flow procedure for the determination of 3-hydroxybutyrate in animal serum and plasma.
An automatic flow procedure based on the multicommutation concept, comprising three-way solenoid valves, for the spectrophotometric determination of 3-hydroxybutyrate in animal serum and plasma is proposed. The 3-hydroxybutyrate was enzymatically converted to acetoacetate with the reduction of NAD+ to NADH monitored at 340 nm. It was possible to carry out up to 600 determinations without a significant decrease in the analytical signal, with 5 mg of 3-hydroxybutyrate dehydrogenase immobilized on porous silica beads and packed in a column. The system enabled 60 determinations/h of 3-hydroxybutyrate in the range of 10-150 mg L(-1), with a consumption of 0.9 mg of NAD+ and 200 microL of sample per determination. A detection limit of 2 mg L(-1) for both animal serum and plasma and coefficients of variation of 1.4% and 1.2% (n = 17), respectively, were determined. Animal serum and plasma samples were analyzed without previous treatment, the results of which agreed with those obtained using the conventional method (UV kit, Sigma).
Pires
Cherrine K
CK
Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, PO Box 96, 13400-970, Piracicaba-SP, Brazil.
Martelli
Patrícia B
PB
Reis
Boaventura F
BF
Lima
José L F C
JL
Saraiva
M Lúcia M F S
ML
D020155:3-Hydroxybutyric Acid / Q000097:blood* / Q000378:metabolism; D000818:Animals; D002417:Cattle / Q000097:blood*; D006863:Hydrogen-Ion Concentration; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism; D008297:Male; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D012756:Sheep / Q000097:blood*; D013053:Spectrophotometry / Q000379:methods*; D013696:Temperature
D006884:Hydroxybutyrate Dehydrogenase; D020155:3-Hydroxybutyric Acid