Simultaneous determinations of liver- and bone-type alkaline phosphatase by curve-fitting of inhibition kinetic data. II. Development and evaluation of a fluorescence-based method.
We describe a kinetic method for the determination of alkaline phosphatase (ALP) isoenzymes, based on fluorescence detection of 4-methylumbelliferone. Several different buffer-inhibitor combinations and substrate concentrations were evaluated. Best results were obtained for inhibition with 2.9 mol/L urea in amino-2-methyl-1-propanol buffer. With this combination, normal concentrations of bone- and liver-type ALP could be determined from kinetic data during an 8-min measurement period. We computed initial velocities from parameters for first-order fits during 1.2 half-lives of the response for liver-type ALP. A linear least-squares fit of initial velocities (y) determined in this way vs results obtained with a comparison procedure (x) gave good correlations. We also estimated total signal changes, delta S, from first-order fits during four half-lives. Isoenzyme content correlated well with parameters computed from the first-order fits. Values for standard errors of the estimates represent 5% and 3% of median responses for activities and isoenzyme content, respectively. When compared with an absorbance-based method described previously, this method had threefold shorter measurement times, but imprecisions were 1.6- to 1.8-fold larger.
Fitzpatrick
C P
CP
Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393.
Pardue
H L
HL
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000097:blood*; D001842:Bone and Bones / Q000201:enzymology*; D002021:Buffers; D006801:Humans; D006923:Hymecromone; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000097:blood*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011412:Propanolamines; D011786:Quality Control; D013050:Spectrometry, Fluorescence / Q000706:statistics & numerical data; D014508:Urea
D002021:Buffers; D007527:Isoenzymes; D011412:Propanolamines; D006923:Hymecromone; D014508:Urea; D000469:Alkaline Phosphatase; C006551:2-amino-2-methyl-1-propanol