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Clinical Chemistry 38: 247-255, 1992;
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Clinical Chemistry, Vol 38, 247-255, Copyright © 1992 by American Association for Clinical Chemistry

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

CP Fitzpatrick and HL Pardue
Department of Chemistry, Purdue University, West Lafayette, IN 47907- 1393.

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.




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Copyright © 1992 by the American Association for Clinical Chemistry.