Fluorometric measurement of 5-aminolevulinic acid in serum

Background: Measurement of 5-aminolevulinic acid (ALA) in serum is potentially useful in acute porphyrias, lead poisoning and hereditary tyrosinemia. Because levels of ALA in serum are about 100 times less than in urine, a highly sensitive method is required. We describe a simple and sensitive fluorometric method that does not require HPLC. Methods: ALA is separated from serum using a cation-exchange resin (DOWEX 50WX8-400), followed by addition of acetylacetone and formaldehyde to produce a fluorescent ALA derivative by the Hantzsch reaction. The fluorescence was measured at an excitation wavelength of 370 nm, and the emission peak was near 463 nm. Results were compared with measurements of ALA by a published HPLC method on the same samples. Results: The fluorometric method was linear for ALA concentrations up to 500 μg/l with a detection limit of about 8.7 μg/l. Within-run variations (N=8) at 25 and 100 μg/l ALA were 3.7% and 3.1%, respectively, and day-to-day variations (N=10) for the same levels were 7.2% and 6.1%, respectively. The regression equation for this method in reference to the HPLC method (Y=0.99X+10.34 for N=34, r=0.98, S_y/x=36.1) had a slope of near unity and an insignificant y-intercept, although values less than 50 μg/l were generally slightly higher by the fluorometric method than by HPLC. The reference range for serum ALA was 0-79.4 μg/l (35.2±22.1, mean±S.D., N=42), with a distribution skewed to the left because levels in eight subjects were below the detection limit. Conclusions: This simple, fluorometric method for serum ALA correlated well with the results of the HPLC method, and may be suitable for assessing biochemical expression of acute porphyrias and response to treatment especially when HPLC is not available.