A method for determining δ-aminolevulinic acid synthase activity in homogenized cells and tissues

Abstract

Objective: In mammalian cells the rate-limiting step in heme biosynthesis is the formation of δ-aminolevulinic acid (ALA). The reaction intermediates, porphyrins and iron and the final product, heme can be highly cytotoxic if allowed to accumulate. The importance of maintaining the levels of metabolic intermediates and heme within a narrow range is apparent based on the complex homeostatic system(s) that have developed. Ultimately, determining the enzymatic activity of ALA synthase (ALAS) present in the mitochondria is highly beneficial to confirm the effects of the transcriptional, translational and post-translational events. The aim of this study was to develop a highly sensitive assay for ALAS that could be used on whole tissue or cellular homogenates.

Design and methods: A systematic approach was used to optimize steps in formation of ALA by ALAS. Reducing the signal to noise ratio for the assay was achieved by derivatizing the ALA formed into a fluorescent product that could be efficiently separated by ultra performance liquid chromatography (UPLC) from other derivatized primary amines. The stability of ALAS activity in whole tissue homogenate and cellular homogenate was determined after extended storage at -80 °C.

Conclusions: A method for assaying ALAS has been developed that can be used with tissue homogenates or cellular lysates. There is no need to purify mitochondria and radiolabeled substrates are not needed for this assay. General laboratory reagents can be used to prepare the samples. Standard UPLC chromatography will resolve the derivatized ALA peak. Samples of tissue homogenate can be stored for approximately one year without significant loss of enzymatic activity.

Keywords: Aminolevulinic acid; Heme biosynthesis; Mitochondrial assay; Mouse erythroleukemia cell.

Figure 1

Influence of buffers on derivatization of ALA. The area under the curve (V*sec) is plotted against ALA concentration in μM. (A) Buffers at a 50 mM concentration are optimal for the initial ALAS reaction, potassium phosphate (KPi). Tris, HEPES and phosphate buffered saline (PBS) were titrated to pH 7.4 and tested to determine if there was any effect on the ability to convert ALA (0.5, 1.0, 1.5 and 2.0 μM) to 2,6-diacetyl-1,5-dimethyl-7-(2-carboxyethyl)-3-pyrrolizine. (B) Assay mixtures from (A) were diluted 10X with water and then derivatized.

Figure 2

Fluorescence UPLC chromatograms of ALA derivative with and without addition of 0.5 μM authentic ALA. The energy of the truncated glycine derivative peak was 121 (Y axis). The energy peak heights for 0.0 and 0.5 μM ALA were at 1.1 and 2.5, respectively.

Figure 3

Stability of the ALA derivative under various conditions between formation and UPLC. Assays for activity of ALAS were set up with a volume of 50 μL ALAS assay buffer (50 mM potassium phosphate pH 7.4, 50 mM glycine, 100 μM succinyl CoA, 40 μM 5′-pyridoxal phosphate and 50 μM succinylacetone). The mixture was spiked with either 0.1 or 1.0 μM ALA, and then diluted with 450 μL ice-cold water. A 50 μL aliquot of the diluted ALAS assay sample was derivatized analyzed by UPLC as described above.

Figure 4

Dependence of ALA synthesis on total liver protein. Livers were homogenized in 0.25 M sucrose and then diluted, 2.5 to 17.5 mg protein/mL. Samples were then analyzed for ALAS activity with the optimal concentrations of reactants (50 mM glycine, 50 μM succinylacetone, 100 μM succinyl CoA, 40 μM pyridoxal 5′-phosphate, 50 mM potassium phosphate pH 7.4).

Figure 5

ALAS activity assay at different concentrations of glycine. Activity of ALAS was measured with 0 mM, 10 mM, 20 mM 40 mM and 80 mM 160 mM added glycine in the reaction mixture. Activity was maximal at 40 mM glycine, with a declining rate after that.

Figure 6

Time course for ALA production by ALAS present in a mouse liver homogenate. ALAS activity was measured with the optimal concentrations of reactants (50 mM glycine, 50 μM succinylacetone, 100 μM succinyl-CoA, 40 μM pyridoxal 5′-phosphate, 50 mM potassium phosphate pH 7.4, 12 mg/mL protein) for 0, 15, 30, 45 and 60 min.

Figure 7

Activity of ALAS in Mouse Erythroleukemia (MEL) cells induced with DMSO. Activity of ALAS was measured in MEL cells at 24, 48, 72 and 96 h after induction with 1.5% DMSO. Each sample was harvested from 30 mL of MEL cells. Activity is calculated as pmol ALA produced per mg total protein per hour. Three independent cultures were induced with DMSO, samples were collected at the indicated time points for each culture.

Scheme 1

Enzymatic synthesis of δ-aminolevulinic acid. Many ALAS assays have been described in the past few decades, but progress in chemical methods coupled with more recent instrumentation technology can give faster, simpler and more sensitive assays. Newer methods for quantification of ALA have also been published, but many of these are suboptimal for use in conjunction with an ALAS assay.

Scheme 2

Hantzsch derivatization of δ-aminolevulinic acid. δ-Aminolevulinic acid reacts with 2 molecules of acetylacetone and 1 molecule of formaldehyde to form the fluorescent derivative 2,6-diacetyl-1,5-dimethyl-7-(2-carboxyethyl)-3-pyrrolizine with an excitation maximum of 370 nm and an emission peak at 460 nm.