Dynamics of absorption, metabolism, and excretion of 5-aminolevulinic acid in human intestinal Caco-2 cells

Abstract

5-Aminolevulinic acid (ALA) is a precursor for the biosynthesis of porphyrins and heme. Although the oral administration of ALA has been widely applied in clinical settings, the dynamics of its absorption, metabolism, and excretion within enterocytes remain unknown. In this study, after enterocytic differentiation, Caco-2 cells were incubated with 200 µM ALA and/or 100 µM sodium ferrous citrate (SFC) for up to 72 h. Both ALA and the combination of ALA and SFC promoted the synthesis of heme, without affecting the expression of genes involved in intestinal iron transport, such as DMT1 and FPN. The enhanced heme synthesis in Caco-2 cells was more pronounced under the effect of the combination of ALA and SFC than under the effect of ALA alone, as reflected by the induced expression of heme oxygenase 1 (HO-1), as well as a reduced protein level of the transcriptional corepressor Bach1. Chromatin immunoprecipitation analysis confirmed Bach1 chromatin occupancy at the enhancer regions of HO-1, which were significantly decreased by the addition of ALA and SFC. Finally, Transwell culture of Caco-2 cells suggested that the administered ALA to the intestinal lumen was partially transported into vasolateral space. These findings enhance our understanding of the absorption and metabolism of ALA in enterocytes, which could aid in the development of a treatment strategy for various conditions such as anemia.

Keywords: 5-Aminolevulinic acid; ALA, 5-aminolevulinic acid; ALAS2, 5-aminolevulinic acid synthase 2; Bach1; CSA, congenital sideroblastic anemia; Caco-2 cell; ChIP, chromatin immunoprecipitation; DMT1, divalent metal transporter 1; FPN, ferroportin; HO-1, heme oxygenase 1; Heme oxygenase 1; PP IX, protoporphyrin IX; RT-PCR, reverse transcription polymerase chain reaction; SFC, sodium ferrous citrate.

Figures

Fig. 1

Differentiation of human intestinal Caco-2 cells. Quantitative RT-PCR analysis of DMT1, FPN, and HO-1 in Caco-2 cells during days 7–21 after seeding. Data are expressed as mean ± standard error (SE). n = 3; *, p

Fig. 2

ALA treatment enhances heme biosynthesis and changes intracellular iron status. A: Cellular pellets (top) and heme levels (bottom) of Caco-2 cells treated with ALA (200 μM) and/or SFC (100 μM) for 72 h. A representative data was shown among 3 independent experiments. B: Western blot analysis of ferritin (left), HO-1 (center), and Bach1 (right). α-tubulin was used as a loading control. C: Quantitative ChIP analysis to detect endogenous Bach1 occupancy at HO-1 enhancers (i.e., the distal E2 and proximal E1 enhancers), based on control and ALA/SFC-treated Caco-2 cells. The RPII215 promoter and the NECDIN promoter were used as negative controls. Data are expressed as mean ± standard error (SE). n = 3; *, p

Fig. 3

ALA did not affect the expression of intestinal iron transporters and minimally changed ALA transporters. A: Quantitative RT-PCR analysis of DMT1, FPN, HO-1, and ALAS1 in Caco-2 cells treated with ALA (200 μM) and/or SFC (100 μM) for 72 h. Data are expressed as mean ± standard error (SE). n = 3; *, p

Fig. 4

Iron administration did not affect heme, ALA, and porphyrin concentrations in medium. A: Chemiluminescent assay to detect heme concentration of medium. HPLC to detect ALA and porphyrin concentrations in medium. N = 3. B: The calculated distribution of ALA in medium after administration. Assuming that the amount of administered ALA is 100%, ALA collection rate is calculated as the percentage of ALA in each compartment 3 days after administration. Total porphyrin equivalent of ALA is the amount of total porphyrin converted to ALA.