Uptake and metabolism of β-apo-8'-carotenal, β-apo-10'-carotenal, and β-apo-13-carotenone in Caco-2 cells

Abstract

β-Apocarotenoids are eccentric cleavage products of carotenoids formed by chemical and enzymatic oxidations. They occur in foods containing carotenoids and thus might be directly absorbed from the diet. However, there is limited information about their intestinal absorption. The present research examined the kinetics of uptake and metabolism of β-apocarotenoids. Caco-2 cells were grown on 6-well plastic plates until a differentiated cell monolayer was achieved. β-Apocarotenoids were prepared in Tween 40 micelles, delivered to differentiated cells in serum-free medium, and incubated at 37°C for up to 8 h. There was rapid uptake of β-apo-8'-carotenal into cells, and β-apo-8'-carotenal was largely converted to β-apo-8'-carotenoic acid and a minor metabolite that we identified as 5,6-epoxy-β-apo-8'-carotenol. There was also rapid uptake of β-apo-10'-carotenal into cells, and β-apo-10'-carotenal was converted into a major metabolite identified as 5,6-epoxy-β-apo-10'-carotenol and a minor metabolite that is likely a dihydro-β-apo-10'-carotenol. Finally, there was rapid cellular uptake of β-apo-13-carotenone, and this compound was extensively degraded. These results suggest that dietary β-apocarotenals are extensively metabolized in intestinal cells via pathways similar to the metabolism of retinal. Thus, they are likely not absorbed directly from the diet.

Keywords: absorption; carotenoid metabolism; cell uptake; dietary lipids; intestine; nutrition/lipids; vitamin A; β-carotene.

Fig. 1.

Structures of the products of the central and eccentric cleavage of β-carotene. All-trans-β-carotene can be cleaved by BCO1 to yield two molecules of retinal, which can be converted to retinol or RA. On the other hand, the main product of the eccentric cleavage pathway of β-carotene is β-apo-10′-carotenal, and this reaction is catalyzed by BCO2. Also, β-carotene can undergo chemical and enzymatic oxidations in vivo and in foods to yield other long-chain and short-chain β-apocarotenoids. The exact mechanisms and enzymes responsible for these products are unknown.

Fig. 2.

Stability of β-apocarotenoids in cell culture conditions. Serum-free medium containing Tween 40 micelles of 1 μM (A) or 5 μM (B) of individual β-apocarotenoids (β-apo-8′-carotenal, β-apo-10′-carotenal, and β-apo-13-carotenone) were incubated at 37°C for time points up to 8 h. Following incubation, β-apocarotenoids were extracted from the medium and analyzed by HPLC-DAD. Percent recovery at each time point was calculated relative to the initial concentration of β-apocarotenoids in cell culture medium at the start of the stability experiment.

Fig. 3.

HPLC-DAD analysis of cell extract after incubation with β-apo-8′-carotenal. A: Representative HPLC-DAD chromatogram of the uptake of 1 μM of β-apo-8′-carotenal into fully differentiated Caco-2 cells after incubation at 37°C for 1 h. B: UV-vis spectra of β-apo-8′-carotenal and its cellular metabolites, the major one of which was confirmed as β-apo-8′-carotenoic acid (compound X) and the other we identified as 5,6-epoxy-β-apo-8′-carotenol (compound Y).

Fig. 4.

UHPLC-MS analysis of cellular metabolites of β-apo-8′-carotenal. A: UHPLC separation of molecules in Caco-2 cells incubated with 1 μM of β-apo-8′-carotenal for 1 h prior to accurate mass analysis. B: Determination of the accurate masses for the cellular metabolites of β-apo-8′-carotenal by QTOF MS. The major peak, compound X, differed from β-apo-8′-carotenal by a mass of +16, while the minor peak, compound Y, differed from β-apo-8′-carotenal by a mass of +18. Based on accurate mass, retention time, and UV absorption spectra, compound X was identified as β-apo-8′-carotenoic acid, while compound Y is 5,6-epoxy- β-apo-8′-carotenol. An * indicates acquisition time, while ** indicates accurate mass for each peak shown.

Fig. 5.

A: Time course of uptake and metabolism of β-apo-8′-carotenal in Caco-2 cells. B: Effect of incubation time on the disappearance of β-apo-8′-carotenal from medium. Differentiated Caco-2 cells were incubated with serum-free medium containing 1 μM of β-apo-8′-carotenal at 37°C for 30 min, 1 h, 4 h, and 8 h. After incubation, the extracts from medium and cells were analyzed using HPLC-DAD. The results shown represent an individual experiment and the points are the mean ± standard deviation of three or more wells. Furthermore, the data shown in A have been confirmed with two independent experiments.

Fig. 6.

Dose-dependent uptake and metabolism of β-apo-8′-carotenal in Caco-2 cells. Differentiated Caco-2 cells were incubated with serum-free medium containing 1, 2, 3, 4, or 5 μM of β-apo-8′-carotenal at 37°C for 4 h. After incubation, the extracts from cells were analyzed using HPLC-DAD. The results shown represent an individual experiment and the points are the mean ± standard deviation of three or more wells. Furthermore, the data shown above have been confirmed with three independent experiments.

Fig. 7.

HPLC-DAD analysis of cell extract after incubation with β-apo-10′-carotenal. A: Representative HPLC-DAD chromatogram of the uptake of 1 μM of β-apo-10′-carotenal into fully differentiated Caco-2 cells after incubation at 37°C for 4 h. B: UV-vis spectra of β-apo-10′-carotenal and its cellular metabolites, the major one of which was identified as 5,6-epoxy-β-apo-10′-carotenol (compound A) and the other is likely a dihydro-β-apo-10′-carotenol (compound B).

Fig. 8.

HPLC-DAD analysis of possible cellular metabolites of β-apo-10ʹ-carotenal. (A): Overlap of HPLC-DAD chromatograms for reference compounds of known retinoids and β-apocarotenoids. (B): UV-vis spectra for the retinoids and β-apocarotenoids analyzed by HPLC-DAD. 13-ketone, β-apo-13-carotenone; atRA, all-trans-RA; atRAL, all-trans-retinal; 12′AL, β-apo-12′-carotenal; 10′CA, β-apo-10′-carotenoic acid; 10′OL, β-apo-10′-carotenol; 10′AL, β-apo-10′-carotenal.

Fig. 9.

A: Time course of uptake and metabolism of β-apo-10′-carotenal in Caco-2 cells. B: Effect of incubation time on the disappearance of β-apo-10′-carotenal from medium. Differentiated Caco-2 cells were incubated with serum-free medium containing 1 μM of β-apo-10′-carotenal at 37°C for 15, 30, 45, and 60 min. After incubation, the extracts from medium and cells were analyzed by HPLC-DAD. The results shown represent an individual experiment and the points are the mean ± standard deviation of three or more wells. Furthermore, the data shown in A have been confirmed with two independent experiments.

Fig. 10.

HPLC-DAD analysis of cell extract after incubation with β-apo-13-carotenone. A: Representative HPLC-DAD chromatogram of the uptake of 5 μM of β-apo-13-carotenone into fully differentiated Caco-2 cells after incubation at 37°C for either 30 min or 8 h. B: UV-vis spectra of β-apo-13-carotenone at both incubation time points. No cellular metabolites were detected.

Fig. 11.

A: Time course of uptake of β-apo-13-carotenone into Caco-2 cells. B: Effect of incubation time on the disappearance of β-apo-13-carotenone from medium. Differentiated Caco-2 cells were incubated with serum-free medium containing 5 μM of β-apo-13-carotenone at 37°C for time points up to 8 h. After incubation, the extracts from medium and cells were analyzed by HPLC-DAD. The inset graph in A shows the uptake of β-apo-13-carotenone into cells at time points up to 1 h. No peaks were detected in medium after 4 or 8 h of incubation. The results shown represent an individual experiment and the points are the mean ± standard deviation of three or more wells. Furthermore, the data shown in A have been confirmed with two independent experiments.

Fig. 12.

Dose-dependent uptake of β-apo-13-carotenone into Caco-2 cells. Differentiated Caco-2 cells were incubated with serum-free medium containing 1, 2, 3, 4, or 5 μM of β-apo-13-carotenone at 37°C for 45 min. After incubation, the extracts from cells were analyzed using HPLC-DAD. The results shown represent an individual experiment and the points are the mean ± standard deviation of three or more wells. Furthermore, the data shown above have been confirmed with two independent experiments.

Fig. 13.

Structures of the metabolites in the metabolic pathway of β-apo-8′-carotenal in Caco-2 cells. There was rapid uptake of β-apo-8′-carotenal into cells, and β-apo-8′-carotenal was largely converted to β-apo-8′-carotenoic acid. Several studies show that this apocarotenoic acid is a major metabolite present in the serum and tissues of animals fed β-apo-8ʹ-carotenal (38, 39). This metabolic conversion is similar to what is observed with retinal, which can be converted to retinol or RA. β-Apo-8′-carotenal was also converted to a minor metabolite that was identified as 5,6-epoxy-β-apo-8′-carotenol, which is analogous to the formation of epoxy metabolites of vitamin A in vivo (41, 42).

Fig. 14.

Structures of the metabolites in the metabolic pathway of β-apo-10′-carotenal in Caco-2 cells. There was rapid uptake of β-apo-10′-carotenal into cells, and β-apo-10′-carotenal was converted to a major metabolite that we identified as 5,6-epoxy-β-apo-10′-carotenol and a minor metabolite that is likely a dihydro-β-apo-10′-carotenol. Previous studies have suggested that β-apo-10′-carotenol, β-apo-10′-carotenoic acid, and β-apo-12′-carotenoic acid are detected in serum and tissues of animals given β-apo-10′-carotenal as the only source of provitamin A activity (39). However, in the present study, we did not detect any of these metabolites.